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WO2023164623A2 - Appareils, procédés et kits pour dosages microfluidiques - Google Patents

Appareils, procédés et kits pour dosages microfluidiques Download PDF

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Publication number
WO2023164623A2
WO2023164623A2 PCT/US2023/063233 US2023063233W WO2023164623A2 WO 2023164623 A2 WO2023164623 A2 WO 2023164623A2 US 2023063233 W US2023063233 W US 2023063233W WO 2023164623 A2 WO2023164623 A2 WO 2023164623A2
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WO
WIPO (PCT)
Prior art keywords
chamber
micro
microfluidic device
microns
area
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2023/063233
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English (en)
Other versions
WO2023164623A9 (fr
WO2023164623A3 (fr
Inventor
Patrick N. INGRAM
Alexander CHIEN
Ke-Chih Lin
Or GADISH
Eric K. SACKMANN
Hansohl E. KIM
Tejasvi JALADI
Grayson Thomas WAWRZYN
Troy A. LIONBERGER
Volker L.S. KURZ
Alexander J. Mastroianni
JR. Randall D. LOWE
Jonathan Cloud Dragon Hubbard
Peyton Shieh
Lily CHAO
Bo Hu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bruker Cellular Analysis Inc
Original Assignee
Berkeley Lights Inc
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Filing date
Publication date
Application filed by Berkeley Lights Inc filed Critical Berkeley Lights Inc
Publication of WO2023164623A2 publication Critical patent/WO2023164623A2/fr
Publication of WO2023164623A3 publication Critical patent/WO2023164623A3/fr
Anticipated expiration legal-status Critical
Publication of WO2023164623A9 publication Critical patent/WO2023164623A9/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

Definitions

  • Oxygen consumption levels and/or acidification of a culture environment may be correlated with the health, viability, and/or productivity of a population of cells. Thus, it may be critical to measure oxygen levels and pH within culture systems and/or monitor cellular oxygen consumption and pH change in order to assess such parameters.
  • current microfluidic cell culture systems generally do not provide the ability to measure and monitor those parameters. Therefore, there is a need for systems and methods for measuring oxygen levels and/or pH, including methods that allow for measuring oxygen levels and/or pH at multiple locations or even throughout such microfluidic cell culture systems. There is also a need for systems and methods for monitoring oxygen consumption and/or acidification by cells being cultured in such microfluidic devices.
  • a method of monitoring a local pH of a medium spatially distributed within a microfluidic device comprises an enclosure, wherein the enclosure comprises a flow region and a chamber having a proximal opening fluidically connecting the chamber to the flow region, the method comprising: introducing a pH-sensitive molecule into the microfluidic device; and detecting a signal associated with the pH-sensitive molecule in an area of interest within the enclosure.
  • a non-transitory computer-readable medium in which a program is stored for causing a system comprising a computer to perform a method of monitoring a local pH of a medium spatially distributed within a microfluidic device of the present disclosure is provided.
  • a method of ranking a micro-object population within a microfluidic device is provided.
  • the method comprises introducing the micro-object population into the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a flow region and a plurality of chambers, wherein each of the plurality of the chamber is fluidically connected through a proximal opening to the flow region; disposing individual micro-object of the micro-object population into a respective chamber of the plurality of chamber resulting in disposed micro-objects within respective chambers; allowing the disposed micro-objects to produce a molecule of interest; introducing a pH- sensitive molecule into the microfluidic device; detecting a first signal associated with the pH- sensitive molecule in a first area of interest within respective chamber; and ranking the disposed micro-objects based on the first signals detected in the respective chambers.
  • a non-transitory computer-readable medium in which a program is stored for causing a system comprising a computer to perform a method of ranking a microobject population within a microfluidic device of the present disclosure is provided.
  • FIG. 1A illustrates a microfluidic device and a system with associated control equipment according to some embodiments of the disclosure.
  • FIG. IB illustrates a microfluidic device with sequestration pens according to an embodiment of the disclosure.
  • FIGs. 2A-2B illustrate a microfluidic device having sequestration pens according to some embodiments of the disclosure.
  • FIG. 2A depicts a vertical cross-section of microfluidic device according to some embodiments of the disclosure.
  • FIG. 2B shows a horizontal cross-section of microfluidic device according to some embodiments of the disclosure.
  • FIG. 2C illustrates a sequestration pen of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 3 illustrates a sequestration pen of a micro fluidic device according to some embodiments of the disclosure.
  • FIGs. 4A-4B illustrate electrokinetic features of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 4A shows a side cross-sectional view of a portion of an enclosure of the microfluidic device according to some embodiments of the disclosure.
  • FIG. 4B shows a top cross-sectional view of a portion of an enclosure of the microfluidic device according to some embodiments of the disclosure.
  • FIG. 5A illustrates a system for use with a microfluidic device and associated control equipment according to some embodiments of the disclosure.
  • FIG. 5B illustrates an imaging device according to some embodiments of the disclosure.
  • FIG. 6 illustrates heatmaps of normalized fluorescence intensity categorized by perfusion conditions at various timestamps of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 7A illustrates heatmaps of normalized oxygen level/consumption at a specified perfusion condition at various timestamps of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 7B illustrates heatmaps of normalized oxygen level/consumption at various perfusion conditions at a fixed perfusion period of time of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 8 is a graphical representation showing normalized oxygen level/consumption as a function of biomass (e.g., a population of biological micro-objects) for sequestration pens of a microfluidic device according to some embodiments of the disclosure.
  • biomass e.g., a population of biological micro-objects
  • FIGs. 9A-9B illustrate normalized fluorescence intensity as a function of oxygen level in channels of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 9A shows a plot of normalized fluorescence intensity taken during standard perfusion in accordance with some embodiments of the present disclosure.
  • FIG. 9B shows a plot of normalized fluorescence intensity taken without perfusion in accordance with some embodiments of the present disclosure.
  • FIGs. 9C-9D illustrate normalized fluorescence intensity as a function of oxygen level in sequestration pens of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 9C shows a plot of normalized fluorescence intensity taken during standard perfusion in accordance with some embodiments of the present disclosure.
  • FIG. 9D shows a plot of normalized fluorescence intensity taken without perfusion in accordance with some embodiments of the present disclosure.
  • FIG. 10 illustrates a flow chart for an example method of determining oxygen consumption level in a population of biological micro-objects in sequestrations pens according to various embodiments of the present disclosure.
  • FIG. 11 illustrates an example approach of converting acquired fluorescence images into data for correlating fluorescence of an AOI to a reference to determine the dissolved oxygen level in accordance with some embodiments of the present disclosure.
  • FIG. 12A illustrates a first example approach of generating a dissolved oxygen (DO) standard curve in accordance with some embodiments of the present disclosure.
  • FIG. 12B illustrates a second example approach of generating a DO standard curve in accordance with some embodiments of the present disclosure.
  • FIG. 13 illustrates exemplary DO standard curves generated by the process of FIG. 12A in accordance with some embodiments of the present disclosure.
  • FIG. 14 illustrates a first example approach of performing a DO perfusion assay in accordance with some embodiments of the present disclosure.
  • FIG. 15 illustrates a second example approach of performing a DO perfusion assay in accordance with some embodiments of the present disclosure.
  • FIG. 16 shows an oxygen delivery system comprising one or more tubes with one or more holes (or lumens) in accordance with some embodiments of the present disclosure.
  • FIG. 17 shows the variability of the normalized fluorescence intensity at a 0.4 mg/mL dye concentration in accordance with some embodiments of the present disclosure.
  • FIGs. 18A-18B show the improvement in dissolved oxygen uniformity achieved by sealing the microfluidic chip from external gas exchange using Parylene in accordance with some embodiments of the present disclosure.
  • FIG. 18A shows an exemplary average normalized intensity of dissolved oxygen fluorescence signal in a sequestration pen of a Parylene-sealed microfluidic device in accordance with some embodiments of the present disclosure.
  • FIG. 18B shows an exemplary average normalized intensity of dissolved oxygen fluorescence signal in a channel of a Parylene- sealed microfluidic device in accordance with some embodiments of the present disclosure.
  • FIGs. 19A-19B show the different performance levels of various sealing techniques in limiting external gas exchange to improve dissolved oxygen uniformity in accordance with some embodiments of the present disclosure.
  • FIG. 19A shows an exemplary average normalized intensity of dissolved oxygen fluorescence signal in sequestration pens of microfluidic chips sealed using Torr Seal®, placed in a gas bath, and sealed using Parylene in accordance with some embodiments of the present disclosure.
  • FIG. 19B shows an exemplary average normalized intensity of dissolved oxygen fluorescence signal in channels of microfluidic chips sealed using Torr Seal®, placed in a gas bath, and sealed using Parylene in accordance with some embodiments of the present disclosure.
  • FIGs. 20A-20B show an example of how the above-described non-uniformities in external gas exchange impact the dissolved oxygen signal as observed over the whole chip in accordance with some embodiments of the present disclosure.
  • FIG. 20A shows an exemplary uniformity of dissolved oxygen fluorescence signal across sequestration pens of microfluidic chips sealed using Torr Seal®, placed in a gas bath, and sealed using Parylene in accordance with some embodiments of the present disclosure.
  • FIG. 20B shows an exemplary uniformity of dissolved oxygen fluorescence signal across channels of microfluidic chips sealed using Torr Seal®, placed in a gas bath, and sealed using Parylene in accordance with some embodiments of the present disclosure.
  • FIG. 21 A shows an exemplary brightfield image of cells in sequestration pens in accordance with some embodiments of the present disclosure.
  • FIG. 2 IB shows an exemplary fluorescence image of dissolved oxygen in the sequestration pens in accordance with some embodiments of the present disclosure.
  • FIG. 22 shows a system configured to implement the methods described herein in accordance with some embodiments of the present disclosure.
  • FIG. 23 illustrates a flow chart for an example method of monitoring a local pH of a medium spatially distributed within a microfluidic device.
  • FIG. 24 illustrates a flow chart for an example method of selecting a biological microobject.
  • FIG. 25A illustrates an exterior appearance and an internal configuration of an example microfluidic chip described herein.
  • FIG. 25B shows the fluorescent intensities of fluorescent signals obtained from twelve microfluidic devices comprising solutions of pH 3, 3.4, 3.6, 3.8, 4, 4.4, 4.6, 4.8, 5, 5.4, 5.6, and 6 respectively.
  • the fluorescent intensities are represented in color intensities at a scale of 0 to 55K.
  • FIG. 25C shows a histogram illustrating the normalized fluorescent intensities derived from the fluorescent signals obtained from the twelve microfluidic devices in FIG.
  • FIG. 26 illustrates the standard curves of pH measurement obtained from the twelve microfluidic devices in FIG. 25B and from a corresponding well plate experiment.
  • FIG. 27A shows a fluorescent image of panels of sequestration pens of a microfluidic device comprising a pH-sensitive molecule and biological micro-objects within the pens before any pH change.
  • FIG. 27B shows a fluorescent image of the same view as the fluorescent image of FIG. 27 A taken after a certain time of culturing.
  • FIG. 28 illustrates the traces of pH changes of every sequestration pen in the experiment of FIG. 27A and FIG. 27B over 15 minutes.
  • FIG. 29 shows a histogram illustrating the fluorescence intensities detected from the culture of the four strains (high production, medium production, low production, zero production) at Min. 0, Min. 9, and Min 14 into the pH acidification assay.
  • FIG. 30 shows a pH standard curve obtained from the on-chip pH measurement of Example 3.
  • FIG. 31A shows a schematic representation of a configuration of a microfluidic channel and a chamber having an in situ-generated barrier formed therewithin.
  • the in situ- generated barrier defines a culture area and an assay area within the chamber. Two areas of interest are indicated as the two dotted rectangles.
  • FIG. 3 IB shows a brightfield image, corresponding to the configuration shown in FIG. 31 A, before disposing a micro-object into the chamber.
  • FIG. 31C shows a brightfield image, corresponding to the configuration shown in FIG. 31 A, after micro-objects are disposed and cultured within the chamber.
  • FIG. 32 shows acidification of media in a time lapse measurement represented by true pH value calculated using the standard curve of FIG. 30.
  • FIG. 33A is a schematic representation of a cross-section view of a chamber showing a height of the chamber and a depth of the area of interest.
  • FIG. 33B is another schematic representation of a cross-section view of a chamber having a micro-object disposed therewithin showing a height of the chamber and a depth of the area of interest.
  • one element e.g., a material, a layer, a substrate, etc.
  • one element can be “on,” “attached to,” “connected to,” or “coupled to” another element regardless of whether the one element is directly on, attached to, connected to, or coupled to the other element or there are one or more intervening elements between the one element and the other element.
  • microfluidic features are described as having a width or an area, the dimension typically is described relative to an x-axial and/or y-axial dimension, both of which lie within a plane that is parallel to the substrate and/or cover of the microfluidic device.
  • the height of a microfluidic feature may be described relative to a z-axial direction, which is perpendicular to a plane that is parallel to the substrate and/or cover of the microfluidic device.
  • a cross sectional area of a microfluidic feature such as a channel or a passageway, may be in reference to a x-axial/z-axial, a y-axial/z-axial, or an x-axial/y-axial area.
  • substantially means sufficient to work for the intended purpose.
  • the term “substantially” thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance.
  • substantially means within ten percent.
  • ones means more than one.
  • the term “plurality” can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • pm means micrometer
  • pm 3 means cubic micrometer
  • pL means picoliter
  • nL means nanoliter
  • pL (or uL) means microliter
  • air refers to the composition of gases predominating in the atmosphere of the earth.
  • gases typically include nitrogen (typically present at a concentration of about 78% by volume, e.g., in a range from about 70-80%), oxygen (typically present at about 20.95% by volume at sea level, e.g. in a range from about 10% to about 25%), argon (typically present at about 1.0% by volume, e.g. in a range from about 0.1% to about 3%), and carbon dioxide (typically present at about 0.04%, e.g., in a range from about 0.01% to about 0.07%).
  • nitrogen typically present at a concentration of about 78% by volume, e.g., in a range from about 70-80%
  • oxygen typically present at about 20.95% by volume at sea level, e.g. in a range from about 10% to about 25%
  • argon typically present at about 1.0% by volume, e.g. in a range from about 0.1% to about 3%
  • carbon dioxide typically present at
  • Air may have other trace gases such as methane, nitrous oxide or ozone, trace pollutants and organic materials such as pollen, diesel particulates and the like. Air may include water vapor (typically present at about 0.25% or may be present in a range from about lOppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein.
  • trace gases such as methane, nitrous oxide or ozone
  • trace pollutants and organic materials such as pollen, diesel particulates and the like.
  • Air may include water vapor (typically present at about 0.25% or may be present in a range from about lOppm to about 5% by volume). Air may be provided for use in culturing experiments as a filtered, controlled composition and may be conditioned as described herein.
  • a “microfluidic device” or “microfluidic apparatus” is a device that includes one or more discrete microfluidic circuits configured to hold a fluid, each microfluidic circuit comprised of fluidically interconnected circuit elements, including but not limited to region(s), flow path(s), channel(s), chamber(s), and/or pen(s), and at least one port configured to allow the fluid (and, optionally, micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device.
  • a microfluidic circuit of a microfluidic device will include a flow region, which may include a microfluidic channel, and at least one chamber, and will hold a volume of fluid of less than about 1 mL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 uL.
  • the microfluidic circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 uL.
  • the microfluidic circuit may be configured to have a first end fluidically connected with a first port (e.g., an inlet) in the microfluidic device and a second end fluidically connected with a second port (e.g., an outlet) in the microfluidic device.
  • a “nanofluidic device” or “nanofluidic apparatus” is a type of microfluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 1 uL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less.
  • a nanofluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more).
  • circuit elements e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more).
  • one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL.
  • one or more (e.g., all) of the at least one circuit elements are configured to hold a volume of fluid of about 20 nL to 200 nL, 100 to 200 nL, 100 to 300 nL, 100 to 400 nL, 100 to 500 nL, 200 to 300 nL, 200 to 400 nL, 200 to 500 nL, 200 to 600 nL, 200 to 700 nL, 250 to 400 nL, 250 to 500 nL, 250 to 600 nL, or 250 to 750 nL.
  • a microfluidic device or a nanofluidic device may be referred to herein as a “microfluidic chip” or a “chip”; or “nanofluidic chip” or “chip”.
  • a “microfluidic channel” or “flow channel” as used herein refers to flow region of a microfluidic device having a length that is significantly longer than both the horizontal and vertical dimensions.
  • the flow channel can be at least 5 times the length of either the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer.
  • the length of a flow channel is about 100,000 microns to about 500,000 microns, including any value therebetween.
  • the horizontal dimension is about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension is about 25 microns to about 200 microns, (e.g., from about 40 to about 150 microns).
  • a flow channel may have a variety of different spatial configurations in a microfluidic device, and thus is not restricted to a perfectly linear element.
  • a flow channel may be, or include one or more sections having, the following configurations: curve, bend, spiral, incline, decline, fork (e.g., multiple different flow paths), and any combination thereof.
  • a flow channel may have different cross-sectional areas along its path, widening and constricting to provide a desired fluid flow therein.
  • the flow channel may include valves, and the valves may be of any type known in the art of microfluidic s. Examples of microfluidic channels that include valves are disclosed in U.S. Patents 6,408,878 and 9,227,200, each of which is herein incorporated by reference in its entirety.
  • the term “obstruction” refers generally to a bump or similar type of structure that is sufficiently large so as to partially (but not completely) impede movement of target micro-objects between two different regions or circuit elements in a microfluidic device.
  • the two different regions/circuit elements can be, for example, a microfluidic sequestration pen and a microfluidic channel, or a connection region and an isolation region of a microfluidic sequestration pen.
  • constriction refers generally to a narrowing of a width of a circuit element (or an interface between two circuit elements) in a microfluidic device.
  • the constriction can be located, for example, at the interface between a microfluidic sequestration pen and a microfluidic channel, or at the interface between an isolation region and a connection region of a microfluidic sequestration pen.
  • transparent refers to a material which allows visible light to pass through without substantially altering the light as is passes through.
  • whitefield illumination and/or image refers to white light illumination of the microfluidic field of view from a broad- spectrum light source, where contrast is formed by absorbance of light by objects in the field of view.
  • structured light is projected light that is modulated to provide one or more illumination effects.
  • a first illumination effect may be projected light illuminating a portion of a surface of a device without illuminating (or at least minimizing illumination of) an adjacent portion of the surface, e.g., a projected light pattern, as described more fully below, used to activate DEP forces within a DEP substrate.
  • the intensity e.g., variation in duty cycle of a structured light modulator such as a DMD, may be used to change the optical power applied to the light activated DEP actuators, and thus change DEP force without changing the nominal voltage or frequency.
  • structured light includes projected light that may be corrected for surface irregularities and for irregularities associated with the light projection itself, e.g., fall-off at the edge of an illuminated field.
  • Structured light is typically generated by a structured light modulator, such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like.
  • a structured light modulator such as a digital mirror device (DMD), a microshutter array system (MSA), a liquid crystal display (LCD), or the like.
  • Illumination of a small area of the surface, e.g., a selected area of interest with structured light improves the signal-to-noise-ratio (SNR), as illumination of only the selected area of interest reduces stray/scattered light, thereby lowering the dark level of the image.
  • SNR signal-to-noise-ratio
  • An important aspect of structured light is that it may be changed quickly over time.
  • a light pattern from the structured light modulator may be used to autofocus on difficult targets such as clean mirrors or surfaces that are far out of focus.
  • a clean mirror a number of self-test features may be replicated such as measurement of modulation transfer function and field curvature/tilt, without requiring a more expensive Shack-Hartmann sensor.
  • spatial power distribution may be measured at the sample surface with a simple power meter, in place of a camera.
  • Structured light patterns may also be used as a reference feature for optical module/system component alignment as well used as a manual readout for manual focus.
  • Another illumination effect made possible by use of structured light patterns is selective curing, e.g., solidification of hydrogels within the microfluidic device.
  • micro-object refers generally to any microscopic object that may be isolated and/or manipulated in accordance with the present disclosure.
  • Nonlimiting examples of micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, glass beads, amorphous solid substrates, LuminexTM beads, or the like); magnetic beads; microrods; microwires; quantum dots, and the like; biological micro-objects such as cells; biological organelles; vesicles, or complexes; synthetic vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts, and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., microbeads attached to cells, liposome-coated micro-beads, liposome-coated magnetic beads, or the like).
  • inanimate micro-objects such as microparticles
  • microbeads e.
  • Beads may include moieties/molecules covalently or non-covalently attached, such as fluorescence labels, proteins (including receptor molecules), carbohydrates, antigens, small molecule signaling moieties, or other chemical/biological species capable of use in an assay.
  • beads/solid substrates including moieties/molecules may be capture beads, e.g., configured to bind molecules including small molecules, peptides, proteins or nucleic acids present in proximity either selectively or nonselectively.
  • a capture bead may include a nucleic acid sequence configured to bind nucleic acids having a specific nucleic acid sequence or the nucleic acid sequence of the capture bead may be configured to bind a set of nucleic acids having related nucleic acid sequences. Either type of binding may be understood to be selective.
  • Capture beads containing moieties/molecules may bind nonselectively when binding of structurally different but physico-chemically similar molecules is performed, for example, size exclusion beads or zeolites configured to capture molecules of selected size or charge. Lipid nanorafts have been described, for example, in Ritchie et al. (2009) “Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,” Methods Enzymol., 464:211-231.
  • biological cells include eukaryotic cells, plant cells, animal cells, such as mammalian cells, reptilian cells, avian cells, fish cells, or the like, prokaryotic cells, bacterial cells, fungal cells, protozoan cells, or the like, cells dissociated from a tissue, such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like, immunological cells, such as T cells, B cells, natural killer cells, macrophages, and the like, embryos (e.g., zygotes), oocytes, ova, sperm cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, and the like.
  • tissue such as muscle, cartilage, fat, skin, liver, lung, neural tissue, and the like
  • immunological cells such as T cells, B cells, natural killer cells, macrophages, and the like
  • embryos e.g., zygotes
  • a mammalian cell can be, for example, from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, a primate, or the like.
  • a colony of biological cells is "clonal" if all of the living cells in the colony that are capable of reproducing are daughter cells derived from a single parent cell. In certain embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 10 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 14 divisions.
  • all the daughter cells in a clonal colony are derived from the single parent cell by no more than 17 divisions. In other embodiments, all the daughter cells in a clonal colony are derived from the single parent cell by no more than 20 divisions.
  • the term "clonal cells" refers to cells of the same clonal colony.
  • a “colony” of biological cells refers to 2 or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or greater than 1000 cells).
  • maintaining (a) cell(s) refers to providing an environment comprising both fluidic and gaseous components and, optionally a surface, that provides the conditions necessary to keep the cells viable and/or expanding.
  • expanding when referring to cells, refers to increasing in cell number.
  • gas permeable means that the material or structure is permeable to at least one of oxygen, carbon dioxide, or nitrogen. In some embodiments, the gas permeable material or structure is permeable to more than one of oxygen, carbon dioxide and nitrogen and may further be permeable to all three of these gases.
  • a “component” of a fluidic medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, or the like.
  • “diffuse” and “diffusion” refer to thermodynamic movement of a component of the fluidic medium down a concentration gradient.
  • flow of a medium means bulk movement of a fluidic medium primarily due to any mechanism other than diffusion, and may encompass perfusion.
  • flow of a medium can involve movement of the fluidic medium from one point to another point due to a pressure differential between the points.
  • Such flow can include a continuous, pulsed, periodic, random, intermittent, or reciprocating flow of the liquid, or any combination thereof.
  • Flowing can comprise pulling solution through and out of the microfluidic channel (e.g., aspirating) or pushing fluid into and through a microfluidic channel (e.g., perfusing).
  • substantially no flow refers to a rate of flow of a fluidic medium that, when averaged over time, is less than the rate of diffusion of components of a material (e.g., an analyte of interest) into or within the fluidic medium.
  • the ratio of a rate of flow of a component in a fluidic medium (i.e., advection) divided by the rate of diffusion of such component can be expressed by a dimensionless Peclet number.
  • the Peclet number associated with a particular region within the microfluidic device can vary with the component or components of the fluidic medium being considered (e.g., the analyte of interest), as the rate of diffusion of a component or components in a fluidic medium can depend on, for example, temperature, the size, mass, and/or shape of the component(s), and the strength of interactions between the component(s) and the fluidic medium.
  • the Peclet number associated with a particular region of the microfluidic device and a component located therein can be 0.95 or less, 0.9 or less, 0.85 or less, 0.8 or less, 0.75 or less, 0.7 or less, 0.65 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, 0.1 or less, 0.05 or less, 0.01 or less, 0.005 or less, or 0.001 or less.
  • fluidically connected means that, when the different regions are substantially filled with fluid, such as fluidic media, the fluid in each of the regions is connected so as to form a single body of fluid. This does not mean that the fluids (or fluidic media) in the different regions are necessarily identical in composition. Rather, the fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, carbohydrates, ions, or other molecules) which are in flux as solutes move down their respective concentration gradients and/or fluids flow through the device.
  • solutes such as proteins, carbohydrates, ions, or other molecules
  • a “flow path” refers to one or more fluidically connected circuit elements (e.g., channel(s), region(s), chamber(s) and the like) that define, and are subject to, the trajectory of a flow of medium.
  • a flow path is thus an example of a swept region of a microfluidic device.
  • Other circuit elements e.g., unswept regions
  • isolation a micro-object confines a micro-object to a defined area within the microfluidic device.
  • pen refers to disposing micro-objects within a chamber (e.g., a sequestration pen) within the microfluidic device.
  • Forces used to pen a microobject may be any suitable force as described herein such as dielectrophoresis (DEP), e.g., an optically actuated dielectrophoretic force (OEP); gravity; magnetic forces; or tilting.
  • DEP dielectrophoresis
  • OEP optically actuated dielectrophoretic force
  • gravity magnetic forces
  • tilting or tilting.
  • penning a plurality of micro-objects may reposition substantially all the microobjects.
  • a selected number of the plurality of micro-objects may be penned, and the remainder of the plurality may not be penned.
  • a DEP force e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects.
  • micro-objects may be introduced to a flow region, e.g., a microfluidic channel, of the microfluidic device and introduced into a chamber by penning.
  • unpen or “unpenning” refers to repositioning micro-objects from within a chamber, e.g., a sequestration pen, to a new location within a flow region, e.g., a microfluidic channel, of the microfluidic device.
  • Forces used to unpen a micro-object may be any suitable force as described herein such as dielectrophoresis, e.g., an optically actuated dielectrophoretic force; gravity; magnetic forces; or tilting.
  • unpenning a plurality of micro-objects may reposition substantially all the micro-objects.
  • a selected number of the plurality of micro-objects may be unpenned, and the remainder of the plurality may not be unpenned.
  • a DEP force e.g., an optically actuated DEP force or a magnetic force may be used to reposition the selected micro-objects.
  • export refers to repositioning micro-objects from a location within a flow region, e.g., a microfluidic channel, of a microfluidic device to a location outside of the microfluidic device, such as a 96 well plate or other receiving vessel.
  • a flow region e.g., a microfluidic channel
  • the orientation of the chamber(s) having an opening to the microfluidic channel permits easy export of micro-objects that have been positioned or repositioned (e.g., unpenned from a chamber) to be disposed within the microfluidic channel.
  • Micro-objects within the microfluidic channel may be exported without requiring disassembly (e.g., removal of the cover of the device) or insertion of a tool into the chamber(s) or microfluidic channel to remove micro-objects for further processing.
  • a microfluidic (or nanofluidic) device can comprise “swept” regions and “unswept” regions.
  • a “swept” region is comprised of one or more fluidically interconnected circuit elements of a microfluidic circuit, each of which experiences a flow of medium when fluid is flowing through the microfluidic circuit.
  • the circuit elements of a swept region can include, for example, regions, channels, and all or parts of chambers.
  • an “unswept” region is comprised of one or more fluidically interconnected circuit element of a microfluidic circuit, each of which experiences substantially no flux of fluid when fluid is flowing through the microfluidic circuit.
  • An unswept region can be fluidically connected to a swept region, provided the fluidic connections are structured to enable diffusion but substantially no flow of media between the swept region and the unswept region.
  • the microfluidic device can thus be structured to substantially isolate an unswept region from a flow of medium in a swept region, while enabling substantially only diffusive fluidic communication between the swept region and the unswept region.
  • a flow channel of a micro-fluidic device is an example of a swept region while an isolation region (described in further detail below) of a microfluidic device is an example of an unswept region.
  • a “non- sweeping” rate of fluidic medium flow means a rate of flow sufficient to permit components of a second fluidic medium in an isolation region of the sequestration pen to diffuse into the first fluidic medium in the flow region and/or components of the first fluidic medium to diffuse into the second fluidic medium in the isolation region; and further wherein the first medium does not substantially flow into the isolation region.
  • capture moiety is a chemical or biological species, functionality, or motif that provides a recognition site for a micro-object.
  • a selected class of micro-objects may recognize the in situ-generated capture moiety and may bind or have an affinity for the in situ- generated capture moiety.
  • Non-limiting examples include antigens, antibodies, and cell surface binding motifs.
  • antibody refers to an immunoglobulin (Ig) and includes both polyclonal and monoclonal antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and may be an intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab, Fab' and F(ab)'2 fragments), or multimers or aggregates of intact molecules and/or fragments; and may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering.
  • an “antibody fragment,” as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which in some embodiments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. This includes, e.g., F(ab), F(ab)'2, scFv, light chain variable region (VL), heavy chain variable region (VH), and combinations thereof.
  • biological micro-objects e.g., biological cells
  • specific biological materials e.g., proteins, such as antibodies
  • sample material comprising biological microobjects (e.g., cells) to be assayed for production of an analyte of interest (e.g., a biomolecule of interest) can be loaded into a swept region of the microfluidic device.
  • biological micro-objects e.g., mammalian cells, such as human cells
  • unswept regions e.g., mammalian cells, such as human cells
  • the remaining sample material can then be flowed out of the swept region and an assay material flowed into the swept region. Because the selected biological micro-objects are in unswept regions, the selected biological micro-objects are not substantially affected by the flowing out of the remaining sample material or the flowing in of the assay material.
  • the selected biological micro-objects can be allowed to produce the analyte of interest, which can diffuse from the unswept regions into the swept region, where the analyte of interest can react with the assay material to produce localized detectable reactions, each of which can be correlated to a particular unswept region. Any unswept region associated with a detected reaction can be analyzed to assess which, if any, of the biological micro-objects in the unswept region are sufficient producers of the analyte of interest.
  • Microfluidic devices/systems featuring cross-applicability may be combinable or interchangeable.
  • features described herein with reference to the microfluidic device 100, 175, 200, 300, 320, 400, 450, 520 and system attributes as described in FIGs. 1A-5B may be combinable or interchangeable.
  • FIG. 1A illustrates an example of a microfluidic device 100.
  • a perspective view of the microfluidic device 100 is shown having a partial cut-away of its cover 110 to provide a partial view into the microfluidic device 100.
  • the microfluidic device 100 generally comprises a microfluidic circuit 120 comprising a flow path 106 through which a fluidic medium 180 can flow, optionally carrying one or more micro-objects (not shown) into and/or through the microfluidic circuit 120.
  • the microfluidic circuit 120 is defined by an enclosure 102. Although the enclosure 102 can be physically structured in different configurations, in the example shown in FIG.
  • the enclosure 102 is depicted as comprising a support structure 104 (e.g., a base), a microfluidic circuit structure 108, and a cover 110.
  • the support structure 104, microfluidic circuit structure 108, and cover 110 can be attached to each other.
  • the microfluidic circuit structure 108 can be disposed on an inner surface 109 of the support structure 104
  • the cover 110 can be disposed over the microfluidic circuit structure 108.
  • the microfluidic circuit structure 108 can define the elements of the microfluidic circuit 120, forming a three-layer structure.
  • the support structure 104 can be at the bottom and the cover 110 at the top of the microfluidic circuit 120 as illustrated in FIG. 1A.
  • the support structure 104 and the cover 110 can be configured in other orientations.
  • the support structure 104 can be at the top and the cover 110 at the bottom of the microfluidic circuit 120.
  • port 107 is a pass-through hole created by a gap in the microfluidic circuit structure 108.
  • the port 107 can be situated in other components of the enclosure 102, such as the cover 110.
  • the microfluidic circuit 120 can have two or more ports 107.
  • a port 107 function as an inlet or an outlet can depend upon the direction that fluid flows through flow path 106.
  • the support structure 104 can comprise one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates.
  • the support structure 104 can comprise one or more semiconductor substrates, each of which is electrically connected to an electrode (e.g., all or a subset of the semiconductor substrates can be electrically connected to a single electrode).
  • the support structure 104 can further comprise a printed circuit board assembly (“PCBA”).
  • PCBA printed circuit board assembly
  • the semiconductor substrate(s) can be mounted on a PCBA.
  • the microfluidic circuit structure 108 can define circuit elements of the microfluidic circuit 120.
  • Such circuit elements can comprise spaces or regions that can be fluidly interconnected when microfluidic circuit 120 is filled with fluid, such as flow regions (which may include or be one or more flow channels), chambers (which class of circuit elements may also include sub-classes including sequestration pens), traps, and the like. Circuit elements can also include barriers, and the like.
  • the microfluidic circuit structure 108 comprises a frame 114 and a microfluidic circuit material 116.
  • the frame 114 can partially or completely enclose the microfluidic circuit material 116.
  • the frame 114 can be, for example, a relatively rigid structure substantially surrounding the microfluidic circuit material 116.
  • the frame 114 can comprise a metal material.
  • the microfluidic circuit structure need not include a frame 114.
  • the microfluidic circuit structure can consist of (or consist essentially of) the microfluidic circuit material 116.
  • the microfluidic circuit material 116 can be patterned with cavities or the like to define the circuit elements and interconnections of the microfluidic circuit 120, such as chambers, pens and microfluidic channels.
  • the microfluidic circuit material 116 can comprise a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like), which can be gas permeable.
  • a flexible polymer e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane (“PDMS”), or the like
  • Other examples of materials that can form the microfluidic circuit material 116 include molded glass, an etchable material such as silicone (e.g., photo-pattemable silicone or “PPS”), photo-resist (e.g., SU8), or the like.
  • microfluidic circuit material 116 can be rigid and/or substantially impermeable to gas. Regardless, microfluidic circuit material 116 can be disposed on the support structure 104 and inside the frame 114.
  • the microfluidic circuit 120 can include a flow region in which one or more chambers can be disposed and/or fluidically connected thereto.
  • a chamber can have one or more openings fluidically connecting the chamber with one or more flow regions.
  • a flow region comprises or corresponds to a microfluidic channel 122.
  • suitable microfluidic devices can include a plurality (e.g., 2 or 3) of such microfluidic circuits.
  • the microfluidic device 100 can be configured to be a nanofluidic device. As illustrated in FIG.
  • the microfluidic circuit 120 may include a plurality of microfluidic sequestration pens 124, 126, 128, and 130, where each sequestration pens may have one or more openings.
  • a sequestration pen may have only a single opening in fluidic communication with the flow path 106.
  • a sequestration pen may have more than one opening in fluidic communication with the flow path 106, e.g., n number of openings, but with n-1 openings that are valved, such that all but one opening is closable. When all the valved openings are closed, the sequestration pen limits exchange of materials from the flow region into the sequestration pen to occur only by diffusion.
  • the sequestration pens comprise various features and structures (e.g., isolation regions) that have been optimized for retaining micro-objects within the sequestration pen (and therefore within a microfluidic device such as microfluidic device 100) even when a medium 180 is flowing through the flow path 106.
  • various features and structures e.g., isolation regions
  • the cover 110 can be an integral part of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 can be a structurally distinct element, as illustrated in FIG. 1A.
  • the cover 110 can comprise the same or different materials than the frame 114 and/or the microfluidic circuit material 116. In some embodiments, the cover 110 can be an integral part of the microfluidic circuit material 116.
  • the support structure 104 can be a separate structure from the frame 114 or microfluidic circuit material 116 as illustrated, or an integral part of the frame 114 or microfluidic circuit material 116.
  • the frame 114 and microfluidic circuit material 116 can be separate structures as shown in FIG. 1A or integral portions of the same structure. Regardless of the various possible integrations, the microfluidic device can retain a three-layer structure that includes a base layer and a cover layer that sandwich a middle layer in which the microfluidic circuit 120 is located.
  • the cover 110 can comprise a rigid material.
  • the rigid material may be glass or a material with similar properties.
  • the cover 110 can comprise a deformable material.
  • the deformable material can be a polymer, such as PDMS.
  • the cover 110 can comprise both rigid and deformable materials.
  • one or more portions of cover 110 e.g., one or more portions positioned over sequestration pens 124, 126, 128, 130
  • Microfluidic devices having covers that include both rigid and deformable materials have been described, for example, in U.S. Patent No.
  • the cover 110 can further include one or more electrodes.
  • the one or more electrodes can comprise a conductive oxide, such as indium-tin-oxide (ITO), which may be coated on glass or a similarly insulating material.
  • the one or more electrodes can be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of electrically conductive nanoparticles, or combinations thereof, embedded in a deformable material, such as a polymer (e.g., PDMS).
  • a polymer e.g., PDMS
  • the cover 110 and/or the support structure 104 can be transparent to light.
  • the cover 110 may also include at least one material that is gas permeable (e.g., PDMS or PPS).
  • the microfluidic circuit 120 is illustrated as comprising a microfluidic channel 122 and sequestration pens 124, 126, 128, 130.
  • Each pen comprises an opening to channel 122, but otherwise is enclosed such that the pens can substantially isolate micro-objects inside the pen from fluidic medium 180 and/or microobjects in the flow path 106 of channel 122 or in other pens.
  • the walls of the sequestration pen extend from the inner surface 109 of the base to the inside surface of the cover 110 to provide enclosure.
  • the opening of the sequestration pen to the microfluidic channel 122 is oriented at an angle to the flow 106 of fluidic medium 180 such that flow 106 is not directed into the pens.
  • the vector of bulk fluid flow in channel 122 may be tangential or parallel to the plane of the opening of the sequestration pen, and is not directed into the opening of the pen.
  • pens 124, 126, 128, 130 are configured to physically isolate one or more micro-objects within the microfluidic circuit 120.
  • Sequestration pens in accordance with the present disclosure can comprise various shapes, surfaces and features that are optimized for use with DEP, OET, OEW, fluid flow, magnetic forces, centripetal, and/or gravitational forces, as will be discussed and shown in detail below.
  • the microfluidic circuit 120 may comprise any number of microfluidic sequestration pens. Although five sequestration pens are shown, microfluidic circuit 120 may have fewer or more sequestration pens. As shown, microfluidic sequestration pens 124, 126, 128, and 130 of microfluidic circuit 120 each comprise differing features and shapes which may provide one or more benefits useful for maintaining, isolating, assaying or culturing biological microobjects. In some embodiments, the microfluidic circuit 120 comprises a plurality of identical microfluidic sequestration pens.
  • a single flow path 106 containing a single channel 122 is shown.
  • the microfluidic circuit 120 further comprises an inlet valve or port 107 in fluid communication with the flow path 106, whereby fluidic medium 180 can access the flow path 106 (and channel 122).
  • the flow path 106 comprises a substantially straight path.
  • the flow path 106 is arranged in a non-linear or winding manner, such as a zigzag pattern, whereby the flow path 106 travels across the microfluidic device 100 two or more times, e.g., in alternating directions.
  • the flow in the flow path 106 may proceed from inlet to outlet or may be reversed and proceed from outlet to inlet.
  • microfluidic device 175 One example of a multi-channel device, microfluidic device 175, is shown in FIG. IB, which may be like microfluidic device 100 in other respects.
  • Microfluidic device 175 and its constituent circuit elements e.g., channels 122 and sequestration pens 128) may have any of the dimensions discussed herein.
  • the microfluidic circuit illustrated in FIG. IB has two inlet/outlet ports 107 and a flow path 106 containing four distinct channels 122.
  • the number of channels into which the microfluidic circuit is sub-divided may be chosen to reduce fluidic resistance.
  • the microfluidic circuit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more channels to provide a selected range of fluidic resistance.
  • Microfluidic device 175 further comprises a plurality of sequestration pens opening off of each channel 122, where each of the sequestration pens is similar to sequestration pen 128 of FIG. 1A, and may have any of the dimensions or functions of any sequestration pen as described herein.
  • the sequestration pens of microfluidic device 175 can have different shapes, such as any of the shapes of sequestration pens 124, 126, or 130 of FIG. 1A or as described anywhere else herein.
  • micro fluidic device 175 can include sequestration pens having a mixture of different shapes.
  • a plurality of sequestration pens is configured (e.g., relative to a channel 122) such that the sequestration pens can be loaded with target micro-objects in parallel.
  • microfluidic circuit 120 further may include one or more optional micro-object traps 132.
  • the optional traps 132 may be formed in a wall forming the boundary of a channel 122, and may be positioned opposite an opening of one or more of the microfluidic sequestration pens 124, 126, 128, 130.
  • the optional traps 132 may be configured to receive or capture a single micro-object from the flow path 106, or may be configured to receive or capture a plurality of micro-objects from the flow path 106.
  • the optional traps 132 comprise a volume approximately equal to the volume of a single target micro-object.
  • the trap 132 comprises a side passage 134 that is smaller than the target micro-object in order to facilitate flow through the trap 132.
  • the microfluidic devices described herein may include one or more sequestration pens, where each sequestration pen is suitable for holding one or more micro-objects (e.g., biological cells, or groups of cells that are associated together).
  • the sequestration pens may be disposed within and open to a flow region, which in some embodiments is a microfluidic channel.
  • Each of the sequestration pens can have one or more openings for fluidic communication to one or more microfluidic channels.
  • a sequestration pen may have only one opening to a microfluidic channel.
  • FIGs. 2A-2C show sequestration pens 224, 226, and 228 of a microfluidic device 200, which may be like sequestration pen 128 of FIG. 1A.
  • Each sequestration pen 224, 226, and 228 can comprise an isolation region 240 and a connection region 236 fluidically connecting the isolation region 240 to a flow region, which may, in some embodiments include a microfluidic channel, such as channel 122.
  • the connection region 236 can comprise a proximal opening 234 to the flow region (e.g., microfluidic channel 122) and a distal opening 238 to the isolation region 240.
  • connection region 236 can be configured so that the maximum penetration depth of a flow of a fluidic medium (not shown) flowing in the microfluidic channel 122 past the sequestration pen 224, 226, and 228 does not extend into the isolation region 240, as discussed below for FIG. 2C. In some embodiments, streamlines from the flow in the microfluidic channel do not enter the isolation region. Thus, due to the connection region 236, a micro-object (not shown) or other material (not shown) disposed in the isolation region 240 of a sequestration pen 224, 226, and 228 can be isolated from, and not substantially affected by, a flow of fluidic medium 180 in the microfluidic channel 122.
  • the sequestration pens 224, 226, and 228 of FIGs. 2A-2C each have a single opening which opens directly to the microfluidic channel 122.
  • the opening of the sequestration pen may open laterally from the microfluidic channel 122, as shown in FIG. 2A, which depicts a vertical cross-section of microfluidic device 200.
  • FIG. 2B shows a horizontal cross-section of microfluidic device 200.
  • An electrode activation substrate 206 can underlie both the microfluidic channel 122 and the sequestration pens 224, 226, and 228.
  • the upper surface of the electrode activation substrate 206 within an enclosure of a sequestration pen, forming the floor of the sequestration pen, can be disposed at the same level or substantially the same level of the upper surface the of electrode activation substrate 206 within the microfluidic channel 122 (or flow region if a channel is not present), forming the floor of the flow channel (or flow region, respectively) of the microfluidic device.
  • the electrode activation substrate 206 may be featureless or may have an irregular or patterned surface that varies from its highest elevation to its lowest depression by less than about 3 micrometers (microns), 2.5 microns, 2 microns, 1.5 microns, 1 micron, 0.9 microns, 0.5 microns, 0.4 microns, 0.2 microns, 0.1 microns or less.
  • the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 10%, 7%, 5%, 3%, 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3% or 0.1% of the height of the walls of the sequestration pen.
  • the variation of elevation in the upper surface of the substrate across both the microfluidic channel 122 (or flow region) and sequestration pens may be equal to or less than about 2%, 1%. 0.9%, 0.8%, 0.5%, 0.3%, 0.2%, or 0.1% of the height of the substrate. While described in detail for the microfluidic device 200, this may also apply to any of the microfluidic devices described herein.
  • the microfluidic channel 122 and connection region 236 can be examples of swept regions, and the isolation regions 240 of the sequestration pens 224, 226, and 228 can be examples of unswept regions.
  • Sequestration pens like 224, 226, 228 have isolation regions wherein each isolation region has only one opening, which opens to the connection region of the sequestration pen. Fluidic media exchange in and out of the isolation region so configured can be limited to occurring substantially only by diffusion.
  • the microfluidic channel 122 and sequestration pens 224, 226, and 228 can be configured to contain one or more fluidic media 180.
  • ports 222 are connected to the microfluidic channel 122 and allow the fluidic medium 180 to be introduced into or removed from the microfluidic device 200.
  • the microfluidic device Prior to introduction of the fluidic medium 180, the microfluidic device may be primed with a gas such as carbon dioxide gas.
  • the flow 242 see FIG. 2C of fluidic medium 180 in the microfluidic channel 122 can be selectively generated and stopped.
  • the ports 222 can be disposed at different locations (e.g., opposite ends) of the flow region (microfluidic channel 122), and a flow 242 of the fluidic medium can be created from one port 222 functioning as an inlet to another port 222 functioning as an outlet.
  • FIG. 2C illustrates a detailed view of an example of a sequestration pen 224, which may contain one or more micro-objects 246, according to some embodiments.
  • the flow 242 of fluidic medium 180 in the microfluidic channel 122 past the proximal opening 234 of the connection region 236 of sequestration pen 224 can cause a secondary flow 244 of the fluidic medium 180 into and out of the sequestration pen 224.
  • the length Leon of the connection region 236 of the sequestration pen 224 should be greater than the penetration depth D p of the secondary flow 244 into the connection region 236.
  • the penetration depth D p depends upon a number of factors, including the shape of the microfluidic channel 122, which may be defined by a width Wcon of the connection region 236 at the proximal opening 234; a width Wch of the microfluidic channel 122 at the proximal opening 234; a height H C h of the channel 122 at the proximal opening 234; and the width of the distal opening 238 of the connection region 236.
  • the width Wcon of the connection region 236 at the proximal opening 234 and the height Hch of the channel 122 at the proximal opening 234 tend to be the most significant.
  • the penetration depth D p can be influenced by the velocity of the fluidic medium 180 in the channel 122 and the viscosity of fluidic medium 180. However, these factors (i.e., velocity and viscosity) can vary widely without dramatic changes in penetration depth D p .
  • the penetration depth D p of the secondary flow 244 ranges from less than 1.0 times W CO n (i.e., less than 50 microns) at a flow rate of 0.1 microliters/sec to about 2.0 times W CO n (i.e., about 100 microns) at a flow rate of 20 microliters/sec, which represents an increase in D p of only about 2.5-fold over a 200-fold increase in the velocity of the fluidic medium 180.
  • the walls of the microfluidic channel 122 and sequestration pen 224, 226, or 228 can be oriented as follows with respect to the vector of the flow 242 of fluidic medium 180 in the microfluidic channel 122: the microfluidic channel width Wch (or cross-sectional area of the microfluidic channel 122) can be substantially perpendicular to the flow 242 of medium 180; the width W CO n (or cross-sectional area) of the connection region 236 at opening 234 can be substantially parallel to the flow 242 of medium 180 in the microfluidic channel 122; and/or the length L con of the connection region can be substantially perpendicular to the flow 242 of medium 180 in the microfluidic channel 122.
  • the foregoing are examples only, and the relative position of the microfluidic channel 122 and sequestration pens 224, 226 and 228 can be in other orientations with respect to each other.
  • the configurations of the microfluidic channel 122 and the opening 234 may be fixed, whereas the rate of flow 242 of fluidic medium 180 in the microfluidic channel 122 may be variable. Accordingly, for each sequestration pen 224, a maximal velocity Vmax for the flow 242 of fluidic medium 180 in channel 122 may be identified that ensures that the penetration depth D p of the secondary flow 244 does not exceed the length L CO n of the connection region 236. When Vmax is not exceeded, the resulting secondary flow 244 can be wholly contained within the connection region 236 and does not enter the isolation region 240.
  • the flow 242 of fluidic medium 180 in the microfluidic channel 122 is prevented from drawing micro-objects 246 out of the isolation region 240, which is an unswept region of the microfluidic circuit, resulting in the micro-objects 246 being retained within the isolation region 240.
  • selection of microfluidic circuit element dimensions and further selection of the operating parameters can prevent contamination of the isolation region 240 of sequestration pen 224 by materials from the microfluidic channel 122 or another sequestration pen 226 or 228.
  • components (not shown) of the second medium 248 in the isolation region 240 can mix with the first medium 180 in the micro fluidic channel 122 substantially only by diffusion of components of the second medium 248 from the isolation region 240 through the connection region 236 and into the first medium 180 in the microfluidic channel 122.
  • the first medium 180 can be the same medium or a different medium than the second medium 248.
  • the first medium 180 and the second medium 248 can start out being the same, then become different (e.g., through conditioning of the second medium 248 by one or more cells in the isolation region 240, or by changing the medium 180 flowing through the microfluidic channel 122).
  • the width W CO n of the connection region 236 can be uniform from the proximal opening 234 to the distal opening 238.
  • the width W CO n of the connection region 236 at the distal opening 238 can be any of the values identified herein for the width Wcon of the connection region 236 at the proximal opening 234.
  • the width of the isolation region 240 at the distal opening 238 can be substantially the same as the width Wcon of the connection region 236 at the proximal opening 234.
  • the width Wcon of the connection region 236 at the distal opening 238 can be different (e.g., larger or smaller) than the width Wcon of the connection region 236 at the proximal opening 234.
  • the width Wcon of the connection region 236 may be narrowed or widened between the proximal opening 234 and distal opening 238.
  • the connection region 236 may be narrowed or widened between the proximal opening and the distal opening, using a variety of different geometries (e.g., chamfering the connection region, beveling the connection region).
  • any part or subpart of the connection region 236 may be narrowed or widened (e.g., a portion of the connection region adjacent to the proximal opening 234).
  • FIG. 3 depicts another exemplary embodiment of a microfluidic device 300 containing microfluidic circuit structure 308, which includes a channel 322 and sequestration pen 324, which has features and properties like any of the sequestration pens described herein for microfluidic devices 100, 175, 200, 400, 520 and any other microfluidic devices described herein.
  • the exemplary microfluidic devices of FIG. 3 include a microfluidic channel 322, having a width Wch, as described herein, and containing a flow 310 of first fluidic medium 302 and one or more sequestration pens 324 (only one illustrated in FIG. 3).
  • the sequestration pens 324 each have a length L s , a connection region 336, and an isolation region 340, where the isolation region 340 contains a second fluidic medium 304.
  • the connection region 336 has a proximal opening 334, having a width Wconi, which opens to the microfluidic channel 322, and a distal opening 338, having a width W CO n2, which opens to the isolation region 340.
  • the width Wconi may or may not be the same as W CO n2, as described herein.
  • the walls of each sequestration pen 324 may be formed of microfluidic circuit material 316, which may further form the connection region walls 330.
  • a connection region wall 330 can correspond to a structure that is laterally positioned with respect to the proximal opening 334 and at least partially extends into the enclosed portion of the sequestration pen 324.
  • the length L con of the connection region 336 is at least partially defined by length L W aii of the connection region wall 330.
  • the connection region wall 330 may have a length L W aii, selected to be more than the penetration depth D p of the secondary flow 344.
  • the secondary flow 344 can be wholly contained within the connection region without extending into the isolation region 340.
  • connection region wall 330 may define a hook region 352, which is a sub-region of the isolation region 340 of the sequestration pen 324. Since the connection region wall 330 extends into the inner cavity of the sequestration pen, the connection region wall 330 can act as a physical barrier to shield hook region 352 from secondary flow 344, with selection of the length of Lwaii, contributing to the extent of the hook region. In some embodiments, the longer the length Lwaii of the connection region wall 330, the more sheltered the hook region 352.
  • the isolation region may have a shape and size of any type, and may be selected to regulate diffusion of nutrients, reagents, and/or media into the sequestration pen to reach to a far wall of the sequestration pen, e.g., opposite the proximal opening of the connection region to the flow region (or microfluidic channel).
  • the size and shape of the isolation region may further be selected to regulate diffusion of waste products and/or secreted products of a biological micro-object out from the isolation region to the flow region via the proximal opening of the connection region of the sequestration pen.
  • the shape of the isolation region is not critical to the ability of the sequestration pen to isolate micro-objects from direct flow in the flow region.
  • the isolation region may have more than one opening fluidically connecting the isolation region with the flow region of the microfluidic device.
  • n- 1 openings can be valved. When the n-1 valved openings are closed, the isolation region has only one effective opening, and exchange of materials into/out of the isolation region occurs only by diffusion.
  • Microfluidic circuit element dimensions Various dimensions and/or features of the sequestration pens and the microfluidic channels to which the sequestration pens open, as described herein, may be selected to limit introduction of contaminants or unwanted microobjects into the isolation region of a sequestration pen from the flow region/microfluidic channel; limit the exchange of components in the fluidic medium from the channel or from the isolation region to substantially only diffusive exchange; facilitate the transfer of micro-objects into and/or out of the sequestration pens; and/or facilitate growth or expansion of the biological cells.
  • Microfluidic channels and sequestration pens for any of the embodiments described herein, may have any suitable combination of dimensions, may be selected by one of skill from the teachings of this disclosure.
  • a microfluidic channel may have a uniform cross sectional height along its length that is a substantially uniform cross sectional height, and may be any cross sectional height as described herein.
  • the substantially uniform cross sectional height of the channel the upper surface of which is defined by the inner surface of the cover and the lower surface of which is defined by the inner surface of the base, may be substantially the same as the cross sectional height at any other point along the channel, e.g., having a cross sectional height that is no more than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2% or about 1% or less, different from the cross-sectional height of any other location within the channel.
  • the chamber(s), e.g., sequestration pen(s), of the microfluidic devices described herein, may be disposed substantially in a coplanar orientation relative to the microfluidic channel into which the chamber(s) open. That is, the enclosed volume of the chamber(s) is formed by an upper surface that is defined by the inner surface of the cover, a lower surface defined by the inner surface of the base, and walls defined by the microfluidic circuit material. Therefore, the lower surface of the chamber(s) may be coplanar to the lower surface of the microfluidic channel, e.g., substantially coplanar.
  • the upper surface of the chamber may be coplanar to the upper surface of the microfluidic channel, e.g., substantially coplanar.
  • the chamber(s) may have a cross-sectional height, which may have any values as described herein, that is the same as the channel, e.g., substantially the same, and the chamber(s) and microfluidic channel(s) within the microfluidic device may have a substantially uniform cross-sectional height throughout the flow region of the microfluidic device, and may be substantially coplanar throughout the microfluidic device.
  • Coplanarity of the lower surfaces of the chamber(s) and the microfluidic channel(s) can offer distinct advantage with repositioning micro-objects within the microfluidic device using DEP or magnetic force. Penning and unpenning of micro-objects, and in particular selective penning/ selective unpenning, can be greatly facilitated when the lower surfaces of the chamber(s) and the microfluidic channel to which the chamber(s) open have a coplanar orientation.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) that is at least as large as the largest dimension of a micro-object (e.g., a biological cell, which may be a plant cell, such as a plant protoplast) for which the sequestration pen is intended.
  • a micro-object e.g., a biological cell, which may be a plant cell, such as a plant protoplast
  • the proximal opening has a width (e.g., Wcon or Wconi) of about 20 microns, about 40 microns, about 50 microns, about 60 microns, about 75 microns, about 100 microns, about 150 microns, about 200 microns, or about 300 microns.
  • the width (e.g., Wcon or Wconi) of a proximal opening can be selected to be a value between any of the values listed above (e.g., about 20- 200 microns, about 20-150 microns, about 20-100 microns, about 20-75 microns, about 20-60 microns, about 50-300 microns, about 50-200 microns, about 50-150 microns, about 50-100 microns, about 50-75 microns, about 75-150 microns, about 75-100 microns, about 100-300 microns, about 100-200 microns, or about 200-300 microns).
  • connection region of the sequestration pen may have a length (e.g., L C on) from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25.
  • L C on a length from the proximal opening to the distal opening to the isolation region of the sequestration pen that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), and the connection region may have a length L CO n that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening.
  • the proximal opening of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), and the connection region may have a length L CO n that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening.
  • the microfluidic channel of a microfluidic device to which a sequestration pen opens may have specified size (e.g., width or height).
  • the height (e.g., H C h) of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be within any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20- 70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns.
  • the height (e.g., H C h) of the microfluidic channel (e.g., 122) can be selected to be between any of the values listed above.
  • the height (e.g., H C h) of the microfluidic channel 122 can be selected to be any of these heights in regions of the microfluidic channel other than at a proximal opening of a sequestration pen.
  • the width (e.g., Wch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen can be within any of the following ranges: about 20- 500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30- 150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-100 microns, 40-80 microns, 40-60 microns, 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-300
  • the width (e.g., Wch) of the microfluidic channel can be a value selected to be between any of the values listed above.
  • the width (e.g., Wch) of the microfluidic channel can be selected to be in any of these widths in regions of the microfluidic channel other than at a proximal opening of a sequestration pen.
  • the width Wch of the microfluidic channel at the proximal opening to the connection region of the sequestration pen e.g., taken transverse to the direction of bulk flow of fluid through the channel
  • a cross-sectional area of the microfluidic channel at a proximal opening to the connection region of a sequestration pen can be about 500-50,000 square microns, 500-40,000 square microns, 500-30,000 square microns, 500-25,000 square microns, 500-20,000 square microns, 500-15,000 square microns, 500-10,000 square microns, 500-7,500 square microns, 500-5,000 square microns, 1,000-25,000 square microns, 1,000-20,000 square microns, 1, GOO- 15, 000 square microns, 1,000-10,000 square microns, 1,000-7,500 square microns, 1,000- 5,000 square microns, 2,000-20,000 square microns, 2,000-15,000 square microns, 2,000- 10,000 square microns, 2,000-7,500 square microns, 2,000-6,000 square microns, 3,000- 20,000 square microns, 3,000-15,000 square microns, 3,000-10,000 square microns, 3,000- 7,500 square microns, or 3,000 to 6,000
  • the cross-sectional area of the microfluidic channel at the proximal opening can be selected to be between any of the values listed above.
  • the cross-sectional area of the microfluidic channel at regions of the microfluidic channel other than at the proximal opening can also be selected to be between any of the values listed above.
  • the cross-sectional area is selected to be a substantially uniform value for the entire length of the microfluidic channel.
  • the microfluidic chip is configured such that the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) from about 20 microns to about 200 microns (e.g., about 50 microns to about 150 microns), the connection region may have a length L CO n (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H C h) at the proximal opening of about 30 microns to about 60 microns.
  • a width e.g., Wcon or Wconi
  • L CO n e.g., 236 or 336
  • the microfluidic channel may have a height (e.g., H C h) at the proximal opening of about 30
  • the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen may have a width (e.g., Wcon or Wconi) from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns), the connection region may have a length L CO n (e.g., 236 or 336) that is at least 1.0 times (e.g., at least 1.5 times, or at least 2.0 times) the width of the proximal opening, and the microfluidic channel may have a height (e.g., H C h) at the proximal opening of about 30 microns to about 60 microns.
  • Wcon or Wconi width from about 20 microns to about 100 microns (e.g., about 20 microns to about 60 microns)
  • the connection region may have a length L CO n (e.g., 236 or 336) that is at least 1.0 times (e.g., at
  • the width (e.g., Wcon or Wconi) of the proximal opening (e.g., 234 or 274), the length (e.g., L CO n) of the connection region, and/or the width (e.g., Wch) of the microfluidic channel (e.g., 122 or 322) can be a value selected to be between any of the values listed above.
  • the width (W CO n or Wconi) of the proximal opening of the connection region of a sequestration pen is less than the width (Wch) of the microfluidic channel.
  • the width (W CO n or Wconi) of the proximal opening is about 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 22%, 24%, 25%, or 30% of the width (Wch) of the microfluidic channel. That is, the width (Wch) of the microfluidic channel may be at least 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times or at least 10.0 times the width (W CO n or Wconi) of the proximal opening of the connection region of the sequestration pen.
  • the size Wc (e.g., cross-sectional width Wch, diameter, area, or the like) of the channel 122, 322, 618, 718 can be about one and a quarter (1.25), about one and a half (1.5), about two, about two and a half (2.5), about three (3), or more times the size Wo (e.g., cross-sectional width W CO n, diameter, area, or the like) of a chamber opening, e.g., sequestration pen opening 234, 334, and the like.
  • a chamber opening e.g., sequestration pen opening 234, 334, and the like.
  • a selected chamber e.g., like sequestration pens 224, 226 of FIG. 2B
  • the rate of diffusion of a molecule is dependent on a number of factors, including (without limitation) temperature, viscosity of the medium, and the coefficient of diffusion Do of the molecule.
  • the Do for an IgG antibody in aqueous solution at about 20°C is about 4.4x1 O’ 7 cm 2 /sec, while the kinematic viscosity of cell culture medium is about 9xl0 -4 m 2 /sec.
  • an antibody in cell culture medium at about 20°C can have a rate of diffusion of about 0.5 microns/sec.
  • a time period for diffusion from a biological micro-object located within a sequestration pen such as 224, 226, 228, 324 into the channel 122, 322, 618, 718 can be about 10 minutes or less (e.g., about 9, 8, 7, 6, 5 minutes, or less).
  • the time period for diffusion can be manipulated by changing parameters that influence the rate of diffusion.
  • the temperature of the media can be increased (e.g., to a physiological temperature such as about 37°C) or decreased (e.g., to about 15°C, 10°C, or 4°C) thereby increasing or decreasing the rate of diffusion, respectively.
  • concentrations of solutes in the medium can be increased or decreased as discussed herein to isolate a selected pen from solutes from other upstream pens.
  • the width (e.g., Wch) of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 50 to 500 microns, about 50 to 300 microns, about 50 to 200 microns, about 70 to 500 microns, about to 70-300 microns, about 70 to 250 microns, about 70 to 200 microns, about 70 to 150 microns, about 70 to 100 microns, about 80 to 500 microns, about 80 to 300 microns, about 80 to 250 microns, about 80 to 200 microns, about 80 to 150 microns, about 90 to 500 microns, about 90 to 300 microns, about 90 to 250 microns, about 90 to 200 microns, about 90 to 150 microns, about 100 to 500 microns, about 100 to 300 microns, about 100 to 250 microns, about 100 to 200 microns, or about 100 to 150 microns.
  • the width Wch of the microfluidic channel at the proximal opening to the connection region of a sequestration pen may be about 70 to 250 microns, about 80 to 200 microns, or about 90 to 150 microns.
  • the width Wcon of the opening of the chamber (e.g., sequestration pen) may be about 20 to 100 microns; about 30 to 90 microns; or about 20 to 60 microns.
  • Wch is about 70-250 microns and Wcon is about 20 to 100 microns; Wch is about 80 to 200 microns and Wcon is about 30 to 90 microns; Wch is about 90 to 150 microns, and Wcon is about 20 to 60 microns; or any combination of the widths of Wch and Wcon thereof.
  • the proximal opening (e.g., 234 or 334) of the connection region of a sequestration pen has a width (e.g., Wcon or Wconi) that is 2.0 times or less (e.g., 2.0, 1.9, 1.8, 1.5, 1.3, 1.0, 0.8, 0.5, or 0.1 times) the height (e.g., H C h) of the flow region/ microfluidic channel at the proximal opening, or has a value that lies within a range defined by any two of the foregoing values.
  • the width Wconi of a proximal opening (e.g., 234 or 334) of a connection region of a sequestration pen may be the same as a width W CO n2 of the distal opening (e.g., 238 or 338) to the isolation region thereof.
  • the width Wconi of the proximal opening may be different than a width W CO n2 of the distal opening, and Wconi and/or Wcon2 may be selected from any of the values described for Wcon or Wconi .
  • the walls (including a connection region wall) that define the proximal opening and distal opening may be substantially parallel with respect to each other. In some embodiments, the walls that define the proximal opening and distal opening may be selected to not be parallel with respect to each other.
  • the length (e.g., L CO n) of the connection region can be about 1-600 microns, 5-550 microns, 10-500 microns, 15-400 microns, 20-300 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, about 100-150 microns, about 20-300 microns, about 20 - 250 microns, about 20-200 microns, about 20-150 microns, about 20-100 microns, about 30- 250 microns, about 30-200 microns, about 30- 150 microns, about 30-100 microns, about 30- 80 microns, about 30-50 microns, about 45-250 microns, about 45-200 microns, about 45-100 microns, about 45- 80 microns, about 45-60 microns, about 60-200 microns, about 60-150 microns, about 60-100 microns or about 60-80 microns.
  • the foregoing are examples only, and
  • connection region wall of a sequestration pen may have a length (e.g., L W aii) that is at least 0.5 times, at least 0.6 times, at least 0.7 times, at least 0.8 times, at least 0.9 times, at least 1.0 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.75 times, at least 2.0 times, at least 2.25 times, at least 2.5 times, at least 2.75 times, at least 3.0 times, or at least 3.5 times the width (e.g., W CO n or Wconi) of the proximal opening of the connection region of the sequestration pen.
  • the width e.g., W CO n or Wconi
  • connection region wall may have a length L W aii of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns.
  • L W aii of about 20-200 microns, about 20-150 microns, about 20-100 microns, about 20-80 microns, or about 20-50 microns.
  • a connection region wall may have a length L W aii selected to be between any of the values listed above.
  • a sequestration pen may have a length L s of about 40-600 microns, about 40-500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40-100 microns or about 40-80 microns.
  • L s length of about 40-600 microns, about 40-500 microns, about 40-400 microns, about 40-300 microns, about 40-200 microns, about 40-100 microns or about 40-80 microns.
  • a sequestration pen may have a specified height (e.g., H s ).
  • a sequestration pen has a height H s of about 20 microns to about 200 microns (e.g., about 20 microns to about 150 microns, about 20 microns to about 100 microns, about 20 microns to about 60 microns, about 30 microns to about 150 microns, about 30 microns to about 100 microns, about 30 microns to about 60 microns, about 40 microns to about 150 microns, about 40 microns to about 100 microns, or about 40 microns to about 60 microns).
  • H s the height of the values listed above.
  • the height H CO n of a connection region at a proximal opening of a sequestration pen can be a height within any of the following heights: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30- 80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns.
  • the foregoing are examples only, and the height H CO n of the connection region can be selected to be between any of the values listed above.
  • the height H CO n of the connection region is selected to be the same as the height H C h of the microfluidic channel at the proximal opening of the connection region.
  • the height H s of the sequestration pen is typically selected to be the same as the height H CO n of a connection region and/or the height H C h of the microfluidic channel.
  • H s , H CO n, and H C h may be selected to be the same value of any of the values listed above for a selected microfluidic device.
  • the isolation region can be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. In other embodiments, the isolation region may contain more than 10, more than 50 or more than 100 micro-objects. Accordingly, the volume of an isolation region can be, for example, at least IxlO 4 , IxlO 5 , 5xl0 5 , 8xl0 5 , IxlO 6 , 2xl0 6 , 4xl0 6 , 6xl0 6 , IxlO 7 , 3xl0 7 , 5xl0 7 IxlO 8 , 5xl0 8 , or 8xl0 8 cubic microns, or more.
  • the isolation region can be configured to contain numbers of micro-objects and volumes selected to be between any of the values listed above (e.g., a volume between IxlO 5 cubic microns and 5xl0 5 cubic microns, between 5xl0 5 cubic microns and IxlO 6 cubic microns, between IxlO 6 cubic microns and 2xl0 6 cubic microns, or between 2xl0 6 cubic microns and IxlO 7 cubic microns).
  • a sequestration pen of a micro fluidic device may have a specified volume.
  • the specified volume of the sequestration pen (or the isolation region of the sequestration pen) may be selected such that a single cell or a small number of cells (e.g., 2-10 or 2-5) can rapidly condition the medium and thereby attain favorable (or optimal) growth conditions.
  • the sequestration pen has a volume of about 5xl0 5 , 6xl0 5 , 8xl0 5 , IxlO 6 , 2xl0 6 , 4xl0 6 , 8xl0 6 , IxlO 7 , 3xl0 7 , 5xl0 7 , or about 8xl0 7 cubic microns, or more.
  • the sequestration pen has a volume of about 1 nanoliter to about 50 nanoliters, 2 nanoliters to about 25 nanoliters, 2 nanoliters to about 20 nanoliters, about 2 nanoliters to about 15 nanoliters, or about 2 nanoliters to about 10 nanoliters.
  • a sequestration pen can have a volume selected to be any value that is between any of the values listed above.
  • the flow of fluidic medium within the microfluidic channel may have a specified maximum velocity (e.g., Vmax).
  • the maximum velocity e.g., Vmax
  • the maximum velocity may be set at around 0.2, 0.5, 0.7, 1.0, 1.3, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.7, 7.0, 7.5, 8.0, 8.5, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, or 25 microliters/sec.
  • the flow of fluidic medium within the microfluidic channel can have a maximum velocity (e.g., Vmax) selected to be a value between any of the values listed above.
  • Vmax maximum velocity
  • the flow of fluidic medium within the microfluidic channel typically may be flowed at a rate less than the Vmax.
  • a fluidic medium may be flowed at about 0.1 microliters/sec to about 20 microliters/sec; about 0.1 microliters/sec to about 15 microliters/sec; about 0.1 microliters/sec to about 12 microliters/sec, about 0.1 microliters/sec to about 10 microliters/sec; about 0.1 microliter/sec to about 7 microliters/sec without exceeding the Vmax.
  • a flow rate of a fluidic medium may be about 0.1 microliters/sec; about 0.5 microliters/sec; about 1.0 microliters/sec; about 2.0 microliters/sec; about 3.0 microliters/sec; about 4.0 microliters/sec; about 5.0 microliters/sec; about 6.0 microliters/sec; about 7.0 microliters/sec; about 8.0 microliters/sec; about 9.0 microliters/sec; about 10.0 microliters/sec; about 11.0 microliters/sec; or any range defined by two of the foregoing values, e.g., 1-5 microliters/sec or 5-10 microliters/sec.
  • the flow rate of a fluidic medium in the microfluidic channel may be equal to or less than about 12 microliters/sec; about 10 microliters/sec; about 8 microliters/sec, or about 6 microliters/sec.
  • the microfluidic device has sequestration pens configured as in any of the embodiments discussed herein where the microfluidic device has about 5 to about 10 sequestration pens, about 10 to about 50 sequestration pens, about 25 to about 200 sequestration pens, about 100 to about 500 sequestration pens, about 200 to about 1000 sequestration pens, about 500 to about 1500 sequestration pens, about 1000 to about 2500 sequestration pens, about 2000 to about 5000 sequestration pens, about 3500 to about 7000 sequestration pens, about 5000 to about 10,000 sequestration pens, about 7,500 to about 15,000 sequestration pens, about 12,500 to about 20,000 sequestration pens, about 15,000 to about 25,000 sequestration pens, about 20,000 to about 30,000 sequestration pens, about 25,000 to about 35,000 sequestration pens, about 30,000 to about 40,000 sequestration pens, about 35,000 to about 45,000 sequestration pens, or about 40,000 to about 50,000 sequest
  • At least one inner surface of the microfluidic device includes a coating material that provides a layer of organic and/or hydrophilic molecules suitable for maintenance, expansion and/or movement of biological micro-object(s) (i.e., the biological micro-object exhibits increased viability, greater expansion and/or greater portability within the microfluidic device).
  • the conditioned surface may reduce surface fouling, participate in providing a layer of hydration, and/or otherwise shield the biological micro-objects from contact with the non-organic materials of the microfluidic device interior.
  • substantially all the inner surfaces of the microfluidic device include the coating material.
  • the coated inner surface(s) may include the surface of a flow region (e.g., channel), chamber, or sequestration pen, or a combination thereof.
  • each of a plurality of sequestration pens has at least one inner surface coated with coating materials.
  • each of a plurality of flow regions or channels has at least one inner surface coated with coating materials.
  • at least one inner surface of each of a plurality of sequestration pens and each of a plurality of channels is coated with coating materials.
  • the coating may be applied before or after introduction of biological micro-object(s), or may be introduced concurrently with the biological microobjects).
  • the biological micro-object(s) may be imported into the microfluidic device in a fluidic medium that includes one or more coating agents.
  • the inner surface(s) of the microfluidic device e.g., a microfluidic device having an electrode activation substrate such as, but not limited to, a device including dielectrophoresis (DEP) electrodes
  • a coating solution comprising a coating agent prior to introduction of the biological micro-object(s) into the microfluidic device.
  • Any convenient coating agent/coating solution can be used, including but not limited to: serum or serum factors, bovine serum albumin (BSA), polymers, detergents, enzymes, and any combination thereof.
  • the at least one inner surface may include a coating material that comprises a polymer.
  • the polymer may be non-covalently bound (e.g., it may be non-specifically adhered) to the at least one surface.
  • the polymer may have a variety of structural motifs, such as found in block polymers (and copolymers), star polymers (star copolymers), and graft or comb polymers (graft copolymers), all of which may be suitable for the methods disclosed herein.
  • alkylene ether containing polymers may be suitable for use in the microfluidic devices described herein, including but not limited to Pluronic® polymers such as Pluronic® L44, L64, P85, and F127 (including F127NF).
  • Pluronic® polymers such as Pluronic® L44, L64, P85, and F127 (including F127NF).
  • F127NF including F127NF
  • suitable coating materials are described in US2016/0312165, the contents of which are herein incorporated by reference in their entirety.
  • the at least one inner surface includes covalently linked molecules that provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) within the microfluidic device, providing a conditioned surface for such cells.
  • the covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device, as described below.
  • the linking group is also covalently linked to a surface modifying moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/ expansion/ movement of biological micro- object(s).
  • the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro- object(s) may include alkyl or fluoroalkyl (which includes perfluoroalkyl) moieties; mono- or polysaccharides (which may include but is not limited to dextran); alcohols (including but not limited to propargyl alcohol); polyalcohols, including but not limited to polyvinyl alcohol; alkylene ethers, including but not limited to polyethylene glycol; polyelectrolytes ( including but not limited to polyacrylic acid or polyvinyl phosphonic acid); amino groups (including derivatives thereof, such as, but not limited to alkylated amines, hydroxyalkylated amino group, guanidinium, and heterocyclic groups containing an unaromatized nitrogen ring atom, such as, but not limited to morpholinyl or piperazinyl); carboxylic acids including but not limited to propi
  • the covalently linked moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device may include non-polymeric moieties such as an alkyl moiety, amino acid moiety, alcohol moiety, amino moiety, carboxylic acid moiety, phosphonic acid moiety, sulfonic acid moiety, sulfamic acid moiety, or saccharide moiety.
  • the covalently linked moiety may include polymeric moieties, which may include any of these moieties.
  • a microfluidic device may have a hydrophobic layer upon the inner surface of the base which includes a covalently linked alkyl moiety.
  • the covalently linked alkyl moiety may comprise carbon atoms forming a linear chain (e.g., a linear chain of at least 10 carbons, or at least 14, 16, 18, 20, 22, or more carbons) and may be an unbranched alkyl moiety.
  • the alkyl group may include a substituted alkyl group (e.g., some of the carbons in the alkyl group can be fluorinated or perfluorinated).
  • the alkyl group may include a first segment, which may include a perfluoroalkyl group, joined to a second segment, which may include a non-substituted alkyl group, where the first and second segments may be joined directly or indirectly (e.g., by means of an ether linkage).
  • the first segment of the alkyl group may be located distal to the linking group, and the second segment of the alkyl group may be located proximal to the linking group.
  • the covalently linked moiety may include at least one amino acid, which may include more than one type of amino acid.
  • the covalently linked moiety may include a peptide or a protein.
  • the covalently linked moiety may include an amino acid which may provide a zwitterionic surface to support cell growth, viability, portability, or any combination thereof.
  • the covalently linked moiety may further include a streptavidin or biotin moiety.
  • a modified biological moiety such as, for example, a biotinylated protein or peptide may be introduced to the inner surface of a microfluidic device bearing covalently linked streptavidin, and couple via the covalently linked streptavidin to the surface, thereby providing a modified surface presenting the protein or peptide.
  • the covalently linked moiety may include at least one alkylene oxide moiety and may include any alkylene oxide polymer as described above.
  • One useful class of alkylene ether containing polymers is polyethylene glycol (PEG M w ⁇ 100,000Da) or alternatively polyethylene oxide (PEO, M w > 100,000).
  • PEG polyethylene glycol
  • PEO polyethylene oxide
  • a PEG may have an M w of about lOOODa, 5000Da, 10,000Da or 20,000Da.
  • the PEG polymer may further be substituted with a hydrophilic or charged moiety, such as but not limited to an alcohol functionality or a carboxylic acid moiety.
  • the covalently linked moiety may include one or more saccharides.
  • the covalently linked saccharides may be mono-, di-, or polysaccharides.
  • the covalently linked saccharides may be modified to introduce a reactive pairing moiety which permits coupling or elaboration for attachment to the surface.
  • One exemplary covalently linked moiety may include a dextran polysaccharide, which may be coupled indirectly to a surface via an unbranched linker.
  • the coating material providing a conditioned surface may comprise only one kind of covalently linked moiety or may include more than one different kind of covalently linked moiety.
  • a polyethylene glycol conditioned surface may have covalently linked alkylene oxide moieties having a specified number of alkylene oxide units which are all the same, e.g., having the same linking group and covalent attachment to the surface, the same overall length, and the same number of alkylene oxide units.
  • the coating material may have more than one kind of covalently linked moiety attached to the surface.
  • the coating material may include the molecules having covalently linked alkylene oxide moieties having a first specified number of alkylene oxide units and may further include a further set of molecules having bulky moieties such as a protein or peptide connected to a covalently attached alkylene oxide linking moiety having a greater number of alkylene oxide units.
  • the different types of molecules may be varied in any suitable ratio to obtain the surface characteristics desired.
  • the conditioned surface having a mixture of first molecules having a chemical structure having a first specified number of alkylene oxide units and second molecules including peptide or protein moieties, which may be coupled via a biotin/streptavidin binding pair to the covalently attached alkylene linking moiety may have a ratio of first molecules: second molecules of about 99:1; about 90:10; about 75:25; about 50:50; about 30:70; about 20:80; about 10:90; or any ratio selected to be between these values.
  • the first set of molecules having different, less sterically demanding termini and fewer backbone atoms can help to functionalize the entire substrate surface and thereby prevent undesired adhesion or contact with the silicon/silicon oxide, hafnium oxide or alumina making up the substrate itself.
  • the selection of the ratio of mixture of first molecules to second molecules may also modulate the surface modification introduced by the second molecules bearing peptide or protein moieties.
  • Conditioned surface properties can alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid phase deposition, spin coating, flooding, and electrostatic coating).
  • the conditioned surface may have a thickness of about Inm to about lOnm.
  • the covalently linked moieties of the conditioned surface may form a monolayer when covalently linked to the surface of the microfluidic device (which may include an electrode activation substrate having dielectrophoresis (DEP) or electrowetting (EW) electrodes) and may have a thickness of less than 10 nm (e.g., less than 5 nm, or about 1.5 to 3.0 nm). These values are in contrast to that of a surface prepared by spin coating, for example, which may typically have a thickness of about 30nm.
  • the conditioned surface does not require a perfectly formed monolayer to be suitably functional for operation within a DEP-configured microfluidic device.
  • the conditioned surface formed by the covalently linked moieties may have a thickness of about 10 nm to about 50 nm.
  • the covalently linked coating material may be formed by reaction of a molecule which already contains the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device, and may have a structure of Formula I, as shown below.
  • the covalently linked coating material may be formed in a two- part sequence, having a structure of Formula II, by coupling the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance and/or expansion of biological micro-object(s) to a surface modifying ligand that itself has been covalently linked to the surface.
  • the surface may be formed in a two-part or three-part sequence, including a streptavidin/biotin binding pair, to introduce a protein, peptide, or mixed modified surface.
  • the coating material may be linked covalently to oxides of the surface of a DEP- configured or EW- configured substrate.
  • the coating material may be attached to the oxides via a linking group (“LG”), which may be a siloxy or phosphonate ester group formed from the reaction of a siloxane or phosphonic acid group with the oxides.
  • LG linking group
  • the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device can be any of the moieties described herein.
  • the linking group LG may be directly or indirectly connected to the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for maintenance/expansion of biological micro-object(s) in the microfluidic device.
  • optional linker (“L”) is not present and n is 0.
  • linker L is present and n is 1.
  • the linker L may have a linear portion where a backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and/or phosphorus atoms, subject to chemical bonding limitations as is known in the art.
  • the coupling group CG represents the resultant group from reaction of a reactive moiety R x and a reactive pairing moiety R px (i.e., a moiety configured to react with the reactive moiety R x ).
  • CG may be a carboxamidyl group, a triazolylene group, substituted triazolylene group, a carboxamidyl, thioamidyl, an oxime, a mercaptyl, a disulfide, an ether, or alkenyl group, or any other suitable group that may be formed upon reaction of a reactive moiety with its respective reactive pairing moiety.
  • CG may further represent a streptavidin/biotin binding pair.
  • Microfluidic device motive technologies can be used with any type of motive technology.
  • the control and monitoring equipment of the system can comprise a motive module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of a microfluidic device.
  • the motive technology(ies) may include, for example, dielectrophoresis (DEP), electrowetting (EW), and/or other motive technologies.
  • the microfluidic device can have a variety of motive configurations, depending upon the type of object being moved and other considerations. Returning to FIG.
  • the support structure 104 and/or cover 110 of the microfluidic device 100 can comprise DEP electrode activation substrates for selectively inducing motive forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 and thereby select, capture, and/or move individual micro-objects or groups of microobjects.
  • motive forces are applied across the fluidic medium 180 (e.g., in the flow path and/or in the sequestration pens) via one or more electrodes (not shown) to manipulate, transport, separate and sort micro-objects located therein.
  • motive forces are applied to one or more portions of microfluidic circuit 120 in order to transfer a single micro-object from the flow path 106 into a desired microfluidic sequestration pen.
  • motive forces are used to prevent a micro-object within a sequestration pen from being displaced therefrom.
  • motive forces are used to selectively remove a micro-object from a sequestration pen that was previously collected in accordance with the embodiments of the current disclosure.
  • the microfluidic device is configured as an optically-actuated electrokinetic device, such as in optoelectronic tweezer (GET) and/or optoelectrowetting (OEW) configured device.
  • OET configured devices e.g., containing optically actuated dielectrophoresis electrode activation substrates
  • U.S. Patent No. RE 44,711 Wang, et al.
  • U.S. Patent No. 7,956,339 Ohta, et al.
  • U.S. Patent No. 9,908,115 Hobbs et al.
  • OEW configured devices can include those illustrated in U.S. Patent No. 6,958,132 (Chiou, et al.), and U.S. Patent Application No. 9,533,306 (Chiou, et al.), each of which is incorporated herein by reference in its entirety.
  • suitable optically-actuated electrokinetic devices that include combined OET/OEW configured devices can include those illustrated in U.S. Patent Application Publication No. 2015/0306598 (Khandros, et al.), U.S. Patent Application Publication No 2015/0306599 (Khandros, et al.), and U.S. Patent Application Publication No. 2017/0173580 (Lowe, et al.), each of which is incorporated herein by reference in its entirety.
  • FIGs. 1-5B may illustrate portions of microfluidic devices while not depicting other portions. Further, Figures 1-5B may be part of, and implemented as, one or more microfluidic systems.
  • FIGs. 4A and 4B show a side cross-sectional view and a top cross- sectional view, respectively, of a portion of an enclosure 102 of the microfluidic device 400 having a region/chamber 402, which may be part of a fluidic circuit element having a more detailed structure, such as a growth chamber, a sequestration pen (which may be like any sequestration pen described herein), a flow region, or a flow channel.
  • microfluidic device 400 may be similar to microfluidic devices 100, 175, 200, 300, 520 or any other microfluidic device as described herein.
  • the microfluidic device 400 may include other fluidic circuit elements and may be part of a system including control and monitoring equipment 152, described above, having one or more of the media module 160, motive module 162, imaging module 164, optional tilting module 166, and other modules 168.
  • Microfluidic devices 175, 200, 300, 520 and any other microfluidic devices described herein may similarly have any of the features described in detail for FIGs. 1A-1B and 4A-4B.
  • the microfluidic device 400 includes a support structure 104 having a bottom electrode 404 and an electrode activation substrate 406 overlying the bottom electrode 404, and a cover 110 having a top electrode 410, with the top electrode 410 spaced apart from the bottom electrode 404.
  • the top electrode 410 and the electrode activation substrate 406 define opposing surfaces of the region/chamber 402.
  • a fluidic medium 180 contained in the region/chamber 402 thus provides a resistive connection between the top electrode 410 and the electrode activation substrate 406.
  • a power source 412 configured to be connected to the bottom electrode 404 and the top electrode 410 and create a biasing voltage between the electrodes, as required for the generation of DEP forces in the region/chamber 402, is also shown.
  • the power source 412 can be, for example, an alternating current (AC) power source.
  • the microfluidic device 200 illustrated in FIGs. 4A and 4B can have an optically-actuated DEP electrode activation substrate. Accordingly, changing patterns of light 418 from the light source 416, which may be controlled by the motive module 162, can selectively activate and deactivate changing patterns of DEP electrodes at regions 414 of the inner surface 408 of the electrode activation substrate 406. (Hereinafter the regions 414 of a microfluidic device having a DEP electrode activation substrate are referred to as “DEP electrode regions.”) As illustrated in FIG.
  • a light pattern 418 directed onto the inner surface 408 of the electrode activation substrate 406 can illuminate select DEP electrode regions 414a (shown in white) in a pattern, such as a square.
  • the non-illuminated DEP electrode regions 414 cross-hatched are hereinafter referred to as “dark” DEP electrode regions 414.
  • the relative electrical impedance through the DEP electrode activation substrate 406 (i.e., from the bottom electrode 404 up to the inner surface 408 of the electrode activation substrate 406 which interfaces with the fluidic medium 180 in the flow region 106) is greater than the relative electrical impedance through the fluidic medium 180 in the region/chamber 402 (i.e., from the inner surface 408 of the electrode activation substrate 406 to the top electrode 410 of the cover 110) at each dark DEP electrode region 414.
  • An illuminated DEP electrode region 414a exhibits a reduced relative impedance through the electrode activation substrate 406 that is less than the relative impedance through the fluidic medium 180 in the region/chamber 402 at each illuminated DEP electrode region 414a.
  • the foregoing DEP configuration creates an electric field gradient in the fluidic medium 180 between illuminated DEP electrode regions 414a and adjacent dark DEP electrode regions 414, which in turn creates local DEP forces that attract or repel nearby micro-objects (not shown) in the fluidic medium 180.
  • DEP electrodes that attract or repel micro-objects in the fluidic medium 180 can thus be selectively activated and deactivated at many different such DEP electrode regions 414 at the inner surface 408 of the region/chamber 402 by changing light patterns 418 projected from a light source 416 into the microfluidic device 400.
  • Whether the DEP forces attract or repel nearby micro-objects can depend on such parameters as the frequency of the power source 412 and the dielectric properties of the fluidic medium 180 and/or micro-objects (not shown).
  • negative DEP forces may be produced. Negative DEP forces may repel the microobjects away from the location of the induced non-uniform electrical field.
  • a microfluidic device incorporating DEP technology may generate negative DEP forces.
  • the square pattern 420 of illuminated DEP electrode regions 414a illustrated in FIG. 4B is an example only. Any pattern of the DEP electrode regions 414 can be illuminated (and thereby activated) by the pattern of light 418 projected into the microfluidic device 400, and the pattern of illuminated/activated DEP electrode regions 414 can be repeatedly changed by changing or moving the light pattern 418.
  • the electrode activation substrate 406 can comprise or consist of a photoconductive material.
  • the inner surface 408 of the electrode activation substrate 406 can be featureless.
  • the electrode activation substrate 406 can comprise or consist of a layer of hydrogenated amorphous silicon (a-Si:H).
  • the a-Si:H can comprise, for example, about 8% to 40% hydrogen (calculated as 100 * the number of hydrogen atoms / the total number of hydrogen and silicon atoms).
  • the layer of a-Si:H can have a thickness of about 500 nm to about 2.0 pm.
  • the DEP electrode regions 414 can be created anywhere and in any pattern on the inner surface 408 of the electrode activation substrate 406, in accordance with the light pattern 418.
  • the number and pattern of the DEP electrode regions 214 thus need not be fixed, but can correspond to the light pattern 418.
  • Examples of microfluidic devices having a DEP configuration comprising a photoconductive layer such as discussed above have been described, for example, in U.S. Patent No. RE 44,711 (Wu, et al.) (originally issued as U.S. Patent No. 7,612,355), each of which is incorporated herein by reference in its entirety.
  • the electrode activation substrate 406 can comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers that form semiconductor integrated circuits, such as is known in semiconductor fields.
  • the electrode activation substrate 406 can comprise a plurality of phototransistors, including, for example, lateral bipolar phototransistors, with each phototransistor corresponding to a DEP electrode region 414.
  • the electrode activation substrate 406 can comprise electrodes (e.g., conductive metal electrodes) controlled by phototransistor switches, with each such electrode corresponding to a DEP electrode region 414.
  • the electrode activation substrate 406 can include a pattern of such phototransistors or phototransistor-controlled electrodes.
  • the pattern for example, can be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns.
  • the pattern can be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes that form a hexagonal lattice.
  • electric circuit elements can form electrical connections between the DEP electrode regions 414 at the inner surface 408 of the electrode activation substrate 406 and the bottom electrode 404, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 418, as described above.
  • microfluidic devices having electrode activation substrates that comprise phototransistors have been described, for example, in U.S. Patent No. 7,956,339 (Ohta et al.) and U.S. Patent No. 9,908,115 (Hobbs et al.), the entire contents of each of which are incorporated herein by reference.
  • Examples of microfluidic devices having electrode activation substrates that comprise electrodes controlled by phototransistor switches have been described, for example, in U.S. Patent No. 9,403,172 (Short et al.), which is incorporated herein by reference in its entirety.
  • the top electrode 410 is part of a first wall (or cover 110) of the enclosure 102, and the electrode activation substrate 406 and bottom electrode 404 are part of a second wall (or support structure 104) of the enclosure 102.
  • the region/chamber 402 can be between the first wall and the second wall.
  • the electrode 410 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 406 and/or the electrode 410 are part of the first wall (or cover 110).
  • the light source 416 can alternatively be used to illuminate the enclosure 102 from below.
  • the motive module 162 of control and monitoring equipment 152 can select a micro-object (not shown) in the fluidic medium 180 in the region/chamber 402 by projecting a light pattern 418 into the microfluidic device 400 to activate a first set of one or more DEP electrodes at DEP electrode regions 414a of the inner surface 408 of the electrode activation substrate 406 in a pattern (e.g., square pattern 420) that surrounds and captures the micro-object.
  • a pattern e.g., square pattern 420
  • the motive module 162 can then move the in situ- generated captured micro-object by moving the light pattern 418 relative to the microfluidic device 400 to activate a second set of one or more DEP electrodes at DEP electrode regions 414.
  • the microfluidic device 400 can be moved relative to the light pattern 418.
  • the microfluidic device 400 may be a DEP configured device that does not rely upon light activation of DEP electrodes at the inner surface 408 of the electrode activation substrate 406.
  • the electrode activation substrate 406 can comprise selectively addressable and energizable electrodes positioned opposite to a surface including at least one electrode (e.g., cover 110).
  • Switches may be selectively opened and closed to activate or inactivate DEP electrodes at DEP electrode regions 414, thereby creating a net DEP force on a micro-object (not shown) in region/chamber 402 in the vicinity of the activated DEP electrodes.
  • the DEP force can attract or repel a nearby micro-object.
  • one or more micro-objects in region/chamber 402 can be selected and moved within the region/chamber 402.
  • the motive module 162 in FIG. 1A can control such switches and thus activate and deactivate individual ones of the DEP electrodes to select, and move particular micro-objects (not shown) around the region/chamber 402.
  • Microfluidic devices having a DEP electrode activation substrate that includes selectively addressable and energizable electrodes are known in the art and have been described, for example, in U.S. Patent No. 6,294,063 (Becker, et al.) and U.S. Patent No. 6,942,776 (Medoro), each of which is incorporated herein by reference in its entirety.
  • a power source 412 can be used to provide a potential (e.g., an AC voltage potential) that powers the electrical circuits of the microfluidic device 400.
  • the power source 412 can be the same as, or a component of, the power source 192 referenced in Fig. 1A.
  • Power source 412 can be configured to provide an AC voltage and/or current to the top electrode 410 and the bottom electrode 404.
  • the power source 412 can provide a frequency range and an average or peak power (e.g., voltage or current) range sufficient to generate net DEP forces (or electrowetting forces) strong enough to select and move individual micro-objects (not shown) in the region/chamber 402, as discussed above, and/or to change the wetting properties of the inner surface 408 of the support structure 104 in the region/chamber 402, as also discussed above.
  • Such frequency ranges and average or peak power ranges are known in the art. See, e.g., U.S. Patent No. 6,958,132 (Chiou, et al.), US Patent No. RE44,711 (Wu, et al.) (originally issued as US Patent No.
  • Other forces may be utilized within the microfluidic devices, alone or in combination, to move selected micro-objects.
  • Bulk fluidic flow within the microfluidic channel may move micro-objects within the flow region.
  • Localized fluidic flow which may be operated within the microfluidic channel, within a sequestration pen, or within another kind of chamber (e.g., a reservoir) can also be used to move selected micro-objects.
  • Localized fluidic flow can be used to move selected micro-objects out of the flow region into a non-flow region such as a sequestration pen or the reverse, from a non-flow region into a flow region.
  • the localized flow can be actuated by deforming a deformable wall of the microfluidic device, as described in U.S. Patent No. 10,058,865 (Breinlinger, et al.), which is incorporated herein by reference in its entirety.
  • Gravity may be used to move micro-objects within the microfluidic channel, into a sequestration pen, and/or out of a sequestration pen or other chamber, as described in U.S. Patent No. 9,744,533 (Breinlinger, et al.), which is incorporated herein by reference in its entirety.
  • Use of gravity e.g., by tilting the microfluidic device and/or the support to which the microfluidic device is attached
  • Magnetic forces may be employed to move microobjects including paramagnetic materials, which can include magnetic micro-objects attached to or associated with a biological micro-object.
  • centripetal forces may be used to move micro-objects within the microfluidic channel, as well as into or out of sequestration pens or other chambers in the microfluidic device.
  • laser-generated dislodging forces may be used to export micro-objects or assist in exporting micro-objects from a sequestration pen or any other chamber in the microfluidic device, as described in International Patent Publication No. WO2017/117408 (Kurz, et al.), which is incorporated herein by reference in its entirety.
  • DEP forces are combined with other forces, such as fluidic flow (e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force), so as to manipulate, transport, separate and sort micro-objects and/or droplets within the microfluidic circuit 120.
  • fluidic flow e.g., bulk fluidic flow in a channel or localized fluidic flow actuated by deformation of a deformable surface of the microfluidic device, laser generated dislodging forces, and/or gravitational force
  • the DEP forces can be applied prior to the other forces.
  • the DEP forces can be applied after the other forces.
  • the DEP forces can be applied in an alternating manner with the other forces.
  • repositioning of microobjects may not generally rely upon gravity or hydrodynamic forces to position or trap microobjects at a selected position.
  • Gravity may be chosen as one form of repositioning force, but the ability to reposition of micro-objects within the microfluidic device does not rely solely upon the use of gravity.
  • fluid flow in the microfluidic channels may be used to introduce micro-objects into the microfluidic channels (e.g., flow region), such regional flow is not relied upon to pen or unpen micro-objects, while localized flow (e.g., force derived from actuating a deformable surface) may, in some embodiments, be selected from amongst the other types of repositioning forces described herein to pen or unpen micro-objects or to export them from the microfluidic device.
  • the electrical power source 192 can provide electric power to the microfluidic device 100, providing biasing voltages or currents as needed.
  • the electrical power source 192 can, for example, comprise one or more alternating current (AC) and/or direct current (DC) voltage or current sources.
  • System 150 can further include a media source 178.
  • the media source 178 e.g., a container, reservoir, or the like
  • the media source 178 can comprise multiple sections or containers, each for holding a different fluidic medium 180.
  • the media source 178 can be a device that is outside of and separate from the microfluidic device 100, as illustrated in FIG. 1A.
  • the media source 178 can be located in whole or in part inside the enclosure 102 of the microfluidic device 100.
  • the media source 178 can comprise reservoirs that are part of the microfluidic device 100.
  • FIG. 1A also illustrates simplified block diagram depictions of examples of control and monitoring equipment 152 that constitute part of system 150 and can be utilized in conjunction with a microfluidic device 100.
  • control and monitoring equipment 152 can include a master controller 154 comprising a media module 160 for controlling the media source 178, a motive module 162 for controlling movement and/or selection of micro-objects (not shown) and/or medium (e.g., droplets of medium) in the microfluidic circuit 120, an imaging module 164 for controlling an imaging device (e.g., a camera, microscope, light source or any combination thereof) for capturing images (e.g., digital images), and an optional tilting module 166 for controlling the tilting of the microfluidic device 100.
  • the control equipment 152 can also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100.
  • the monitoring equipment 152 can further include a display device 170 and an input/output device 172.
  • the master controller 154 can comprise a control module 156 and a digital memory 158.
  • the control module 156 can comprise, for example, a digital processor configured to operate in accordance with machine executable instructions (e.g., software, firmware, source code, or the like) stored as non-transitory data or signals in the memory 158.
  • the control module 156 can comprise hardwired digital circuitry and/or analog circuitry.
  • the media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 can be similarly configured.
  • functions, processes acts, actions, or steps of a process discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus can be performed by any one or more of the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 configured as discussed above.
  • the master controller 154, media module 160, motive module 162, imaging module 164, optional tilting module 166, and/or other modules 168 may be communicatively coupled to transmit and receive data used in any function, process, act, action or step discussed herein.
  • the media module 160 controls the media source 178.
  • the media module 160 can control the media source 178 to input a selected fluidic medium 180 into the enclosure 102 (e.g., through an inlet port 107).
  • the media module 160 can also control removal of media from the enclosure 102 (e.g., through an outlet port (not shown)).
  • One or more media can thus be selectively input into and removed from the microfluidic circuit 120.
  • the media module 160 can also control the flow of fluidic medium 180 in the flow path 106 inside the microfluidic circuit 120.
  • the media module 160 may also provide conditioning gaseous conditions to the media source 178, for example, providing an environment containing 5% CO2 (or higher).
  • the media module 160 may also control the temperature of an enclosure of the media source, for example, to provide feeder cells in the media source with proper temperature control.
  • the motive module 162 can be configured to control selection and movement of micro-objects (not shown) in the microfluidic circuit 120.
  • the enclosure 102 of the microfluidic device 100 can comprise one or more electrokinetic mechanisms including a dielectrophoresis (DEP) electrode activation substrate, optoelectronic tweezers (OET) electrode activation substrate, electrowetting (EW) electrode activation substrate, and/or an opto-electrowetting (OEW) electrode activation substrate, where the motive module 162 can control the activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects and/or droplets in the flow path 106 and/or within sequestration pens 124, 126, 128, and 130.
  • DEP dielectrophoresis
  • OET optoelectronic tweezers
  • EW electrowetting
  • OEW opto-electrowetting
  • the electrokinetic mechanism(s) may be any suitable single or combined mechanism as described within the paragraphs describing motive technologies for use within the microfluidic device.
  • a DEP configured device may include one or more electrodes that apply a non-uniform electric field in the microfluidic circuit 120 sufficient to exert a dielectrophoretic force on micro-objects in the microfluidic circuit 120.
  • An OET configured device may include photo-activatable electrodes to provide selective control of movement of micro-objects in the microfluidic circuit 120 via light-induced dielectrophoresis.
  • the imaging module 164 can control the imaging device.
  • the imaging module 164 can receive and process image data from the imaging device.
  • Image data from the imaging device can comprise any type of information captured by the imaging device (e.g., the presence or absence of micro-objects, droplets of medium, accumulation of label, such as fluorescence label, etc.).
  • the imaging module 164 can further calculate the position of objects (e.g., micro-objects, droplets of medium) and/or the rate of motion of such objects within the microfluidic device 100.
  • the imaging device (part of imaging module 164, discussed below) can comprise a device, such as a digital camera, for capturing images inside microfluidic circuit 120.
  • the imaging device further comprises a detector having a fast frame rate and/or high sensitivity (e.g., for low light applications).
  • the imaging device can also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein).
  • the emitted light beams may be in the visible spectrum and may, e.g., include fluorescence emissions.
  • the reflected light beams may include reflected emissions originating from an LED or a wide spectrum lamp, such as a mercury lamp (e.g., a high-pressure mercury lamp) or a Xenon arc lamp.
  • the imaging device may further include a microscope (or an optical train), which may or may not include an eyepiece.
  • System 150 may further comprise a support structure 190 configured to support and/or hold the enclosure 102 comprising the microfluidic circuit 120.
  • the optional tilting module 166 can be configured to activate the support structure 190 to rotate the microfluidic device 100 about one or more axes of rotation.
  • the optional tilting module 166 can be configured to support and/or hold the microfluidic device 100 in a level orientation (i.e., at 0° relative to x- and y-axes), a vertical orientation (i.e., at 90° relative to the x-axis and/or the y-axis), or any orientation therebetween.
  • support structure 190 can optionally be used to tilt the microfluidic device 100 (e.g., as controlled by optional tilting module 166) to 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 90° relative to the x-axis or any degree therebetween.
  • the support structure 190 can hold the microfluidic device 100 at a fixed angle of 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0.8°, 0.9°, 1°, 2°, 3°, 4°, 5°, or 10° relative to the x-axis (horizontal), so long as DEP is an effective force to move micro-objects out of the sequestration pens into the microfluidic channel. Since the surface of the electrode activation substrate is substantially flat, DEP forces may be used even when the far end of the sequestration pen, opposite its opening to the microfluidic channel, is disposed at a position lower in a vertical direction than the microfluidic channel.
  • the microfluidic device 100 may be disposed in an orientation such that the inner surface of the base of the flow path 106 is positioned at an angle above or below the inner surface of the base of the one or more sequestration pens opening laterally to the flow path.
  • the term “above” as used herein denotes that the flow path 106 is positioned higher than the one or more sequestration pens on a vertical axis defined by the force of gravity (i.e., an object in a sequestration pen above a flow path 106 would have a higher gravitational potential energy than an object in the flow path), and inversely, for positioning of the flow path 106 below one or more sequestration pens.
  • the support structure 190 may be held at a fixed angle of less than about 5°, about 4°, about 3° or less than about 2 0 relative to the x-axis (horizontal), thereby placing the sequestration pens at a lower potential energy relative to the flow path.
  • the device when long term culturing (e.g., for more than about 2, 3, 4, 5, 6, 7 or more days) is performed within the microfluidic device, the device may be supported on a culturing support and may be tilted at a greater angle of about 10°, 15°, 20°, 25°, 30°, or any angle therebetween to retain biological micro-objects within the sequestration pens during the long-term culturing period.
  • the microfluidic device containing the cultured biological micro-objects may be returned to the support 190 within system 150, where the angle of tilting is decreased to values as described above, affording the use of DEP to move the biological micro-objects out of the sequestration pens.
  • Further examples of the use of gravitational forces induced by tilting are described in U.S. Patent No. 9,744,533 (Breinlinger et al.), the contents of which are herein incorporated by reference in its entirety.
  • the support structure 190 is further configured to support and/or hold an oxygen delivery module, such as an oxygen delivery module described herein with respect to FIG. 16.
  • the support structure is configured to support and/or hold the oxygen delivery module in proximity to the microfluidic device.
  • the support structure is configured to support and/or hold the oxygen delivery module such that the oxygen delivery module surrounds the microfluidic device.
  • the support structure is configured to support and/or hold the oxygen delivery module a distance of at least about 1 millimeter (mm), 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or more from the microfluidic device.
  • the support structure is configured to support and/or hold the oxygen delivery module a distance of at most about 10 mm, 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, or less from the microfluidic device. In some embodiments, the support structure is configured to support and/or hold the oxygen delivery module a distance from the microfluidic device that ranges between any two of the preceding values.
  • the system 150 can include a structure (also referred to as a “nest”) 500 configured to hold a microfluidic device 520, which may be like microfluidic device 100, 200, or any other microfluidic device described herein.
  • the nest 500 can include a socket 502 capable of interfacing with the microfluidic device 520 (e.g., an optically actuated electrokinetic device 100, 200, etc.) and providing electrical connections from power source 192 to microfluidic device 520.
  • the nest 500 can further include an integrated electrical signal generation subsystem 504.
  • the electrical signal generation subsystem 504 can be configured to supply a biasing voltage to socket 502 such that the biasing voltage is applied across a pair of electrodes in the microfluidic device 520 when it is being held by socket 502.
  • the electrical signal generation subsystem 504 can be part of power source 192.
  • the ability to apply a biasing voltage to microfluidic device 520 does not mean that a biasing voltage will be applied at all times when the microfluidic device 520 is held by the socket 502. Rather, in most cases, the biasing voltage will be applied intermittently, e.g., only as needed to facilitate the generation of electrokinetic forces, such as dielectrophoresis or electro-wetting, in the microfluidic device 520.
  • the nest 500 can include a printed circuit board assembly (PCBA) 522.
  • the electrical signal generation subsystem 504 can be mounted on and electrically integrated into the PCBA 522.
  • the exemplary support includes socket 502 mounted on PCBA 522, as well.
  • the nest 500 can comprise an electrical signal generation subsystem 504 configured to measure the amplified voltage at the microfluidic device 520 and then adjust its own output voltage as needed such that the measured voltage at the microfluidic device 520 is the desired value.
  • the waveform amplification circuit can have a +6.5V to -6.5V power supply generated by a pair of DC-DC converters mounted on the PCBA 322, resulting in a signal of up to 13 Vpp at the microfluidic device 520.
  • the nest 500 further comprises a controller 508, such as a microprocessor used to sense and/or control the electrical signal generation subsystem 504.
  • a controller 508 such as a microprocessor used to sense and/or control the electrical signal generation subsystem 504.
  • suitable microprocessors include the chickenTM microprocessors, such as the PC NanoTM.
  • the controller 508 may be used to perform functions and analysis or may communicate with an external master controller 154 (shown in FIG. 1A) to perform functions and analysis. In the embodiment illustrated in FIG. 3 the controller 308 communicates with the master controller 154 (of FIG. 1A) through an interface (e.g., a plug or connector).
  • the support structure 500 can further include a thermal control subsystem 506.
  • the thermal control subsystem 506 can be configured to regulate the temperature of microfluidic device 520 held by the support structure 500.
  • the thermal control subsystem 506 can include a Peltier thermoelectric device (not shown) and a cooling unit (not shown).
  • the support structure 500 comprises an inlet 516 and an outlet 518 to receive cooled fluid from an external reservoir (not shown) of the cooling unit, introduce the cooled fluid into the fluidic path 514 and through the cooling block, and then return the cooled fluid to the external reservoir.
  • the Peltier thermoelectric device, the cooling unit, and/or the fluidic path 514 can be mounted on a casing 512 of the support structure 500.
  • the thermal control subsystem 506 is configured to regulate the temperature of the Peltier thermoelectric device so as to achieve a target temperature for the microfluidic device 520. Temperature regulation of the Peltier thermoelectric device can be achieved, for example, by a thermoelectric power supply, such as a PololuTM thermoelectric power supply (Pololu Robotics and Electronics Corp.).
  • the thermal control subsystem 506 can include a feedback circuit, such as a temperature value provided by an analog circuit. Alternatively, the feedback circuit can be provided by a digital circuit.
  • the nest 500 can include a serial port 524 which allows the microprocessor of the controller 508 to communicate with an external master controller 154 via the interface.
  • the microprocessor of the controller 508 can communicate (e.g., via a Plink tool (not shown)) with the electrical signal generation subsystem 504 and thermal control subsystem 506.
  • the electrical signal generation subsystem 504 and the thermal control subsystem 506 can communicate with the external master controller 154.
  • the master controller 154 can, among other things, assist the electrical signal generation subsystem 504 by performing scaling calculations for output voltage adjustments.
  • GUI Graphical User Interface
  • a display device 170 coupled to the external master controller 154, can be configured to plot temperature and waveform data obtained from the thermal control subsystem 506 and the electrical signal generation subsystem 504, respectively.
  • the GUI can allow for updates to the controller 508, the thermal control subsystem 506, and the electrical signal generation subsystem 504.
  • FIG. 5B is a schematic of an optical sub-system 550 having an optical apparatus 510 for imaging and manipulating micro-objects in a microfluidic device 520, which can be any microfluidic device described herein.
  • the optical apparatus 510 can be configured to perform imaging, analysis and manipulation of one or more micro-objects within the enclosure of the microfluidic device 520.
  • the optical apparatus 510 may have a first light source 552, a second light source 554, and a third light source 556.
  • the first light source 552 can transmit light to a structured light modulator 560, which can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical apparatus 510.
  • a structured light modulator 560 which can include a digital mirror device (DMD) or a microshutter array system (MSA), either of which can be configured to receive light from the first light source 552 and selectively transmit a subset of the received light into the optical apparatus 510.
  • DMD digital mirror device
  • MSA microshutter array system
  • the structured light modulator 560 can include a device that produces its own light (and thus dispenses with the need for a light source 552), such as an organic light emitting diode display (OLED), a liquid crystal on silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive liquid crystal display (LCD).
  • OLED organic light emitting diode display
  • LCOS liquid crystal on silicon
  • FLCOS ferroelectric liquid crystal on silicon device
  • LCD transmissive liquid crystal display
  • the structured light modulator 560 can be, for example, a projector.
  • the structured light modulator 560 can be capable of emitting both structured and unstructured light.
  • an imaging module and/or motive module of the system can control the structured light modulator 560.
  • the modulator when the structured light modulator 560 includes a mirror, the modulator can have a plurality of mirrors. Each mirror of the plurality of mirrors can have a size of about 5 microns x 5 microns to about 10 microns x 10 microns, or any values therebetween.
  • the structured light modulator 560 can include an array of mirrors (or pixels) that is 2000 x 1000, 2580 x 1600, 3000 x 2000, or any values therebetween. In some embodiments, only a portion of an illumination area of the structured light modulator 560 is used.
  • the structured light modulator 560 can transmit the selected subset of light to a first dichroic beam splitter 558, which can reflect this light to a first tube lens 562.
  • the first tube lens 562 can have a large clear aperture, for example, a diameter larger than about 40 mm to about 50 mm, or more, providing a large field of view.
  • the first tube lens 562 can have an aperture that is large enough to capture all (or substantially all) of the light beams emanating from the structured light modulator 560.
  • the structured light 515 having a wavelength of about 400 nm to about 710 nm may alternatively or in addition, provide fluorescence excitation illumination to the microfluidic device.
  • the second light source 554 may provide unstructured brightfield illumination.
  • the brightfield illumination light 525 may have any suitable wavelength, and in some embodiments, may have a wavelength of about 400 nm to about 760 nm.
  • the second light source 554 can transmit light to a second dichroic beam splitter 564 (which also may receive illumination light 535 from the third light source 556), and the second light, brightfield illumination light 525, may be transmitted therefrom to the first dichroic beam splitter 558.
  • the second light, brightfield illumination light 525 may then be transmitted from the first dichroic beam splitter 558 to the first tube lens 562.
  • the third light source 556 can transmit light through a matched pair relay lens (not shown) to a mirror 566.
  • the third illumination light 535 may therefrom be reflected to the second dichroic beam splitter 5338 and be transmitted therefrom to the first beam splitter 5338, and onward to the first tube lens 5381.
  • the third illumination light 535 may be a laser and may have any suitable wavelength. In some embodiments, the laser illumination 535 may have a wavelength of about 350 nm to about 900 nm.
  • the laser illumination 535 may be configured to heat portions of one or more sequestration pens within the microfluidic device.
  • the laser illumination 535 may be configured to heat fluidic medium, a micro-object, a wall or a portion of a wall of a sequestration pen, a metal target disposed within a microfluidic channel or sequestration pen of the microfluidic channel, or a photoreversible physical barrier within the microfluidic device, and described in more detail in U. S. Application Publication Nos. 2017/0165667 (Beaumont, et al.) and 2018/0298318 (Kurz, et al.), each of which disclosure is herein incorporated by reference in its entirety.
  • the laser illumination 535 may be configured to initiate photocleavage of surface modifying moieties of a modified surface of the microfluidic device or photocleavage of moieties providing adherent functionalities for micro-objects within a sequestration pen within the microfluidic device. Further details of photocleavage using a laser may be found in International Application Publication No. W02017/205830 (Lowe, Jr. et al.), which disclosure is herein incorporated by reference in its entirety.
  • the light from the first, second, and third light sources (552, 554, 556) passes through the first tube lens 562 and is transmitted to a third dichroic beam splitter 568 and filter changer 572.
  • the third dichroic beam splitter 568 can reflect a portion of the light and transmit the light through one or more filters in the filter changer 572 and to the objective 570, which may be an objective changer with a plurality of different objectives that can be switched on demand.
  • Some of the light (515, 525, and/or 535) may pass through the third dichroic beam splitter 568 and be terminated or absorbed by a beam block (not shown).
  • the light reflected from the third dichroic beam splitter 568 passes through the objective 570 to illuminate the sample plane 574, which can be a portion of a microfluidic device 520 such as the sequestration pens described herein.
  • the nest 500 as described in FIG. 5A, can be integrated with the optical apparatus 510 and be a part of the apparatus 510.
  • the nest 500 can provide electrical connection to the enclosure and be further configured to provide fluidic connections to the enclosure. Users may load the microfluidic apparatus 520 into the nest 500.
  • the nest 500 can be a separate component independent of the optical apparatus 510.
  • Light can be reflected off and/or emitted from the sample plane 574 to pass back through the objective 570, through the filter changer 572, and through the third dichroic beam splitter 568 to a second tube lens 576.
  • the light can pass through the second tube lens 576 (or imaging tube lens 576) and be reflected from a mirror 578 to an imaging sensor 580.
  • Stray light baffles (not shown) can be placed between the first tube lens 562 and the third dichroic beam splitter 568, between the third dichroic beam splitter 568 and the second tube lens 576, and between the second tube lens 576 and the imaging sensor 580.
  • the optical apparatus can comprise the objective lens 570 that is specifically designed and configured for viewing and manipulating of micro-objects in the microfluidic device 520.
  • the objective lens 570 is specifically designed and configured for viewing and manipulating of micro-objects in the microfluidic device 520.
  • conventional microscope objective lenses are designed to view micro-objects on a slide or through 5mm of aqueous fluid, while micro-objects in the microfluidic device 520 are inside the plurality of sequestration pens within the viewing plane 574 which have a depth of 20, 30, 40, 50, 60 70, 80 microns or any values therebetween.
  • a transparent cover 520a for example, glass or ITO cover with a thickness of about 750 microns, can be placed on top of the plurality of sequestration pens, which are disposed above a microfluidic substrate 520c.
  • the objective lens 570 of the optical apparatus 510 can be configured to correct the spherical and chromatic aberrations in the optical apparatus 1350.
  • the objective lens 570 can have one or more magnification levels available such as, 4X, 10X, 20X.
  • the structured light modulator 560 can be configured to modulate light beams received from the first light source 552 and transmits a plurality of illumination light beams 515, which are structured light beams, into the enclosure of the microfluidic device, e.g., the region containing the sequestration pens.
  • the structured light beams can comprise the plurality of illumination light beams.
  • the plurality of illumination light beams can be selectively activated to generate a plurality of illuminations patterns.
  • the structured light modulator 560 can be configured to generate an illumination pattern, similarly as described for FIGs. 4A-4B, which can be moved and adjusted.
  • the optical apparatus 560 can further comprise a control unit (not shown) which is configured to adjust the illumination pattern to selectively activate the one or more of the plurality of DEP electrodes of a substrate 520c and generate DEP forces to move the one or more micro-objects inside the plurality of sequestration pens within the microfluidic device 520.
  • the plurality of illuminations patterns can be adjusted over time in a controlled manner to manipulate the micro-objects in the microfluidic device 520.
  • Each of the plurality of illumination patterns can be shifted to shift the location of the DEP force generated and to move the structured light for one position to another in order to move the micro-objects within the enclosure of the microfluidic apparatus 520.
  • the optical apparatus 510 may be configured such that each of the plurality of sequestration pens in the sample plane 574 within the field of view is simultaneously in focus at the image sensor 580 and at the structured light modulator 560.
  • the structured light modulator 560 can be disposed at a conjugate plane of the image sensor 580.
  • the optical apparatus 510 can have a confocal configuration or confocal property.
  • the optical apparatus 510 can be further configured such that only each interior area of the flow region and/or each of the plurality of sequestration pens in the sample plane 574 within the field of view is imaged onto the image sensor 580 in order to reduce overall noise to thereby increase the contrast and resolution of the image.
  • the first tube lens 562 can be configured to generate collimated light beams and transmit the collimated light beams to the objective lens 570.
  • the objective 570 can receive the collimated light beams from the first tube lens 562 and focus the collimated light beams into each interior area of the flow region and each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510.
  • the first tube lens 562 can be configured to generate a plurality of collimated light beams and transmit the plurality of collimated light beams to the objective lens 570.
  • the objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and converge the plurality of collimated light beams into each of the plurality of sequestration pens in the sample plane 574 within the field of view of the image sensor 580 or the optical apparatus 510.
  • the optical apparatus 510 can be configured to illuminate the at least a portion of sequestration pens with a plurality of illumination spots.
  • the objective 570 can receive the plurality of collimated light beams from the first tube lens 562 and project the plurality of illumination spots, which may form an illumination pattern, into each of the plurality of sequestration pens in the sample plane 574 within the field of view.
  • each of the plurality of illumination spots can have a size of about 5 microns X 5 microns; 10 microns X 10 microns; 10 microns X 30 microns, 30 microns X 60 microns, 40 microns X 40 microns, 40 microns X 60 microns, 60 microns X 120 microns, 80 microns X 100 microns, 100 microns X 140 microns and any values there between.
  • the illumination spots may individually have a shape that is circular, square, or rectangular.
  • the illumination spots may be grouped within a plurality of illumination spots (e.g., an illumination pattern) to form a larger polygonal shape such as a rectangle, square, or wedge shape.
  • the illumination pattern may enclose (e.g., surround) an unilluminated space that may be square, rectangular or polygonal.
  • each of the plurality of illumination spots can have an area of about 150 to about 3000, about 4000 to about 10000, or 5000 to about 15000 square microns.
  • An illumination pattern may have an area of about 1000 to about 8000, about 4000 to about 10000, 7000 to about 20000, 8000 to about 22000, 10000 to about 25000 square microns and any values there between.
  • the optical system 510 may be used to determine how to reposition micro-objects and into and out of the sequestration pens of the microfluidic device, as well as to count the number of micro-objects present within the microfluidic circuit of the device. Further details of repositioning and counting micro-objects are found in U. S. Application Publication No. 2016/0160259 (Du); U. S. Patent No. 9,996,920 (Du et al.); and International Application Publication No. WO2017/102748 (Kim, et al.). The optical system 510 may also be employed in assay methods to determine concentrations of reagents/assay products, and further details are found in U. S. Patent Nos.
  • Additional system components for maintenance of viability of cells within a chamber of a microfluidic device In order to promote growth and/or expansion of cell populations, environmental conditions conducive to maintaining healthy, functional cells may be provided by additional components of the system.
  • additional components can provide nutrients, cell growth signaling species, pH modulation, gas exchange, temperature control, and removal of waste products from cells.
  • the disclosure provides methods, systems and devices for determining the level of oxygen and pH in a medium disposed within a microfluidic device.
  • developing a new antibody production line can take many months of work and cost millions of dollars in personnel, equipment and materials.
  • the ability to screen and identify promising clones within a microfluidic device, very early in expanding populations, such as 3, 4, 5, 6, or 7 days after seeding individual founding cells, as described herein, can offer significant time and cost advantages.
  • the nanofluidic environment particularly one based on sequestration pens, as described herein, provides exemplary isolation of clonal populations from each other, while permitting manipulation of the isolated populations in a manner similar to fed-batch bioreactors and the ability to obtain assay results from each individual clonal population without contamination from other clonal populations located within the microfluidic device.
  • Parameters such as dissolved oxygen and pH value of the medium in which the biological cells are can provide insights into oxygen consumption and acidification, which can be correlated to productivity of the biological cells at more typical macroscale scale of expansion (e.g, shake flasks, etc.). It has been discovered that the assays taught by the present disclosure are able to determine the relative or absolute value of dissolved oxygen or pH even at early state of clonal expansion. Further, the ability to screen individual clones at such an early stage can also permit identification of desired clones meeting specific requirements of growth rate and/or more robust production (for example, highly productive clones which are more resistant to levels of a material in the culturing environment such as metabolic waste products or exhausted nutrients).
  • the productivity of the biological cells can be measured in terms of grams of a biomolecule of interest produced per liter of culture, or any comparable metric suitable quantifying productivity.
  • nanofluidic chambers e.g., sequestration pens
  • the nanofluidic environment described here permits examination of the effects of specific conditions upon cells, with feedback from repeated assays.
  • conditions and materials such as culture medium, more closely related to large scale production of a cellular product may be used to find and characterize the most suitable clones for further examination.
  • diverse stimulation protocols for B-cell antibody stimulation may be examined in a more reproducible manner, and may be assayed in order to more comparably assess the benefits of one protocol over another.
  • Biomolecules of interest can comprise any molecule produced by a biological cell that a user of the methods, systems, and kits disclosed herein may wish to utilize for a particular purpose.
  • a biomolecule of interest can include a cellular product generated and used internally or targeted to the cell membrane (in both cases “non- secreted”) or secreted by a biological micro-object, and may be a protein, a saccharide, a nucleic acid, an organic molecule having a molecular weight of less than 3 kDa, a vesicle, a virus, or any combination thereof.
  • a biomolecule of interest may be a naturally expressed biomolecule (e.g., natively expressed) or may be a bioengineered biomolecule (e.g., a product resulting from gene insertion, deletion, modification and the like).
  • a biomolecule of interest that is a nucleic acid may be a ribonucleic or a deoxynucleic acid, may include natural or unnatural nucleotides.
  • a biomolecule of interest that is a virus may be a viral particle, a vector or a phage.
  • a biomolecule of interest that is a saccharide may be a mono-, di- or polysaccharide.
  • Non-limiting examples of saccharides may include glucose, trehalose, mannose, arabinose, fructose, ribose, xanthan or chitosan.
  • a small, organic molecule may include but is not limited to biofuels, oils, polymers, or pharmaceutics such as macrolide antibiotics.
  • a biomolecule of interest that is a protein can be an antibody or fragment of an antibody.
  • a biomolecule of interest that is a protein can be a blood protein, such as an albumin, a globulin (e.g., alpha2-macroglobulin, gamma globulin, beta-2 microglobulin, haptoglobulin), a complement protein (e.g., component 3 or 4), transferrin, prothrombin, alpha 1 antitrypsin, and the like; a hormone, such as insulin, glucagon, somatostatin, growth hormone, growth factors (e.g., FGF, HGF, NGF, EGF, PDGF, TGF, Erythropoietin, IGF, TNF), follicle stimulating hormone, luteinizing hormone, leptin, and the like; a fibrous protein, such as a silk or an extracellular matrix protein (e.g., a fibronectin, laminin, collagen, elastin, vitronectin, tenascin, versican, bone sialoprotein
  • a biomolecule of interest that is a protein can be an antibody, fragment of an antibody, an enzyme (including but not limited to a proteolytic enzyme), an engineered (normally intracellular protein) protein, such as for example, albumin, and/or a structural protein including but not limited to silkworm silk or spider silk). This list is not limiting and any protein that may be engineered may be produced by cells that are evaluated by the methods.
  • the biomolecule of interest may be an antibody-drug conjugate.
  • a non-limiting example of a biomolecule of interest that may have a combination of a protein, a saccharide, a nucleic acid, an organic molecule having a molecular weight of less than 3 kDa, and/or a virus, can include a proteoglycan or glycoprotein.
  • the methods according to the various embodiments herein can allow for subcloning and comparative analysis of subclones, by further expanding and assaying the resultant subclone populations selected according to their oxygen consumption levels and/or the acidification of the medium in which the subclone populations are disposed using the methods and systems described herein. This may be accomplished, for example, by moving one or more selected clonal populations to other sets of chambers (e.g., sequestration pens) within the microfluidic device and expanding each individual cell of the selected population again.
  • sets of chambers e.g., sequestration pens
  • the method may further include a step of exporting the selected biological micro-object or the population of biological micro-objects generated therefrom to the flow region (or channel) and, optionally, out of the microfluidic device.
  • the step of export from either the chamber (e.g., sequestration pens) to the channel or from the chamber and/or channel out of the microfluidic device may be performed on each selected chamber individually (e.g., cells from a set of selected chamber may be exported in a series of export steps, one chamber at a time).
  • biological micro-objects from multiple chambers can be exported simultaneously.
  • the cells which are disposed within a chamber can come from a previously assayed chamber, allowing for subcloning and comparative analysis of subclones.
  • an absolute or relative value of oxygen consumption may be used to select and expand cells.
  • an absolute or relative pH value indicating the acidification of a medium can be used to select and expand cells.
  • both values of the oxygen consumption and pH value of the medium can be used to select and expand cells.
  • all the cells from a chamber associated with a relative or absolute value representing the amount of oxygen consumption and/or a relative or absolute value representing the acidification of a medium can be selected and expanded in the same chamber or other contained area of the chip.
  • one or more of the cells from the same chamber associated with a relative or absolute value representing the amount of oxygen consumption and/or a relative or absolute value representing the acidification of a medium will be selected and expanded in different chambers.
  • generating a relative or absolute value of oxygen consumption and/or a relative or absolute value of acidification of a medium may be repeatedly performed (IX, 2X, 3X, 4X, or more times) on the expanded cells.
  • application of the disclosed methods may permit examination of the effects of specific conditions upon cells, with feedback from repeated assays.
  • conditions and materials related to large scale production of a biomolecule of interest may be used, in order to find and characterize the most suitable clones for further examination.
  • diverse stimulation protocols for B-cell antibody stimulation may be examined in a more reproducible manner, and may be assayed in order to more comparably assess the benefits of one protocol over another. pH measurement and acidification
  • a culture environment is constantly acidified by the wastes produced by a biological micro-object cultured therein, the acidification can be viewed as an indicator of a biological micro-object’s rate of metabolism, which can provide information for evaluating a productivity of the biological micro-object in producing a biomolecule of interest.
  • biological micro-objects of a population can be ranked based on a level of local acidification in media in which each biological micro-objects of the population are cultured so that a better producer can be identified.
  • Monitoring pH condition in a microfluidic device In accordance with various embodiments, methods of monitoring a local pH of a medium spatially distributed within a microfluidic device are provided.
  • Monitoring a local pH refers to monitoring a pH of a medium spatially distributed within a microfluidic device at a selected area of interest (AOI) within the microfluidic device.
  • the microfluidic device can be as described herein having a microfluidic circuit comprising a flow region and a chamber fluidically connected to the flow region.
  • the local pH refers to a pH of the medium within a selected location within the microfluidic device, for instance, within a particular chamber. Therefore, monitoring a local pH provides valuable information that cannot be obtained when using an integrated pH sensor which can only obtain an overall pH readout of the medium within the microfluidic circuit.
  • the microfluidic device does not comprise an integrated pH sensor.
  • FIG. 23 shows an embodiment of the methods of monitoring a local pH of a medium spatially distributed within a microfluidic device.
  • the microfluidic device for conducting the methods of the present disclosure can comprise an enclosure comprising a flow region and a chamber fluidically connected to the flow region.
  • the method can start with preparation of the microfluidic device (Box 2310) and then comprises introducing a pH-sensitive molecule into the microfluidic device (Box 2320), preferably, into the flow region of the microfluidic device; and detecting a signal associated with the pH-sensitive molecule in an area of interest within the enclosure (Box 2330).
  • the methods of the present disclosure can be used to select a micro-object within the microfluidic device.
  • the method can further comprise introducing a micro-object into the microfluidic device and disposing the micro-object into the chamber.
  • the micro-object is a biological micro-object.
  • the micro-object is disposed into the chamber by gravity or DEP force.
  • the microfluidic device can be as described above or herein.
  • the methods can comprise: introducing a pH-sensitive molecule into the flow region of the microfluidic device and allowing the pH- sensitive molecule to diffuse into the chamber (Box 2420); detecting a signal associated with the pH-sensitive molecule in an area of interest within the chamber (Box 2430); comparing the detected signal associated with the pH-sensitive molecule with a reference signal (Box 2440); and selecting the biological micro-object based on the comparison between the detected signal and the reference signal (Box 2450). For example, the micro-object can be selected if the detected signal associated with the pH-sensitive molecule is greater than (or lower than) the reference signal.
  • the area of interest is an area from which a signal associated with a pH-sensitive molecule is detected.
  • the signal associated with a pH sensitive molecule is only generated within the area of interest instead of the entire microfluidic device or field of view.
  • the pH-sensitive molecule is a fluorescent dye
  • only the area of interest is exposed to excitatory electromagnetic waves (e.g., light) and is therefore capable of emitting the fluorescent signal.
  • excitatory electromagnetic waves e.g., light
  • the background noise resulting from, for example, auto-fluorescence from the microfluidic circuit materials can be minimized. This is different from a detection using a standard light microscope in which the entire field of view is exposed excitatory electromagnetic waves, resulting in potential widespread emissions of fluorescent signals.
  • the area of interest comprises at least a portion of the chamber (e.g., a sequestration pen).
  • the AOI is within a connection region of a sequestration pen.
  • the AOI is within an isolation region of the sequestration pen.
  • the AOI is an area covering the entire chamber.
  • the microfluidic circuit materials might have auto-fluorescent characteristics or surface features of the enclosure of the microfluidic device might interfere with accurate detection
  • the AOI does not overlay an area of a wall of the chamber and/or an in situ-generated barrier.
  • the area of interest comprises about 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 12,000, 15,000 square microns or any value therebetween.
  • the area of interest comprises about 50 to 15,000 square microns, 50 to 10,000 square microns, 50 to 9,000 square microns, 50 to 8,000 square microns, 50 to 7,000 square microns, 50 to 6,000 square microns, 50 to 5,000 square microns, 50 to 3,000 square microns, 50 to 1,500 square microns, 50 to 1,000 square microns, 50 to 500 square microns, 50 to 200 square microns, 200 to 15,000 square microns, 200 to 10,000 square microns, 200 to 9,000 square microns, 200 to 8,000 square microns, 200 to 7,000 square microns, 200 to 6,000 square microns, 200 to 5,000 square microns, 200 to 3,000 square microns, 200 to 1,500 square microns, 200 to 1,000 square microns, 200 to 500 square microns, 700 to 15,000 square microns, 700 to 10,000 square microns, 700 to 9,000 square microns, 700 to 8,000 square microns, 700 to 7,000 square microns,
  • a boundary of the AOI is distanced from a wall of the chamber.
  • the distance between the boundary and the wall of the chamber can be about 0.1 um, 0.2 um, 0.3 um, 0.4 um, 0.5 um, 0.6 um, 0.7 um, 0.8 um, 0.9 um, 1.0 um, 1.1 um, 1.2 um, 1.3 um, 1.4 um, 1.5 um, 1.6 um, 1.7 um, 1.8 um, 1.9 um, 2.0 um, or any value therebetween.
  • the distance is about 0.1 to 2.0 um, 0.5 to 2.0 um, 1.0 to 2.0 um, 1.5 to 2.0 um, 0.1 to 1.5 um, 0.5 to 1.5 um, or 1.0 to 1.5 um.
  • Detecting the signal associated with the pH-sensitive molecule from an area of interest may comprise obtaining an image of the area of interest and analyzing the image with respect to the area of interest for the signal associated with the pH-sensitive molecule.
  • the image of the area of interest is not limited to only comprise the area of interest. In other words, the image of the area of interest can comprise other areas in the field of view as long as the area of interest is within the image.
  • Detecting a signal associated with the pH-sensitive molecule from an area of interest is not necessarily limited such that only signal generated within the area of interest is detected.
  • “from an area of interest” refers to setting the area of interest as a focal plane for taking the image and for detecting collective signal from a region adjacent thereto.
  • the region can comprise a volume of media, which, in various embodiments, can be presented as an area (i.e., the area of interest), when the chamber has a uniform cross-sectional height along its length. That said, the larger the area, the larger the volume of media.
  • the collective signal is detected from a region comprising a volume of media of about 5 pL (5xl0 A 3 cubic micron), 10 pL, 15 pL, 20 pL, 25 pL, 30 pL, 35 pL, 40 pL, 45 pL, 50 pL, 60 pL, 70 pL, 80 pL, 90 pL, 100 pL, 150 pL, 200 pL, 250 pL, 300 pL, 350 pL, 400 pL, 450 pL, 500 pL, 600 pL, 700 pL, 800 pL, 900 pL, 1000 pL (1 nL), or more, or any value therebetween.
  • the collective signal is detected from a region comprising a volume of media of about 5 pL to about 1000 pL, about 10 pL to about 950 pL, about 15 pL to about 900 pL, about 20 pL to about 850 pL, about 25 pL to about 800 pL, about 30 pL to about 750 pL, about 35 pL to about 700 pL, about 40 pL to about 650 pL, about 45 pL to about 600 pL, about 50 pL to about 550 pL, about 60 pL to about 500 pL, about 70 pL to about 450 pL, about 80 pL to about 400 pL, about 90 pL to about 350 pL, about 100 pL to about 300 pL, about 150 pL to about 750 pL, about 200 pL to about 600 pL, about 250 pL to about 500 pL, about 300 pL to about 1000
  • a microfluidic device used in the present disclosure can have a three-layer structure comprising a cover, a substrate (e.g., a support structure), and a microfluidic circuit structure in between.
  • a chamber can have a height defined in a z-axial direction from the bottom of the cover to the upper surface of the substrate.
  • the area of interest which is a plane in-focus for taking images and for signal detection, can be set at a mid-level with respect to the height thereby having a larger volume of media adjacent to the area of interest and maximizing the collective signal detected from the area of interest.
  • the area of interest can be set at a depth which substantially bisects the chamber into an upper portion and a bottom portion with respect to the height thereof.
  • the area of interest can be set at a depth of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or any value therebetween of the height, wherein 50% is the mid-level of the height.
  • the area of interest can be set at a depth of about 20% to 80%, 25% to 75%, 30% to 70%, 40% to 60%, or 45% to 55% of the height of the chamber, or any range defined by two of the foregoing endpoints.
  • the area of interest can be set at a depth of about 6 um to about 10 um, about 7 um to about 12 um, about 8 um to about 14 um, about 9 um to about 15 um, about 10 um to about 17 um, about 11 um to about 19 um, about 12 um to about 20 um, about 13 um to about 22 um, about 14 um to about 21 um, about 15 um to about 25 um, about 16 um to about 27 um, about 17 to about 28 um, about 18 um to about 30 um, or any range defined by two of the foregoing endpoints.
  • FIG. 33A is a schematic representation of a cross-section view of a chamber showing a height of the chamber and a depth of the area of interest.
  • the chamber has a height 3330 defined from a bottom 3311 of a cover 3310 and a surface 3321 of a substrate 3320.
  • the area of interest 3340 has a depth 3341 of about 50% of the height 3330.
  • the area of interest can be set at a depth so that the area of interest does not comprise the location of the micro-object. In other embodiments, the area of interest can be set at a depth so that the area of interest keeps a distance from the location of the micro-object.
  • the location of the micro-object is with respect to the z-axis direction in this consideration and can be defined as an imaging depth used for cell detection in a brightfield image.
  • the area of interest can be set at a depth that is 1 um, 2 um, 3 um, 4 um, 5 um, 6 um, 7 um, 8 um, 9 um, 10 um, 12 um, 14 um, 16 um, 18 um, 20 um, 22 um, 24 um, 26 um, or 28 um, or any value therebetween, or 1 um to 28 um, 2 to 26 um, 5 um to 20 um, 5 um to 16 um, or 10 um to 20 um offset from an imaging depth for cell detection in a brightfield image.
  • FIG. 33B is a schematic representation of a cross-section view of a chamber showing a height of the chamber and a depth of the area of interest.
  • the chamber has a height 3330 defined from a bottom 3311 of a cover 3310 and a surface 3321 of a substrate 3320.
  • the chamber has a micro-object 3350 disposed therewithin.
  • the area of interest 3340 has a depth 3341 offset from an imaging depth for cell detection in a brightfield image.
  • the microfluidic device can have a plurality of chambers.
  • the microfluidic device can have a first chamber and a second chamber, each fluidically connected to the flow region via a proximal opening.
  • introducing a pH- sensitive molecule into the microfluidic device comprises allowing the pH- sensitive molecule to diffuse into both the first chamber and the second chamber.
  • detecting the signal associated with the pH-sensitive molecule in the area of interest comprises detecting a first signal associated with the pH-sensitive molecule within the first chamber and a second signal associated with the pH-sensitive molecule within the second chamber.
  • the first signal and the second signal are detected respectively from an area of interest within the first chamber and the second chamber.
  • the area of interest within the first chamber and the area of interest within the second chamber can be located at a corresponding location within the first chamber and the second chamber respectively.
  • the area of interest for detecting the first signal can be located within the isolation region of the first chamber, and correspondingly, the area of interest for detecting the second signal can be located within the isolation region of the second chamber.
  • the AOI described in the DO measurement herein is also applicable for the pH measurement discussed herein.
  • the pH measurement of the present disclosure can be performed in three modes depending on the needs or purposes of the assays.
  • Mode#l the pH measurement is mainly performed to monitor the pH value of the media spatially distributed within the chamber and the microfluidic device.
  • the perfusion of the medium comprising the pH-sensitive molecule is continued throughout the measurement, and a in situ-generated barrier (as will be described in further detail in following paragraphs) is formed within the chamber to define a culture area and an assay area.
  • the area of interest can be within and smaller than the assay area.
  • the area of interest comprises 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or any value therebetween of the assay area (e.g., a horizonal cross-sectional area of the assay area).
  • the area of interest comprises 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 30% to 70%, 40% to 70%, 50% to 70%, or 60% to 70% of the assay area (e.g., a horizonal cross sectional area of the assay area).
  • the assay area e.g., a horizonal cross sectional area of the assay area.
  • the detection is performed from the top of the device (i.e., the image taken for signal detection is a top-view image).
  • the pH measurement is mainly performed to observe the acidification of a media spatially distributed within the chamber while maintaining a micro-object therewithin.
  • a water immiscible fluidic medium can be introduced to seal the chamber.
  • the area of interest can be the entire chamber (e.g., the horizontal cross-sectional area of the chamber).
  • the micro-object does not affect the pH measurement, so the area of interest can comprise one or more micro-objects.
  • the area of interest does not overlay a wall of the chamber.
  • the area of interest has an area comprising 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any value therebetween of the chamber (e.g., the horizontal cross-sectional area of the chamber).
  • the area of interest comprises about 5% to 100%, 5% to 90%, 5%, to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 5% to 90%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 5% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, or 70% to 80%, of the chamber (e.g., the horizontal cross-sectional area of the chamber).
  • the chamber e.g., the horizontal cross-sectional area of the chamber.
  • Mode#3 can be an integration assay of pH measurement and dissolved oxygen measurement of the present disclosure.
  • a water immiscible fluidic medium can be introduced to seal the chamber.
  • An area within the chamber can be chosen as an area of interest for both pH measurement and dissolved oxygen measurement.
  • an area free of micro-object(s) e.g., cells is chosen as the micro-object(s) can affect the detection of the oxygen- sensitive molecule.
  • the area of interest has an area comprising 5%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any value therebetween of a micro-object free area within the chamber (e.g., a horizonal cross sectional area of the micro-object free area).
  • the area of interest comprises about 5% to 100%, 5% to 90%, 5%, to 80%, 5% to 70%, 5% to 60%, 5% to 50%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 5% to 90%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 5% to 80%, 10% to 80%, 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, or 70% to 80%, of the micro-object free area within the chamber (e.g., a horizonal cross sectional area of the micro-object free area).
  • the micro-object free area within the chamber e.g., a horizonal cross sectional area of the micro-object free area.
  • an area of interest for the dissolved oxygen measurement can be an area within the chamber that is free of micro-object.
  • An area of interest for the pH measurement can be the entire chamber area as described above.
  • an in situ-generated barrier can be introduced to define a culture assay and an assay area.
  • the assay area in this mode can be an area proximal to the in situ-generated barrier, located between the in situ-generated barrier and a proximal opening of the chamber, and the culture area can be an area proximal to the in situ-generated barrier, located between the in situ-generated barrier and a distal end of the chamber.
  • the in situ- generated barrier restricts passage of the micro-object thereby retaining the micro-object within the culture area.
  • the area of interest can be within the assay area.
  • the area of interest has an area comprising 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any value therebetween of the assay area (e.g., a horizonal cross sectional area of the assay area).
  • the area of interest comprises 5% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 30% to 90%, 40% to 90%, 50% to 90%, 60% to 90%, 70% to 90%, 80% to 90%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 30% to 70%, 40% to 70%, 50% to 70%, or 60% to 70% of the assay area (e.g., a horizonal cross sectional area of the assay area).
  • the assay area e.g., a horizonal cross sectional area of the assay area.
  • the method further comprises an in situ-generated barrier formed within the chamber.
  • an “in situ-generated barrier” refers to a structure that is formed in a selected area while the microfluidic device is in operation. The structure is not formed while manufacturing the microfluidic device or does not exist before the microfluidic device is used for experiments or research.
  • the term “barrier” refers to a physical structure that is formed and fixed, at least for a certain period of time, in a selected area and is capable of restricting, impeding or blocking passage of a particle (e.g., a micro-object or a molecule) through the structure.
  • the in situ- generated structure will be described in detail hereafter and can be those described in U.S. Patent Application Publication No. 20170165667, filed on November 22, 2016, which is incorporated by reference herein in their entirety.
  • the barrier formed in situ within the chamber can separate the inner space thereof into two areas on each side of the barrier.
  • the in situ-generated barrier can define an assay area and a culture area within the chamber.
  • the area of interest is within the assay area while the culture area is designed to maintain a micro-object.
  • the micro-object is a cell, which can grow and proliferate within the culture area but will not enter the assay area. Therefore, detection of a signal associated with an assay reagent (the pH- sensitive molecule) can be performed in the assay area and will not be interfered with by the cells.
  • the method of the present invention is not limited to detecting the signal within the assay area (i.e., it is not limited that the area of interest must be within the assay area).
  • the area of interest can also be within a culture area.
  • the area of interest is an area free of the micro-object within the culture area.
  • the assay area is located proximal to the in situ-generated barrier, between the in situ-generated barrier and a distal end of the chamber, and the culture area is located proximal to the other side of the in situ-generated barrier, between the in situ- generated barrier and the proximal opening of the chamber.
  • a microfluidic channel 3101 and a sequestration pen 3102 are illustrated.
  • the sequestration pen 3102 has a proximal opening 3103 to the microfluidic channel 3101.
  • An in situ-generated barrier 3110 was formed within the sequestration pen 3102 to divide it into a culture area 3104 and an assay area 3105.
  • the assay area 3105 is located at a distal side of the in situ-generated barrier 3110, and the culture area 3104 is located to the other side of the in situ-generated barrier 3110, closer to the proximal opening 3103 of the sequestration pen 3102.
  • the in situ-generated barrier 3110 can separate the culture area 3104 and a microobject maintained therein from the distal end of the sequestration pen 3102 (See FIG. 31C).
  • the assay area is located to one side of the in situ- generated barrier, closer to the proximal opening of the chamber, and the culture area is located to the other side of the in situ-generated barrier, closer to the distal end of the chamber (for example, for DO measurement as described herein).
  • This configuration can be beneficial for the dissolved oxygen measurement as described herein, because, in various embodiments, the microfluidic circuit material of the microfluidic device can be highly gas permeable, so that the assay area and the area of interest are better off away from the distal end of the chamber to maximize signal changes of the oxygen- sensitive molecule used for the assay.
  • the in situ-generated barrier can be formed in a central portion of the chamber (i.e., mid-chamber or mid-pen).
  • the central portion of the chamber can be, with respect to a long axis of the chamber, for example, with respect to an axis of the chamber that is substantially perpendicular to a direction of intended flow of medium within the flow region (e.g., a barrier that bisects the chamber into a proximal region and a distal region relative to the opening to the flow region).
  • the in situ-generated barrier can be formed in the central portion of the chamber and divide the chamber into a first area that is close to a distal end of the chamber and a second area that is close to a proximal end of the chamber; wherein the first area is the assay area, and the second area is the culturing area; and wherein the distal end and the proximal end is defined with respect to the proximal opening of the chamber to the flow region.
  • the in situ-generated barrier defines within the chamber an enclosed assay area.
  • enclosed assay area refers to an area pre-determined for performing an assay or for detecting a signal associated with an assay, but the methods of the present disclosure are not limited to being performed (or signal detected) within the assay area.
  • the term “enclosed” used herein describes a region having one or more walls and/or barriers (e.g., in situ-generated barrier) that limit the movement of a particle or substance of interest (e.g., a micro-object or a molecule) into or out of the enclosed region (e.g., a particle/substance outside of the region cannot easily enter the region, and a particle/substance inside the region cannot easily leave the region).
  • a particle or substance of interest e.g., a micro-object or a molecule
  • the term “enclosed” is not limited to require that the area is completely closed or sealed. While a substance might be blocked from entering or leaving the area being enclosed, other substances might still be able to move in and out. For example, nutrients, waste, or an assay reagent can still enter or leave the area.
  • the term “enclosed” can also comprise the situation in which a micro-object is not completely blocked but restricted from entering or leaving the area.
  • the in situ-generated barrier does not completely seal the assay area, for example, there can be a gap between the edge of an in situ-generated barrier and a wall of the chamber where the in situ-generated barrier is located.
  • the gap can have a width that is smaller than a dimension of a micro-object so that the micro-object still cannot enter the assay area without passing through the in situ-generated barrier, or the gap can have a width that is about the same size as a dimension of a micro-object so that even if the micro-object is not completely blocked from passing through, its movement is restricted.
  • the in situ-generated barrier can comprise a gap having a width that is smaller than or about the same size as a dimension of a micro-object so that the micro-object is either blocked or restricted from passing through the barrier.
  • the in situ-generated barrier has a porosity restricting passage of the micro-object.
  • the in situ-generated barrier has a porosity blocking passage of the micro-object.
  • the in situ-generated barrier can be porous to a fluidic medium within the microfluidic device (e.g., within the enclosure, or within the chamber).
  • the in situ-generated barrier can be porous to an assay reagent within a fluidic medium being introduced into the microfluidic device.
  • the in situ-generated barrier can be porous to the pH-sensitive molecule used in the methods of the present disclosure.
  • the in situ-generated barrier can be formed by introducing a polymer solution into the flow region of the microfluidic device; allowing the polymer solution to diffuse into the chamber; and solidifying the polymer solution thereby forming the in situ- generated barrier within the chamber.
  • pH-sensitive molecule used herein is referred to a molecule that is able to provide, directly or indirectly, a detectable signal or change in signal reflecting a pH condition of an environment.
  • the pH-sensitive molecule can be a pH indicator that significantly increase in fluorescence as the pH of its environment becomes more acidic. In other words, in such examples, the stronger the signal, the more acid the environment is. Given that acidification of the media can be resulted from metabolism of a biological micro-object cultured within that environment, a more acidic media can indicate a more robust metabolism. In certain embodiments that the biological micro-object is cultured to produce a molecule of interest, a more acid media can suggest a higher productivity.
  • the pH-sensitive molecule is soluble and/or diffusible in a fluidic medium perfused to introduce the pH-sensitive molecule into the microfluidic device or any fluidic medium within the enclosure of the microfluidic device.
  • the pH- sensitive molecule is a fluorescent dye, and a signal associated with the pH-sensitive molecule is referred to a signal (e.g., a fluorescent signal) emitted by the pH-sensitive molecule.
  • the pH-sensitive molecule is an acidotropic dye.
  • the pH- sensitive molecule is soluble and diffusible in a medium spatially distributed within an environment to be monitored.
  • the pH-sensitive molecule is responsive to a pH range from 1 to 9, 2 to 8, 3 to 7, or 3 to 6.
  • introducing the pH-sensitive molecule into the microfluidic device comprises: introducing the pH-sensitive molecule into the flow region and allowing the pH-sensitive molecule to diffuse into the chamber.
  • the pH-sensitive molecule is introduced within a fluidic medium.
  • introducing the pH- sensitive molecule into the microfluidic device comprises perfusing the fluidic medium comprising the pH-sensitive molecule through the flow region.
  • the perfusion of the fluidic medium comprising the pH-sensitive molecule can be stopped while detecting the signal associated with the pH-sensitive molecule.
  • the fluidic medium is a culture medium for maintaining a biological micro-object within the microfluidic device.
  • the fluidic medium can also be a medium that is configured to induce a biological micro-object maintained in the chamber to produce a biomolecule of interest.
  • the fluidic medium has a buffer at a concentration of about 0 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2.0 M, 5M, or any value therebetween.
  • the buffer is not limited and can include a citrate buffer or a phosphate buffer.
  • the fluidic medium can be non-buffered.
  • the pH-sensitive molecule is allowed to reach an equilibrated concentration across the flow region and the chamber before detecting the signal associated with the pH-sensitive molecule. Reaching an equilibrium concentration across the flow region and the chamber is referred to as a situation where the difference in concentration between the flow region and the chamber(s) is insignificant (e.g., the concentration in the flow region and the concentration is substantially the same).
  • the method does not require that the concentration of the pH-sensitive molecule reaches equilibrium throughout the flow region and chamber(s), but may be performed at any time point when the reagent has reached a concentration that is sufficient to permit detection.
  • the fluidic medium for introducing the pH- sensitive molecule is flowed at a flow rate of at least about 0.1 microliters per second (uL/s), 0.2 uL/s, 0.3 uL/s , 0.4 uL/s, 0.5 uL/s, 0.6 uL/s, 0.7 uL/s, 0.8 uL/s, 0.9 uL/s, 1 uL/s, 2 uL/s, 3 uL/s, 4 uL/s, 5 uL/s, 6 uL/s, 7 uL/s, 8 uL/s, 9 uL/s, 10 uL/s, or more.
  • the fluidic medium is flowed at a flow rate of at most about 10 uL/s, 9 uL/s, 8 uL/s, 7 uL/s, 6 uL/s, 5 uL/s, 4 uL/s, 3 uL/s, 2 uL/s, 1 uL/s, 0.9 uL/s, 0.8 uL/s, 0.7 uL/s, 0.6 uL/s, 0.5 uL/s, 0.4 uL/s, 0.3 uL/s, 0.2 uL/s, 0.1 uL/s, or less.
  • the fluidic medium is flowed at a flow rate ranging between any two of the preceding values. In accordance with various embodiments, the fluidic medium is flowed at a flow rate ranging between 0.1 microliter/s and 10 microliter s/s.
  • the pH-sensitive molecule includes but not limited to LysoSensorTM, LysoTrackerTM probe, or pHrodoTM. Any suitable pH-sensitive molecule, as is known in the art, may be used.
  • detecting the signal associated with the pH-sensitive molecule may comprise: obtaining an image of the area of interest and analyzing the image with respect to the area of interest for the signal associated with the pH-sensitive molecule.
  • analyzing the image may comprise determining the intensity of the signal. The intensity can be used to represent a pH of a medium within the first area of interest.
  • the detected signal associated with the pH-sensitive molecule may be normalized with a reference signal to determine a relative pH in.
  • the reference signal can be a signal detected right before the pH-sensitive molecule is introduced or after the pH-sensitive molecule is introduced and before any substantial change of pH can take place.
  • a relative pH value can be determined to evaluate a pH change.
  • the reference signal can be a pre-determined threshold value representing an upper limit or a bottom limit of a tolerable pH range for a biological microobject.
  • a relative pH value compared with the threshold value can be determined to evaluate whether the environment within the first area of interest is suitable for culturing the biological micro-object.
  • the microfluidic device may comprise a first chamber and a second chamber.
  • a first signal associated with the pH-sensitive molecule in a first area of interest within the first chamber and a second signal associated with the pH-sensitive molecule in a second area of interest within the second chamber are detected respectively.
  • a geographic pH stability within the microfluidic device can be evaluated.
  • the method optionally includes taking a plurality of fluorescence images at a plurality of times/timestamps correlating a respective fluorescence of each fluorescence image to determine a respective observed pH in the AOI at the respective timestamp.
  • a first signal associated with the pH-sensitive molecule in the AOI can be detected at a first time point
  • a second signal associated with the pH-sensitive molecule in the same AOI can be detected at a second time point, subsequent to the first time point.
  • a relative pH value can be determined to evaluate the pH stability (e.g., a temporal pH change) of the medium within the AOI.
  • analyzing the image with respect to the first area of interest for the signal associated with the pH-sensitive molecule can comprise quantifying an amount of the signal associated with the pH-sensitive molecule.
  • the amount of the signal associated with the pH-sensitive molecule can be quantified by a reference signal, which can be detected before the pH-sensitive molecule is introduced or right after the pH-sensitive molecule is introduced and before any substantial change of pH can take place.
  • the quantified signal can then be compared with a standard curve to determine an observed pH value.
  • the standard curve can be obtained by introducing solutions of various predetermined pH comprising the same kind of pH-sensitive molecule into respective microfluidic devices and detecting signals respectively.
  • the image obtained for the first area of interest for the signal associated with the pH-sensitive molecule is further normalized by a fixed-point signal, which is generated via fluorescent exposure of the pH-sensitive solution at an alternative wavelength where the corresponding fluorescent intensity measured is insensitive to pH change.
  • the fixed- point signal instead varies with a concentration of the pH-sensitive molecule existing in the medium/solution, and the wavelength of the fixed-point signal is dependent on the nature of the pH-sensitive molecule.
  • LysoSensorTM has a fixed-point signal emission at 490 nm.
  • the signal associated with pH-sensitive molecule is normalized per pixel, per area of interest, or per sequestration pen.
  • flowing the fluidic medium and taking the fluorescence image are performed at a selected temperature.
  • the temperature is from about 20°C to about 40°C.
  • the temperature is from about 28°C to about 30°C.
  • the cells may alternatively be cultured at other temperatures, such as at least about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or higher.
  • the cells may be cultured at temperature of at most about 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, or less.
  • the cells may be cultured at a temperature that ranges between any two of the preceding values.
  • the in situ-generated barrier is a hydrogel.
  • the in situ-generated barrier comprises a solidified polymer network.
  • the solidified polymer network comprises a synthetic polymer, a modified synthetic polymer, or a biological polymer.
  • the solidified polymer network comprises at least one of a polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N-isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, poly aery lie acid (PAA), modified poly aery lie acid, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination.
  • the solidified polymer network does not include a silicone polymer.
  • Physical and chemical characteristics determining suitability of a polymer for use in the solidified polymer network may include molecular weight, hydrophobicity, solubility, rate of diffusion, viscosity (e.g., of the medium), excitation and/or emission range (e.g., of fluorescent reagents immobilized therein), known background fluorescence, characteristics influencing polymerization, and pore size of a solidified polymer network.
  • the solidified polymer network is formed upon polymerization or thermal gelling of a flowable polymer solution containing at least one of a polyethylene glycol, modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N- isopropylacrylamide, polyvinyl alcohol (PVA), modified polyvinyl alcohol, poly aery lie acid (PAA), modified poly aery lie acid, fibronectin, modified fibronectin, collagen, modified collagen, laminin, modified laminin, polysaccharide, modified polysaccharide, or a co-polymer in any combination.
  • a polyethylene glycol modified polyethylene glycol, polyglycolic acid (PGA), modified polyglycolic acid, polyacrylamide (PAM), modified polyacrylamide, poly-N-isopropylacrylamide (PNIPAm), modified poly-N- isopropyl
  • co-polymer classes may be used, including but not limited to any of the above listed polymers, or biological polymers such as fibronectin, collagen or laminin. Polysaccharides such as dextran or modified collagens may be used.
  • the flowable polymer may be referred alternatively here as a pre-polymer, in the sense that the flowable polymer is crosslinked in-situ. Biological polymers having photoactivatable functionalities for polymerization may also be used.
  • a polymer may include a cleavage motif.
  • a cleavage motif may include a peptide sequence inserted into the polymer that is a substrate for one or more proteases, including but not limited to a matrix metalloproteinase, a collagenase, or a serine proteinase such as Proteinase K.
  • Another category of cleavage motif may include a photocleavable motif such as a nitrobenzyl photocleavable linker which may be inserted into selected locations of the prepolymer.
  • a nitrobenzyl photocleavable linker may include a l- methinyl, 2-nitrobenzyl moiety configured to be photocleavable.
  • the photocleavable linker may include a benzoin moiety, a 1, 3 nitrophenolyl moiety, a coumarin-4-ylmethyl moiety or a 1-hydroxy 2- cinnamoyl moiety.
  • a cleavage motif may be utilized to remove the solidified polymer network of an isolation structure.
  • the polymer may include cell recognition motifs including but not limited to a RGD peptide motif, which is recognized by integrins.
  • PEGDA polyethylene glycol diacrylate
  • c diacrylamide, multi-armed acrylamide or substituted versions as described herein.
  • Photoactivated polymerization may be accomplished using a free radical initiator Igracure® 2959 (BASF), a highly efficient, non-yellowing radical, alpha hydroxy ketone photoinitiator, is typically used for initiation at wavelengths in the UV region (e.g., 365nm), but other initiators may be used.
  • An example of another useful photoinitiator class for polymerization reactions is the group of lithium acyl phosphinate salts, of which lithium phenyl 2, 4, 6, - trimethylbenzolylphosphinate (LAP) has particular utility due to its more efficient absorption at longer wavelengths (e.g., 405nm) than that of the alpha hydroxy ketone class.
  • LAP lithium phenyl 2, 4, 6, - trimethylbenzolylphosphinate
  • Another initiator that may be used are water soluble azo initiators, such as 2, 2-Azobis [2- methyl-N-(2-hydroxyethyl)propionamide].
  • the initiator may be present within the flowable polymer solution at a concentration of about 5 millimolar, about 8 millimolar, about 10 millimolar, about 12 millimolar, about 15 millimolar, about 18 millimolar, about 20 millimolar, about 22 millimolar, about 25 millimolar, about 28 millimolar, about 30 millimolar, about 35 millimolar, or about 40 millimolar.
  • Crosslinking may be performed by photopatterning of linear or branched PEG polymers, free radical polymerization of PEG acrylates or PEG acrylamides, and specifically tailored chemical reactions such as Michael addition, condensation, Click chemistry, native chemical ligation and/or enzymatic reactions.
  • photopatteming of crosslinking may be used to gain precise control of extent of the physical extent of the hydrogel barrier as well as the degree of crosslinking, as described in the following section and in the Examples.
  • Inhibitors may be included within the flowable polymer solution to ensure precise control of photopatteming and to prevent extraneous or undesired polymerization.
  • One useful inhibitor is hydroquinone monomethyl ether, MEHQ, but other suitable inhibitors may be used.
  • the inhibitor may be present in the flowable polymer solution at a concentration of about 1 millimolar, about 2 millimolar, about 5 millimolar, about 10 millimolar, about 15 millimolar, about 20 millimolar, about 25 millimolar, about 30 millimolar, about 35 millimolar, about 40 millimolar or more, as needed to provide the photopatteming control desired.
  • the hydrogel may be a polyethylene glycol polymer or a modified polyethylene glycol polymer.
  • a wide range of molecular weights of the flowable polymer may be suitable, depending upon the structure of the polymer.
  • the flowable polymer may have a molecular weight of about 500 Da to about 20kDA, or about 500Da, about IkDa, about 3kDa, about 5kDa, about 10 kDa, about 12kDa, about 15kDa, about 20 kDa or any value therebetween.
  • a useful star type polymer may have Mw (weight average molecular weight) in a range from about 500Da to about 20kDa (e.g., four arm polymer), or up to about 5kDa for each arm, or any value therebetween.
  • a polymer having a higher molecular weight range may be used at lower concentrations in the flowable polymer, and still provide an in situ-generated barrier or isolation structure that may be used in the methods described herein.
  • Reversing/removing/minimizing the in situ-generated isolation structure A number of mechanisms may be used to remove or reduce the in situ-generated hydrogel barrier when there is no further purpose for it. For example, once an assay is completed and desirable biological cells have been identified, it may be useful to remove the hydrogel barrier in order to export the cells and/or continue culturing and expanding the biological cell demonstrating desirable activities or properties.
  • laser initiated bubbles may provide forces that can deform or disrupt the hydrogel barrier, permitting export of the cells.
  • Porogens including polymers which are incapable of being chemically linked to the photoinitiated polymer(s), may be included when forming the hydrogel barrier.
  • the degree/size of openings within the formed hydrogel can customize the hydrolysis rate via accessibility within the hydrogel barrier).
  • the pores formed may be employed to permit secreted materials or chemical reagents to pass through the hydrogel barrier but prevent a cell from moving into, out of, and/or through the isolation structure.
  • degradability of these polymers may be increased by introducing degradable segments such as polyester, acetal, fumarate, poly(propylene fumarate) or polyhydroxyacids into polymers (e.g., PEG polymers).
  • PEG may be formed with disulfide linkages at intervals along the macromere, which may be random or predetermined.
  • the disulfide bonds may be broken by Dithiothreitol (DTT), mercaptoethanol, or TCEP.
  • DTT Dithiothreitol
  • TCEP TCEP
  • PNIPAm poly N-isopropylacrylamide
  • suitable LCST polymers may be used to introduce hydrogel barriers upon heating. They may be removed by decreasing the temperature of the formed polymer hydrogel barrier.
  • the polymers may include ELPs or other motifs that also permit removal by other mechanisms such as hydrolysis or proteolysis.
  • PNIPAm may be used to create a surface for adherent cells, but then switched to permit export.
  • Hydrogels may have any sort of peptide sequence engineered in, such that selective proteolysis upon a selected motif by a selected protease can remove/reverse/or minimize a hydrogel isolation structure.
  • Some classes of modified PEG include PEG having elastin like peptide (ELP) motifs and/or having peptide motifs for susceptibility to a variety of proteases (enzyme sensitive peptide ESP). A large number of these motifs are known.
  • ELP elastin like peptide
  • ESP enzyme sensitive peptide ESP
  • Osmotic susceptibility Calcium concentration/other osmotic strategies can be employed to degrade and remove a hydrogel barrier. As above, changes of media flowed through the channel or flow region may dimensionally swell or de-swell hydrogel barriers.
  • Photocleavage As described above, if a polymer of the solidified polymer network includes a pho tocleav able moiety, directing illumination of an exciting wavelength to the solidified polymer network will cause cleavage within sections of the solidified polymer network. This cleavage may provide complete or partial disruption of the solidified polymer network, thereby removing or reducing the hydrogel barrier.
  • the hydrogel barrier may not be removed but may simply be swelled or de-swelled using light or media ⁇ solvent changes.
  • Some types of hydrogels may incorporate moieties that respond reversibly to light (for example, change regiochemistry about a rigid bond; form reversible crosslinks within the polymer, or form/break ion pairs).
  • Detecting acidification for selection Local medium acidification can be detected to provide information for selecting a biological micro-object among a cultured population.
  • the fluidic medium with the enclosure of the microfluidic device does not comprise a pH buffer (nonbuffered) so that pH change can be observed effectively.
  • the flow of the first fluidic medium comprising the pH-sensitive molecule is stopped before detecting the signal associated with the pH-sensitive molecule so that the supply of the pH-sensitive molecule into the microfluidic circuit is paused, which is favorable for detecting the local pH within the microfluidic device.
  • enclose can have the same definition as described herein. Enclosing the chamber from the flow region can prevent cross-talking between adjacent chambers.
  • a chamber of a microfluidic device described herein can be designed to have an unswept region.
  • the unswept region can be an optimized space for maintaining micro-objects and for performing assays because there is substantially no flow of media and only diffusion is allowed as the only fluidic communication between it and a flow region. Nevertheless, smaller molecules can move in or out of the unswept region relatively quickly because they diffuse at a faster rate. There is a possibility that a small molecule can move from one chamber into another chamber of the microfluidic device during assaying. Therefore, enclosing the chamber from the flow region can be helpful in retaining a small molecule within the chamber.
  • enclosing the chamber from the flow region can prevent the cell from migrating from the chamber into the flow region.
  • the cell can grow and proliferate within the chamber into a colony. When the cell number of the colony continuously grows, the colony might grow into an area close to the proximal opening of the chamber to the flow region. A possibility for the cells to assess the flow region might increase, and consequently, cells might enter the flow region and be brought away by a flow of media. In those situations, enclosing the chamber from the flow region can be helpful in retaining the cells within the chamber.
  • a chamber can be enclosed from the flow region by introducing a in situ-generated barrier, a water immiscible fluidic medium, or a gaseous fluid.
  • An in situ-generated barrier can be formed in a selected area within the microfluidic device or within the chamber of the microfluidic device as described herein.
  • a water immiscible fluidic medium is introduced into the microfluidic circuit to fill a portion of the flow region adjacent to the chamber with the water immiscible fluidic medium, e.g. adjacent to the proximal opening of the chamber to the flow region.
  • a gaseous fluid can be introduced into the microfluidic circuit to fill a portion of the flow region adjacent to the chamber with the gaseous fluid.
  • the water immiscible fluidic medium and the gaseous fluid are introduced to seal, e.g., disrupt, substantially the fluidic connection between the flow region and the chamber, which can assist localizing the signal associated with the pH-sensitive molecule within the chamber.
  • the water immiscible fluidic medium and/or the gaseous fluid may occupy substantially the entire microfluidic channel.
  • the water immiscible fluidic medium may include but is not limited to a mineral oil, HFE (hydrofluoroether) oil, or a silicon oil.
  • the water immiscible fluidic medium can be 3MTM NovecTM 7500 Engineered Fluid.
  • a reference signal can be a threshold value representing a level of acidification.
  • the threshold value can be obtained from a verified biological micro-object having a desired productivity; therefore, a biological micro-object can be selected if the detected signal is equal to or greater than the reference signal, indicating the biological microobject exhibits a comparable productivity as that of the verified biological micro-object.
  • a biological micro-object is selected if the detected signal is equal to or lower than a reference signal.
  • the microfluidic device can comprise a first chamber and a second chamber, and the method can comprises introducing a first micro-object and a second micro-object and disposing the first micro-object and the second micro-object respectively into the first chamber and the second chamber.
  • detecting the signal associated with the pH-sensitive molecule in the area of interest comprises detecting a first signal associated with the pH-sensitive molecule within the first chamber and detecting a second signal associated with the pH-sensitive molecule within the second chamber.
  • the area of interest within the first chamber and the area of interest within the second chamber can be located at a same corresponding location within the first chamber and the second chamber respectively.
  • the area of interest for detecting the first signal can be located within the isolation region of the first chamber, and correspondingly, the area of interest for detecting the second signal can be located within the isolation region of the second chamber.
  • the method further comprises selecting the first micro-object or the second micro-object or both by comparing the first signal with the second signal or by comparing the first signal and/or the second signal with a pre-determined threshold value.
  • the first signal and the second signal can be compared with each other or respectively with a pre-determined threshold value.
  • the method can further comprise selecting a desired micro-object from the first micro-object, the second microobject, or both. For example, if the first signal has a higher intensity indicating lower pH value, the first biological micro-object might exhibit a better productivity as it causes higher acidification to the medium within the chamber.
  • the desired microobject can be exported out from the microfluidic device and collected for further evaluation or uses.
  • a first signal associated with the pH-sensitive molecule in an area of interest within a chamber comprising a biological micro-object can be detected at a first time point, and a second signal associated with the pH-sensitive molecule in the same area of interest can be detected at a second time point. While the second time point is after the first time point, by recording the first signal and the second signal, a change of the productivity can be observed.
  • a plurality of signals associated with the pH-sensitive molecule can be detected and recorded over time so that a trace of pH change, which infers a productivity change of the biological micro-object, can be obtained.
  • the method further comprises determining a level of oxygen in the microfluidic device for selecting a biological micro-object.
  • the determination of the level of oxygen can be performed by the methods described herein.
  • determining the level of oxygen comprises introducing an oxygen- sensitive molecule into the microfluidic circuit of the microfluidic device, and detecting a signal associated with the oxygen-sensitive molecule in an area of interest.
  • the oxygen- sensitive molecule is introduced in a fluidic medium different from the fluidic medium comprising the pH-sensitive molecule (i.e., the oxygen-sensitive molecule is introduced separately with the pH-sensitive molecule).
  • the oxygen-sensitive molecule is introduced in the same fluidic medium comprising the pH-sensitive molecule.
  • the area of interest is as described herein (i.e., the oxygen- sensitive molecule is introduced together with the pH- sensitive molecule).
  • the signal associated with the pH-sensitive molecule and the signal associated with the oxygen- sensitive molecule are detected sequentially.
  • a fluidic medium comprising the pH-sensitive molecule is introduced, and a signal associated with the pH-sensitive molecule is detected.
  • a fluidic medium comprising the oxygensensitive molecule is introduced, and a signal associated with the oxygen- sensitive molecule is detected.
  • a fluidic medium comprising both the pH-sensitive molecule and the oxygen-sensitive molecule is introduced, and a signal associated with the pH-sensitive molecule and a signal associated with the oxygen-sensitive molecule are detected respectively.
  • the area of interest is the same as the area of interest for detecting a signal associated with the pH-sensitive molecule.
  • a water immiscible fluidic medium can be introduced to seal the chamber from the flow region.
  • a signal associated with the pH- sensitive molecule and a signal associated with the oxygen-sensitive molecule can be respectively detected from the same area within the chamber.
  • the area is free of micro-object (e.g., free of cells).
  • the area of interest is different from the area of interest for detecting a signal associated with the pH-sensitive molecule.
  • a signal associated with the pH- sensitive molecule can be detected from an area within the assay area, while a signal associated with the oxygen-sensitive molecule can be detected from an area within the culture area, which is free of micro-object (e.g., free of cells).
  • a micro-object e.g., a biological micro-object used as a producer takes in nutrition such as carbon sources and amino acids, consumes oxygen from culture medium, and utilizes the resources for maintaining viability, proliferation, and producing various biomolecules, including one or more molecules of interest.
  • biomass is increasing, molecules of interest are being produced, and metabolic waste is released causing acidification of the culture medium. Therefore, instead of detecting the molecules of interest directly, local acidification of the culture medium can be informative for ranking a micro-object in terms of its productivity. Conversly, a faster grower, which proliferates robustly, may be channeling excessive amounts of energy into cell growth rather than production of a molecule of interest, resulting in a producer with lower overall production. Therefore, biomass (as well as oxygen consumption) can also be included to provide a more accurate ranking.
  • a method of ranking a micro-object population within a microfluidic device comprises introducing the micro-object population into the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a flow region and a plurality of chambers, wherein each of the plurality of the chamber is fluidically connected through a proximal opening to the flow region; disposing individual micro-objects of the micro-object population into respective chambers of the plurality of chambers, resulting in disposed micro-objects within respective chambers; allowing the disposed micro-objects to produce a molecule of interest; introducing a pH-sensitive molecule into the microfluidic device; detecting a first signal associated with the pH-sensitive molecule in a first area of interest within respective chamber; and ranking the disposed micro-objects based on the first signals detected in the respective chambers.
  • single micro-objects of the micro-object population are disposed into the respective chambers whereby each chamber comprises a single micro-object. Therefore, the single micro-object can grow into a clonal population within each respective chamber.
  • the method further comprises culturing the disposed microobjects in the respective chambers. Culturing the disposed micro-objects can comprise allowing each disposed micro-object grows into a colony thereof within the respective chambers. Each colony can comprise a single clonal population.
  • the method further comprises measuring a biomass of the disposed micro-objects in the respective chambers, Optionally, ranking the disposed micro-objects is based on the pH and the biomass measured in the respective chambers. Biomass measurement is described in WO2022216778, filed on April 6, 2022, its disclosure is herein incorporated by reference in its entirety.
  • the method further comprises introducing an oxygen- sensitive molecule into the microfluidic device, and detecting a second signal associated with the oxygen-sensitive molecule within the respective chamber (e.g., in a second area of interest).
  • ranking the plurality of micro-objects is based on the pH and the second signal detected in the respective chambers.
  • ranking the plurality of micro-objects is based on the biomass and the second signal detected in the respective chambers.
  • ranking the plurality of micro-objects is based on the pH, the biomass, and the second signal detected in the respective chambers.
  • the oxygen-sensitive molecule, detecting a signal associated with the oxygen-sensitive molecule, and other features of the methods related to using the oxygensensitive molecule can be as described herein.
  • the detected signals associated with the pH-sensitive molecule, the biomass, and/or the detected signals associated with the oxygen-sensitive molecule can be processed by an algorithm to perform the ranking.
  • the algorithm can be hierarchical agglomerative clustering, e.g., using the UPGMA algorithm in SciPy.
  • the algorithm can include a Principal Component Analysis.
  • the micro-object of the present disclosure can be a biological microobject.
  • the biological micro-object described herein can be any kind of cells that produce or is configured to produce a biomolecule of interest.
  • the biological microobject may be genetically engineered to produce a biomolecule of interest.
  • the production of the biomolecule of interest by the biological micro-object can be indirectly evaluated by conducting pH measurement as disclosed herein because higher production is associated with higher metabolism resulting in higher secretion of metabolic wastes that could acidify the environment where the biological -micro-object is.
  • the biomolecule of interest produced can also acidify the environment where the biological microobject is.
  • the biological micro-object can be an animal cell, a plant cell, or a bacteria cell.
  • the biological micro-object can be a fungus cell.
  • the fungus cell is a yeast cell, such as a Saccharomyces cell (e.g. Saccharomyces cerevisiae) or a Pichia cells (e.g. Pichia pasloris).
  • the micro-object is a non-biological micro-object (for example, a bead) having biological molecules attached on surface thereof.
  • the biological molecules are configured to generate a biomolecule of interest.
  • the biomolecule of interest can acidify the environment where the micro-object is, therefore, the production of the biomolecule of interest can be evaluated indirectly by conducting pH measurement as disclosed herein.
  • the disclosure further provides machine-readable storage devices for storing non-transitory machine-readable instructions for carrying out the foregoing methods.
  • Kits for monitoring a local pH of a medium spatially distributed within a microfluidic device are also provided.
  • the kit comprises a microfluidic device as described herein and a pH-sensitive molecule.
  • Kits for selecting a biological micro-object are also provided.
  • the kid comprises a microfluidic device as described herein, a pH-sensitive molecule, and a fluidic medium for introducing the pH-sensitive molecule into the microfluidic device, wherein the fluidic medium does not comprise a pH buffer.
  • a kit for monitoring a local pH of a medium spatially distributed within a microfluidic device may include a pH- sensitive molecule and instructions for its use.
  • the kit may further include a fluidic medium, which may be any fluidic medium suitable for performing the methods described herein.
  • the fluidic medium may be a mineral oil, HFE (hydrofluoroether) oil, or a silicon oil.
  • a kit for selecting a biological micro-object may be provided, and may include a pH-sensitive molecule and instructions for its use.
  • the kit may further include a fluidic medium, which may be any fluidic medium suitable for performing the methods of selecting a biological micro-object described herein.
  • the fluidic medium may be a mineral oil, HFE (hydrofluoroether) oil, or a silicon oil
  • systems and methods of monitoring dissolved oxygen (DO) within a microfluidic device are disclosed.
  • the disclosed systems for monitoring dissolved oxygen during cell culture and disclosed methods for using analytical and measurement results from the assay are used for optimizing culturing and perfusion parameters for a given cell culture.
  • Monitoring dissolved oxygen is performed by monitoring fluorescence associated with an oxygen sensitive dye, which is environmentally sensitive to oxygen.
  • the oxygen sensitive dye may provide a detectable signal or change in fluorescence upon binding to oxygen.
  • the oxygen sensitive dye need not require a discrete binding reaction by oxygen in order to obtain a detectable signal or change in fluorescence.
  • microfluidic -based cell cultures e.g., a microfluidic device having chambers (e.g., sequestration pens) which open to flow regions or channels comprised by such flow regions.
  • determining the dissolved oxygen within an individual chamber (e.g., sequestration pen) in the microfluidic device, relative to that in the flow region, e.g., channel, to which the chamber is fluidically connected can be an indicator of relative amounts of oxygen consumption. Measurements of such oxygen consumption levels can be used to provide a correlation with the size of the clonal population within the chamber, e.g., more consumption correlates with more cells using oxygen.
  • a correlation between the amount of oxygen being consumed by the living cells can provide a quick metric for detecting the largest number of viable, growing cells.
  • the results from such correlation can help focus a cell culture experiment, for example, by guiding unpenning and export of cells from only the pens having oxygen demand above a user-defined level, e.g., according to the expected phenotype of the cells being cultured.
  • the results obtained using the aforementioned approach can be applied to maintain a certain dissolved oxygen concentration level to enhance cell expansion and viability in a given cell culture, for example, through feedback control of a rate of media perfusion, increasing the rate of media perfusion if the DO drops below a first setpoint DO level or decreasing the rate if the DO rises above a second setpoint DO level, wherein the first setpoint is lower than the second setpoint, and wherein the first and second setpoint levels may be determined according to required environmental conditions in the microfluidic device, for example, for growing cells.
  • a method for determining oxygen consumption level in a population of biological micro-objects is provided.
  • the oxygen consumption level can be detected by diffusing soluble reporter molecules, such as a dye molecule, into the population of biological micro -objects.
  • the dye can include, but is not limited to oxygen sensitive dye “RTDP” (2 mg/L; Aldrich Cat. No. 544981-1G; CAS Registry No. 50525-27-4; Tris(2,2'-bipyridyl)-dichlororuthenium(II) hexahydrate; (Ru(BPY)3)).
  • the dye can be used as the soluble, diffusible reporter molecule.
  • the RTDP ruthenium complex is oxygen sensitive.
  • the RTDP complex is a lumiphore and produces fluorescence when not quenched by local concentrations of oxygen.
  • the dye’s fluorescence is diminished in the presence of dissolved oxygen, via a radiationless deactivation involving molecular interaction between oxygen and the ruthenium complex, e.g., collisional quenching, which is diffusion limited.
  • a sufficient concentration of oxygen is present in the local environment, e.g., in proximity to the dye, the dye’s fluorescence is disrupted or quenched.
  • fluorescence of the dye changes based on availability of oxygen proximate to the dye.
  • the oxygen consumption level may be measured by noting the difference in fluorescence intensity observed between a region (such as a chamber or sequestration pen described herein) in which biological micro-objects are growing (and therefore consuming oxygen) and a region (such as a flow region or channel described herein) in which biological micro-objects are not growing.
  • the dye can include RTDP, a polycyclic aromatic hydrocarbon, a fluoranthene, a pyrene, a decacyclene, a camphorquinone, an erythrosine, a fullerene, pyrene- 1 -butyric acid, pyrenedecanoic acid, perfluorodecanoic acid, perylene dibutyrate, erythrosine B, fluorescent yellow, Ceo fullerene, C70 fullerene, a ligandmetal complex, a ruthenium(II) ligand-metal complex, an iridium(III) ligand-metal complex, an osmium(II) ligand-metal complex, a rhenium(II) ligand-metal complex, a trivalent lanthanide, a metalloporphyrin, 8-hydroxy-7-iodo-5-quinoline sulfonate (“RTDP), a polycyclic aromatic hydrocarbon
  • the disclosed method begins with introducing the population of biological micro-objects into a chamber of a microfluidic device having a flow region (which may include a channel) and the chamber.
  • the chamber is connected or opens to the flow region/channel.
  • the microfluidic device can include a single chamber or a plurality of chambers, and/or a single flow region/channel or a plurality of channels.
  • the chamber can be a sequestration pen or any form or type of container.
  • the method also includes flowing a fluidic medium containing a dye, such as RTDP, and a supplied partial pressure of oxygen into the microfluidic device for a period of time.
  • fluorescence of the dye changes when the dye is in proximity to a local concentration of oxygen, for example, fluorescence level changes depending on the amount of dissolved oxygen.
  • fluorescence of the dye diminishes when the dye is quenched by a local concentration of oxygen molecules.
  • the method includes taking a fluorescence image of an area of interest (AOI) within the chamber at a time associated with a particular timestamp.
  • the area of interest may comprise one or more portions of a fluorescence image.
  • “Heatmaps”, as shown and described with respect to FIGs. 6, 7A, and 7B, may include data from such fluorescence images.
  • FIG. 6 illustrates heatmaps 600 showing normalized fluorescence intensity categorized by perfusion conditions at various times/timestamps for a microfluidic device including a plurality of chambers (e.g. sequestration pens) and channels according to some embodiments of the disclosure.
  • the heatmaps 600 shown in FIG. 6 are of a microfluidic device that is similar to the microfluidic device shown in FIG. IB.
  • the culture media perfused into the microfluidic device may include any suitable culture medium, as is known in the art, for the cells under investigation, which may be any type of cells described elsewhere in this disclosure, for example animal, mammalian, human, immunological, bacterial or fungal cells.
  • perfusion may include flowing a gaseous medium.
  • the gaseous medium may include a specified percentage of oxygen or other gases providing either optimized or test conditions for culturing the cells of interest.
  • the gaseous medium may include a percentage of oxygen similar to that of a standard atmosphere, e.g. about 21% oxygen (Clean Dry Air, CD A).
  • the gaseous medium may include a concentration of oxygen that is greater than that of CDA, such as about 25%, about 30%, about 35%, about 40%, about 45%, about 50% or more oxygen in the gaseous medium.
  • perfusion may be performed using a mixture of liquid medium and gaseous medium.
  • the mixture may include a mixture of liquid: gaseous media that may have a ratio of about 90:10; about 80:20; about 70:30; about 60:40; about 50:50; about 40:60; about 30:70; about 20:80, about 10:90 v/v.
  • perfusion may be performed with a mixture of liquid medium and gaseous medium which includes 80% CDA, or any percentage of oxygen as described above.
  • perfusion may be performed by performing one or more alternating perfusions of liquid medium and gaseous medium.
  • the alternating perfusions may have a duty cycle of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, wherein the percentage given represents the “on time” for perfusion of liquid medium.
  • the alternating perfusions may have a duty cycle of at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less, wherein the percentage given represents the “on time” for perfusion of liquid medium.
  • the alternating perfusions may have a duty cycle that ranges between any two of the preceding values.
  • the alternating perfusions may produce a 10% liquid to 90% gas mixture by flowing liquid medium for a period of 1 minute and then flowing gas for 9 minutes. Such a mixture would constitute a 10% duty cycle.
  • the liquid medium may be sparged or bubbled with gas in a reagent bay to equilibrate it to a proper set point (such as 2% O2, 21% O2, or 40% O2) prior to flowing the liquid medium into the microfluidic device.
  • the media source 178 comprises a sparging component in fluidic communication with a gas source providing a gas mixture with a supplied partial pressure of O2, wherein the sparging component is operable to sparge the liquid medium with the gas mixture to provide sparged liquid medium.
  • the different perfusion conditions shown in the heatmaps 600 include various perfusion rates, including perfusion at 0.1 microliter/s at top row 610, 1 microliter/s at middle row 620, and 5 microliter/s at bottom row 630, introducing a culture medium which causes an increase in metabolic rate in the cells under culture, e.g., causing an increase in oxygen demand by the cells.
  • the various timestamps illustrated in the heatmaps 600 are taken at time 0 hour, 0.33 hour, 0.67 hour, 1 hour, 1.33 hour, 1.67 hour, 2 hour, and 2.33 hour.
  • the change in metabolic rate becomes apparent after about 1.5 hour of perfusion.
  • the normalized fluorescence intensity increases sharply from left to right, indicating that the perfusion rate at 0.1 microliter/s does not adequately provide sufficient oxygen content to the living cells, having more oxygen demand. The lack of oxygen becomes more severe as the experiment progresses.
  • the normalized fluorescence intensity does not vary as sharply from left to right and is less intense than that of the fluorescence seen in row 610 at 2.33 hours. This indicates that the perfusion rate at 1 microliter/s corrects some of the deficiency and provides a more suitable amount of oxygen content to the living cells.
  • FIG. 7A illustrates the same data from FIG. 6, e.g., row 610 but the heat map presents the extent of oxygen saturation.
  • FIG. 7B illustrates heatmaps 700b of normalized oxygen level/consumption at various perfusion conditions at a fixed perfusion period of time of a microfluidic device including a plurality of chambers (e.g., sequestration pens) and channels according to some embodiments of the disclosure.
  • the heatmaps 700b show dissolved oxygen levels across the microfluidic device for a period of time (e.g., 1.33 hours) for different perfusion rates: 0.1 microliter/s (left heatmap), 1 microliter/s (middle heatmap), and 5 microliter/s (right heatmap), taken from the data of rows 610, 620, and 630 respectively.
  • FIG. 8 is a graphical representation 800 showing normalized oxygen level/consumption as a function of biomass (e.g., a population of biological micro-objects) for each of the chambers (e.g. sequestration pens) of a microfluidic device according to some embodiments of the disclosure.
  • the graphical representation 800 includes the dissolved oxygen level shown as a function of the biomass of the colonies in the corresponding sequestration pen, where a high dissolved oxygen level indicates a low oxygen utilization (810) by the biomass and a low dissolved oxygen level indicates a high oxygen utilization (820).
  • a clear pattern of oxygen consumption can be correlated with greater biomass, e.g., greater viable clonal population, as illustrated for example in FIG. 8.
  • the cells from preferred sequestration pens may be either the colonies having the highest biomass or may be from sequestration pens where the cells have the highest O2 consumption per mass unit, e.g. where the colony may not have the most number of cells but the individual cells are consuming oxygen at the highest rate per cell.
  • Oxygen consumption may be related to the rate of growth per cell or may be related to the rate of a cellular process such as production of a gene product or other cellular product.
  • FIGs. 9A-9D illustrate the changes in observed normalized fluorescence intensity depending on perfusion conditions between the chambers and the channels.
  • FIGs. 9A-9B illustrate normalized fluorescence intensity as a function of oxygen level in the channels of a microfluidic device according to some embodiments of the disclosure.
  • FIG. 9A shows a plot 900a of normalized fluorescence intensity taken during standard perfusion
  • FIG. 9B shows a plot 900b of normalized fluorescence intensity taken without perfusion.
  • FIGs. 9C-9D illustrate normalized fluorescence intensity as a function of oxygen level in sequestration pens of a microfluidic device according to some embodiments of the disclosure.
  • the x axis for FIGs. 9C and 9D are oxygen level in percentage, similar to those of FIGs. 9A and 9B.
  • FIG. 9C shows a plot 900c of normalized fluorescence intensity taken during standard perfusion
  • FIG. 9D shows a plot 900d of normalized fluorescence intensity taken without perfusion.
  • FIG. 10 illustrates a flow chart for an example method 1000 of determining oxygen consumption level in a population of biological micro-objects, according to various embodiments of the present disclosure.
  • the population of biological micro-objects are located in sequestration pens.
  • the method 1000 includes locating the population of biological microobjects into a chamber of a microfluidic device comprising a channel and the chamber, wherein the chamber opens to the channel, at step 1010.
  • locating the population of biological micro-objects into the chamber comprises introducing the population of biological micro-objects into the chamber.
  • the method 1000 includes flowing a fluidic medium containing a dye and a supplied partial pressure of oxygen into the microfluidic device for a period of time, wherein fluorescence emitted by the dye changes when the dye encounters oxygen in the local environment, at step 1020.
  • the method 1000 includes taking a fluorescence image of an area of interest (AOI) within the chamber, at step 1030.
  • the method 1000 includes correlating fluorescence intensity of the fluorescence image of the AOI to a reference to determine an observed partial pressure of the oxygen in the AOI, thereby determining the oxygen consumption level, at step 1040.
  • AOI area of interest
  • the fluorescence intensity may comprise a sum of the fluorescence intensity over the AOI, a mean of the fluorescence intensity over the AOI, a median of the fluorescence intensity over the AOI, a maximum of the fluorescence intensity over the AOI, a minimum of the fluorescence intensity over the AOI, a gradient of fluorescence intensity over the AOI, or any function of fluorescence intensity over the AOI.
  • the dye includes a soluble and diffusible dye.
  • the dye is a ruthenium complex.
  • the dye is RTDP, as described herein.
  • the dye is any dye described herein.
  • fluorescence emitted by the dye is quenched when the dye encounters oxygen in the local environment and fluoresces when the dye is not experiencing collisional quenching by oxygen in its local environment.
  • the oxygen consumption level corresponds to a decrease in the observed partial pressure of the oxygen compared to the supplied partial pressure.
  • the supplied partial pressure is measured in an area of the microfluidic device in which biological micro-objects are not growing, such as a channel described herein.
  • the fluidic medium is flowed at a flow rate of at least about 0.1 microliters per second (uL/s), 0.2 uL/s, 0.3 uL/s , 0.4 uL/s, 0.5 uL/s, 0.6 uL/s, 0.7 uL/s, 0.8 uL/s, 0.9 uL/s, 1 uL/s, 2 uL/s, 3 uL/s, 4 uL/s, 5 uL/s, 6 uL/s, 7 uL/s, 8 uL/s, 9 uL/s, 10 uL/s, or more.
  • the fluidic medium is flowed at a flow rate of at most about 10 uL/s, 9 uL/s, 8 uL/s, 7 uL/s, 6 uL/s, 5 uL/s, 4 uL/s, 3 uL/s, 2 uL/s, 1 uL/s, 0.9 uL/s, 0.8 uL/s, 0.7 uL/s, 0.6 uL/s, 0.5 uL/s, 0.4 uL/s, 0.3 uL/s, 0.2 uL/s, 0.1 uL/s, or less.
  • the fluidic medium is flowed at a flow rate ranging between any two of the preceding values. In accordance with various embodiments, the fluidic medium is flowed at a flow rate ranging between 0.1 microliter/s and 10 microliters/s. In accordance with various embodiments, the population of biological micro-objects in the chamber of the microfluidic device is cultured under aerobic conditions comprising flowing a fluidic medium comprising a supplied partial pressure of oxygen.
  • the supplied partial pressure of oxygen is at least about 0.01 bar, 0.02 bar, 0.3 bar, 0.04 bar, 0.05 bar, 0.06 bar, 0.07 bar, 0.08 bar, 0.09 bar, 0.1 bar, 0.15 bar, 0.2 bar, 0.25 bar, 0.3 bar, 0.4 bar, 0.5 bar, 0.6 bar, 0.7 bar, 0.8 bar, 0.9 bar, 1 bar, or more.
  • the supplied partial pressure of oxygen is at most about 1 bar, 0.9 bar, 0.8 bar, 0.7 bar, 0.6 bar, 0.5 bar, 0.4 bar, 0.3 bar, 0.25 bar, 0.2 bar, 0.15 bar, 0.1 bar, 0.09 bar, 0.08 bar, 0.07 bar, 0.06 bar, 0.05 bar, 0.04 bar, 0.03 bar, 0.02 bar, 0.01 bar, or less.
  • the supplied partial pressure of oxygen ranges between any two of the preceding values.
  • the AOI is disposed within the chamber at a location wherein transference of components of the fluidic medium flowing in the channel is dominated by diffusion.
  • “dominated by diffusion” means that diffusion is the primary mechanism for transference of components of the fluidic medium flowing in the channel, as compared to all other non-diffusive transport mechanisms.
  • “dominated by diffusion” means that diffusion contributes at least about 75%, 80%, 85%, 90%, 95%, 99%, or more of the transference of components of the fluidic medium flowing in the channel.
  • “dominated by diffusion” means that diffusion contributes at most about 99%, 95%, 90%, 85%, 80%, 75%, or less of the transference of components of the fluidic medium flowing in the channel. In accordance with various embodiments, “dominated by diffusion” means that diffusion contributes a range of the transference of components of the fluidic medium flowing in the channel that is defined by any two of the preceding values. In accordance with various embodiments, “dominated by diffusion” means that transference of components of the fluidic medium flowing in the channel occurs substantially only by diffusion.
  • the AOI may contain no biological microobjects.
  • the fluidic medium includes a liquid medium, a gaseous medium or a mixture thereof.
  • the fluidic medium includes a mixture of a liquid medium and a gaseous medium.
  • the mixture of the liquid medium and the gaseous medium includes at least a 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, or 40:60 v/v ratio of the liquid medium to the gaseous medium.
  • the mixture of the liquid medium and the gaseous medium includes at most a 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, or 10:90 v/v ratio of the liquid medium to the gaseous medium.
  • the mixture of the liquid medium and the gaseous medium includes a v/v ratio that ranges between any two of the preceding values.
  • the medium includes a liquid medium saturated with a selected supplied partial pressure of the oxygen. In some embodiments, alternating perfusions of liquid medium and gaseous medium are supplied.
  • the alternating perfusions may have a duty cycle of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the alternating perfusions may have a duty cycle of at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less. In some embodiments, the alternating perfusions may have a duty cycle that ranges between any two of the preceding values. Additional details can be found below in the Experimental section, in Example 1.
  • correlating fluorescence of the fluorescence image of the AOI to determine an observed partial pressure of the oxygen at the AOI further comprises identifying an intensity of the fluorescence on a reference curve correlating fluorescence intensities of the dye with supplied partial pressures of the oxygen.
  • the method 1000 further includes constructing a reference curve correlating an observed intensity of fluorescence of the dye with a supplied partial pressure of oxygen.
  • constructing the reference curve further includes flowing a liquid medium through the channel of the microfluidic device for a first reference period of time, wherein the liquid medium comprises the dye and a first supplied partial pressure of the oxygen, detecting a first fluorescence intensity of the dye within a selected region of the microfluidic device, flowing a second liquid medium through the channel of the microfluidic device for a second reference period of time, wherein the second liquid medium comprises the dye and a second supplied partial pressure of the oxygen, detecting a second fluorescence intensity of the dye within the selected region of the microfluidic device, and correlating each of the first and the second fluorescence intensities with the supplied partial pressure of the oxygen.
  • constructing the reference curve further includes flowing liquid media comprising the dye and successive supplied partial pressures of the oxygen at successive points in time; detecting successive fluorescence intensities of the dye at the successive points in time; and correlating each of the fluorescence intensities with a supplied partial pressure of the oxygen.
  • the microfluidic device does not contain any biological micro-objects while constructing the reference curve.
  • the selected supplied partial pressure of oxygen is at least about 0.01 bar, 0.02 bar, 0.03 bar, 0.04 bar, 0.05 bar, 0.06 bar, 0.07 bar, 0.08 bar, 0.09 bar, 0.1 bar, 0.11 bar, 0.12 bar, 0.13 bar, 0.14 bar, 0.15 bar, 0.16 bar, 0.17 bar, 0.18 bar, 0.19 bar, 0.2 bar, 0.21 bar, 0.22 bar, 0.23 bar, 0.24 bar, 0.25 bar, or more.
  • the selected supplied partial pressure of oxygen is at most about 0.25 bar, 0.24 bar, 0.23 bar, 0.22 bar, 0.21 bar, 0.2 bar, 0.19 bar, 0.18 bar, 0.17 bar, 0.16 bar, 0.15 bar, 0.14 bar, 0.13 bar, 0.12 bar, 0.11 bar, 0.1 bar, 0.09 bar, 0.08 bar, 0.07 bar, 0.06 bar, 0.05 bar, 0.04 bar, 0.03 bar, 0.02 bar, 0.01 bar, or less.
  • the selected supplied partial pressure of oxygen ranges between any two of the preceding values. In accordance with various embodiments, the selected supplied partial pressure of oxygen ranges from about 0.02 bar to about 0.21 bar.
  • the method 1000 includes detecting fluorescence intensities associated with at least about three, four, five, or more different supplied partial pressures of the oxygen. In accordance with various embodiments, the method 1000 includes detecting fluorescence intensities associated with at most about five, four, three, or fewer supplied different partial pressures of the oxygen. In accordance with various embodiments, the method 1000 includes detecting a number of fluorescence intensities associated with a number of different supplied partial pressures of the oxygen that ranges between any two of the preceding values. In accordance with various embodiments, the fluorescence image is taken under a perfusion condition. In accordance with various embodiments, the perfusion condition comprises a constant flow, a steady state flow, a preprogrammed flow with one or more defined flow rates, or zero flow after equilibrium.
  • the microfluidic device includes a plurality of chambers, and the method 1000 further includes introducing the population of biological micro-objects into the plurality of chambers.
  • the microfluidic device includes a plurality of channels, and the method 1000 further includes introducing the population of biological micro-objects into the plurality of channels.
  • flowing the fluidic medium and taking the fluorescence image are performed at a selected temperature.
  • the temperature is from about 20°C to about 40°C.
  • the temperature is from about 28°C to about 30°C.
  • the cells may alternatively be cultured at other temperatures, such as at least about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or higher.
  • the cells may be cultured at temperature of at most about 70°C, 65°C, 60°C, 55°C, 50°C, 45°C, 40°C, 35°C, 30°C, 25°C, 20°C, 15°C, 10°C, or less.
  • the cells may be cultured at a temperature that ranges between any two of the preceding values.
  • flowing the fluidic medium and taking the fluorescence image is performed at a selected pH.
  • the pH may be at least about 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9,0, or more.
  • the pH may be at most about 9.0, 8.0, 7.0, 6,0, 5.0, 4.0, 3.0, or less. In accordance with various embodiments, the pH may range between any two of the preceding values. In accordance with various embodiments, the pH is from about 3.0 to about 9.0.
  • the method 1000 optionally includes taking a plurality of fluorescence images at a plurality of times/timestamps correlating a respective fluorescence of each fluorescence image to determine a respective observed partial pressure of the oxygen in the AOI at the respective timestamp, at step 1050. In accordance with various embodiments, the method 1000 optionally includes taking a plurality of fluorescence images at a plurality of points within the AOI, at step 1060.
  • the chamber includes a sequestration pen, wherein the sequestration includes an isolation region and a connection region, and wherein the connection region has a proximal opening to the channel and a distal opening to the isolation region.
  • the isolation region includes a single opening to the connection region.
  • the population of biological micro-objects is disposed within the isolation region of the sequestration pen.
  • the method 1000 optionally includes taking a plurality of fluorescence images at a plurality of points extending from the isolation region of the sequestration pen along an axis of diffusion towards the channel, at step 1070.
  • the AOI includes at least part of the connection region.
  • the area of interest comprises at least a portion of the sequestration pen aligned along an axis of diffusion from within the sequestration pen out into the flow region.
  • the area of interest can be partitioned into a plurality of segments and, in some embodiments, an average signal can be computed for each of the segments.
  • FIG. 11 illustrates an example approach 1100 of converting acquired fluorescence images into data for correlating fluorescence of an AOI to a reference to determine the dissolved oxygen level accordance with some embodiments of the present disclosure.
  • the approach 1100 includes performing dissolved oxygen (DO) Standard Testing with multiple oxygen setpoints as described with respect to FIGs. 6-10.
  • DO dissolved oxygen
  • the fluorescence images taken in the Standard Testing is labeled as DO Standard Images 1110 as shown in FIG. 11.
  • the DO Standard Images of air saturated RTDP media are selected to be used for normalization for imaging correction at a later time. Such images are named Normalization Reference Images 1120.
  • the approach 1100 includes performing the DO assay with RTDP during cell culture as described with respect to FIGs. 6-10. Such data are referred to as RTDP Assay Images 1130, as shown in FIG. 11.
  • the approach 1100 includes using an Offline Analysis operation 1140 to measure the mean intensities in the AOIs for all fluorescence images.
  • an Offline Analysis operation 1140 to measure the mean intensities in the AOIs for all fluorescence images.
  • the DO Standard Images 1110, Normalization Reference Images 1120, and RTDP Assay Images 1130 are converted into DO Standard Data (Raw) 1112, Normalization Reference Data 1122, and RTDP Assay Data (Raw) 1132, respectively.
  • the customized AOIs and configurable parameters used in the Offline Analysis operation 1140 are identical so that the processed data from the three individual sequences are comparable and can be further processed and analyzed.
  • the approach 1100 further includes normalizing the DO Standard Data (Raw) 1112 and RTDP Assay Data (Raw) 1132 by the Normalization Reference Data 1122 via Normalization 1150.
  • the approach 1100 further includes dividing the mean intensity of DO Standard Data (Raw) 1112 and RTDP Assay Data (Raw) 1132 in the AOIs in each chamber (e.g., sequestration pen) by the corresponding mean intensity in the Normalization Reference Data 1122 to generate a Normalized DO Standard Curve 1114 and Normalized Assay Data 1134.
  • the normalization process can suppress most of the imaging artifacts, such as for example, nonuniform illumination, flat-field effect, etc.
  • the approach 1100 further includes conversion of the Normalized Assay Data 1134 into percentage dissolved oxygen (%DO) using the generated Normalized DO Standard Curve 1114 via DO Conversion 1160. Once the DO Conversion 1160 is obtained, the DO Distribution 1170 is calculated.
  • %DO percentage dissolved oxygen
  • FIG. 12A illustrates a first example approach 1200a of generating a DO standard curve.
  • the approach 1200a includes an instrument preparation operation 1210a.
  • the instrument preparation operation includes a full clean of the flow system, microfluidic device or chip wetting.
  • the approach 1200a includes a priming operation 1220a.
  • the priming operation includes a flow system line priming operation and flushing of the microfluidic device or chip with RTDP.
  • the approach 1200a includes a RTDP equilibration operation 1230a.
  • the RTDP equilibration operation includes allowing the RTDP to equilibrate within the microfluidic device or chip.
  • the approach 1200a includes an oxygen setpoint determination operation 1240a.
  • the oxygen setpoint determination operation includes determining whether the current oxygen setpoint is approximately 21%. In accordance with various embodiments, if the current oxygen setpoint is approximately 21%, the flow system is connected to a 21% O2 source (such as a 21% O2 gas cylinder) at operation 1242a. In accordance with various embodiments, if the current oxygen setpoint is not approximately 21%, the flow system is connected to an O2 source having an O2 concentration different from 21% at operation 1244a.
  • the approach 1200a includes a media sparging operation 1252a, a gas flush operation 1254a, and/or a gas bath operation 1256a.
  • the media sparging operation includes sending a gas mixture containing oxygen from the O2 source to a liquid medium and allowing the gas mixture to bubble into the liquid medium for a first period of time until the liquid medium attains a desired equilibrium oxygen setpoint.
  • the first period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more. In accordance with various embodiments, the first period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less. In accordance with various embodiments, the first period of time ranges between any two of the preceding values.
  • the gas flush operation includes flushing O2 through channels of the microfluidic device or chip for a second period of time.
  • the second period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more.
  • the second period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the second period of time ranges between any two of the preceding values.
  • the gas bath operation comprises surrounding the microfluidic device or chip in an O2 gas bath, as described herein, for a third period of time.
  • the third period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more. In accordance with various embodiments, the third period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less. In accordance with various embodiments, the third period of time ranges between any two of the preceding values.
  • the approach 1200a includes a time-lapse imaging operation 1260a.
  • the time-lapse imaging operation comprises acquiring a plurality of fluorescence images of the microfluidic device or chip in the presence of a liquid flush.
  • the liquid flush includes flowing liquid through the microfluidic device or chip at a flow rate for a fourth period of time.
  • the flow rate is at least about 1 microliter per second (uL/s), 2 uL/s, 3 uL/s, 4 uL/s, uL/s, 5 uL/s, 6 uL/s, 7 uL/s, 8 uL/s, 9 uL/s, 10 uL/s, or more. In accordance with various embodiments, the flow rate is at most about 10 uL/s, 9 uL/s, 8 uL/s, 7 uL/s, 6 uL/s, 5 uL/s, 4 uL/s, 3 uL/s, 2 uL/s, 1 uL/s, or less.
  • the flow rate ranges between any two of the preceding values.
  • the fourth period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more.
  • the fourth period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the fourth period of time ranges between any two of the preceding values.
  • the process 1200a includes a dye replenishment operation 1272a.
  • the dye replenishment operation comprises perfusing fresh dye (such as RTDP) through the microfluidic device or chip.
  • the process 1200a includes repeating any of operations 1240a, 1242a, 1244a, 1252a, 1254a, 1260a, 1262a, and 1272a.
  • the process includes repeating any of operations 1240a, 1242a, 1244a, 1252a, 1254a, 1260a, 1262a, and 1272a at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.
  • the process includes repeating any of operations 1240a, 1242a, 1244a, 1252a, 1254a, 1260a, 1262a, and 1272a at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times.
  • the process includes repeating any of operations 1240a, 1242a, 1244a, 1252a, 1254a, 1260a, 1262a, and 1272a a number of times that ranges between any two of the preceding values.
  • the RTDP fluorescence signal may be imaged for a variety of desired oxygen setpoints.
  • the fluorescence signals associated with each oxygen setpoint may be collected and used to fit a DO standard curve.
  • the RTDP fluorescence signals in areas surrounding biological micro-objects may then be compared to this DO standard curve to determine the associated DO level in the areas surrounding the biological micro-objects, as described herein.
  • FIG. 12B illustrates a second example approach 1200b of generating a DO standard curve.
  • the approach 1200b includes an instrument preparation operation 1210b.
  • the instrument preparation operation 1210b is similar to instrument preparation operation 1210a described herein with respect to FIG. 12A.
  • the instrument preparation operation includes a full clean of the flow system, microfluidic device or chip wetting, and calibration.
  • the approach 1200b includes a priming operation 1220b.
  • the priming operation 1220b is similar to priming operation 1220a described herein with respect to FIG. 12A.
  • the priming operation includes a flow system line priming operation and flushing of the microfluidic device or chip with RTDP.
  • the approach 1200b includes a RTDP equilibration operation 1225b.
  • the RTDP equilibration operation 1225b is similar to RTDP equilibration operation 1230a described herein with respect to FIG. 12A.
  • the RTDP equilibration operation includes allowing the RTDP to equilibrate within the microfluidic device or chip.
  • the RTDP equilibration operation includes slowly perfusing RTDP through the microfluidic device or chip to allow RTDP to diffuse into sequestration pens until equilibrium RTDP concentration is reached.
  • the approach 1200b includes an oxygen setpoint operation 1230b.
  • the oxygen setpoint operation 1230b includes receiving an oxygen setpoint supplied by a user.
  • the oxygen setpoint operation includes sending a signal to a multi-gas controller to mix gases from supply tanks until the oxygen content of the gas mixture reaches the desired oxygen setpoint.
  • the oxygen setpoint operation includes sending the gas mixture to the microfluidic device or chip, or elsewhere in the system, for example, for liquid media sparging.
  • the approach 1200b includes a media sparging operation 1235b.
  • the media sparging operation 1235b is similar to media sparging operations 1252a described herein with respect to FIG. 12A.
  • the media sparging operation includes sending the gas mixture to a liquid medium (which includes RTDP) and allowing the gas mixture to bubble into the liquid medium for a first period of time until the liquid medium attains a desired equilibrium oxygen setpoint.
  • the first period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more. In accordance with various embodiments, the first period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less. In accordance with various embodiments, the first period of time ranges between any two of the preceding values.
  • the approach 1200b includes a first chip flush operation 1240b.
  • the chip flush operation comprises flushing the liquid medium (which has been oxygenated by the gas sparging operation) through the microfluidic device or chip.
  • the approach 1200b includes a gas flush operation 1245b.
  • the gas flush operation 1245b is similar to gas flush operation 1254a described herein with respect to FIG. 12A.
  • the gas flush operation includes flushing the gas mixture through channels of the microfluidic device or chip for a second period of time.
  • the second period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more.
  • the second period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less. In accordance with various embodiments, the second period of time ranges between any two of the preceding values.
  • the approach 1200b includes a second chip flush operation 1250b.
  • the second chip flush operation 1250b is similar to first chip flush operation 1240b described herein with respect to FIG. 12B.
  • the approach 1200b comprises a time-lapse imaging operation 1255b.
  • the time-lapse imaging operation 1255b is similar to time-lapse imaging operation 1260a described herein with respect to FIG. 12A.
  • the time-lapse imaging operation comprises acquiring a plurality of fluorescence images of the microfluidic device or chip in the presence of a liquid flush.
  • the liquid flush includes flowing liquid through the microfluidic device or chip at a flow rate for a third period of time.
  • the flow rate is at least about 1 microliter per second (uL/s), 2 uL/s, 3 uL/s, 4 uL/s, uL/s, 5 uL/s, 6 uL/s, 7 uL/s, 8 uL/s, 9 uL/s, 10 uL/s, or more. In accordance with various embodiments, the flow rate is at most about 10 uL/s, 9 uL/s, 8 uL/s, 7 uL/s, 6 uL/s, 5 uL/s, 4 uL/s, 3 uL/s, 2 uL/s, 1 uL/s, or less.
  • the flow rate ranges between any two of the preceding values.
  • the third period of time is at least about 1 minute (min), 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, or more.
  • the third period of time is at most about 60 min, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the third period of time ranges between any two of the preceding values.
  • the approach 1200b includes an oxygen setpoint feedback operation 1260b.
  • the oxygen setpoint feedback operation includes determining whether all desired oxygen setpoints have been imaged. If no, the operations 1230b, 1235b, 1240b, 1245b, 1250b, and 1255b are repeated one or more times for one or more desired oxygen setpoints. In this manner, the RTDP fluorescence signal may be imaged for a variety of desired oxygen setpoints. If yes, the microfluidic device or chip may be flushed with RTDP at operation 1270b. The fluorescence signals associated with each oxygen setpoint may be collected and used to fit a DO standard curve. The RTDP fluorescence signals in areas of interest proximate to biological micro-objects may then be compared to this DO standard curve to determine the associated DO level in the areas surrounding the biological micro-objects, as described herein.
  • FIG. 14 illustrates a first example approach 1400 of performing a DO perfusion assay.
  • the approach 1400 includes a cell loading operation 1410.
  • the cell loading operation comprises loading cells into sequestration pens of the microfluidic device or chip.
  • the approach 1400 includes a first culture operation 1420.
  • the first culture operation comprises culturing the cells in the presence of BMGY (buffered glycerol complex medium) growth medium, or other liquid growth medium sufficient to support growth of cells.
  • BMGY growth medium comprises peptone, yeast extract, biotin, yeast nitrogen base, potassium phosphate monobasic, potassium phosphate dibasic, and glycerol.
  • the first culture operation comprises culturing the cells in the presence of BMMY (buffered methanol complex medium) growth medium.
  • BMMY growth medium comprises peptone, yeast extract, biotin, yeast nitrogen base, potassium phosphate monobasic, potassium phosphate dibasic, and methanol.
  • the first culture operation comprises culturing the cells in the presence of Bird growth medium.
  • Bird growth medium comprises ammonium sulfate, monopotassium phosphate, magnesium sulfate heptahydrate, succinic acid, biotin, calcium pantothenate, nicotinic acid, myoinositol, thiamine hydrochloride, pyridoxol hydrochloride, p-aminobenzoic acid, ethylenediaminetetraacetic acid (EDTA), zinc sulfate heptahydrate, copper sulfate anhydrous, manganese chloride tetrahydrate, cobalt chloride hexahydrate, sodium molybdenite dihydrate, iron sulfate heptahydrate, iron chloride hexahydrate, calcium chloride dihydrate, and lysine.
  • EDTA ethylenediaminetetraacetic acid
  • the first culture operation comprises culturing the cells in the presence of Delft growth medium.
  • Delft growth medium comprises ammonium sulfate, monopotassium phosphate, magnesium sulfate heptahydrate, glucose, EDTA, zinc sulfate heptahydrate, manganese chloride dihydrate, cobalt chloride hexahydrate, copper sulfate pentahydrate, sodium molybdenite dihydrate, calcium chloride dihydrate, iron sulfate heptahydrate, boric acid, potassium iodide, biotin, p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, thiamine hydrochloride, and myoinositol.
  • the first culture operation comprises culturing the cells in LSM (lymphocyte separation medium) growth medium.
  • LSM growth medium comprises potassium phosphate monobasic, ammonium sulfate, calcium sulfate dihydrate, magnesium sulfate heptahydrate, sodium citrate, glycerol or methanol, vitamin mix, and PTM4 (Pichia trace minerals 4) solution.
  • the first culture operation comprises culturing the cells in FM22 growth medium.
  • FM22 growth medium comprises potassium phosphate monobasic, ammonium sulfate, calcium sulfate dihydrate, magnesium sulfate heptahydrate, PTM4 solution, and dextrose or glycerol.
  • the first culture operation comprises culturing the cells for at least about 1 hour (h), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, or more.
  • the first culture operation comprises culturing the cells for at most about 24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h, 16 h, 15 h, 14 h, 13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, or less.
  • the first culture operation comprises culturing the cells for a period of time that ranges between any two of the preceding values.
  • the approach 1400 includes a second culture operation 1430.
  • the second culture operation comprises culturing the cells in minimal growth medium with RTDP.
  • the second culture operation comprises culturing the cells for at least about 1 hour (h), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, or more.
  • the second culture operation comprises culturing the cells for at most about 24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h, 16 h, 15 h, 14 h, 13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, or less.
  • the second culture operation comprises culturing the cells for a period of time that ranges between any two of the preceding values.
  • the process 1400 includes determining whether the cells require DO monitoring at operation 1440. In accordance with various embodiments, if the cells require DO monitoring, the approach 1400 includes a time-lapse imaging operation 1442. In accordance with various embodiments, the time-lapse imaging operation 1442 comprises the time-lapse imaging operation 1260 described herein with respect to FIG. 12A and/or operation 1255 described herein with respect to FIG. 12B.
  • the approach 1400 includes continuing the second culture operation 1430.
  • the approach 1400 includes repeating any of operations 1440, 1442, and 1430.
  • the approach 1400 includes repeating any of operations 1440, 1442, and 1430 at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.
  • the approach 1400 includes repeating any of operations 1440, 1442, and 14300 at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times.
  • the approach 1400 includes repeating any of operations 1440, 1442, and 1430 a number of times that ranges between any two of the preceding values.
  • FIG. 15 illustrates a second example approach 1500 of performing a DO perfusion assay.
  • the approach 1500 includes a pre-loading operation 1510.
  • the pre-loading operation comprises wetting and optically calibrating a microfluidic device or chip, flushing the microfluidic device or chip with RTDP solution, equilibrating the microfluidic device or chip to an oxygen setpoint, and imaging the microfluidic device or chip to create a normalization reference image.
  • the approach 1500 includes a cell loading operation 1520.
  • the cell loading operation comprises loading cells into sequestration pens of the microfluidic device or chip.
  • the cell loading operation may be similar to the cell loading operation 1410 described herein with respect to FIG. 14.
  • the approach 1500 includes a batch culture operation 1530.
  • the batch culture operation comprises culturing the cells in the presence of BMGY growth medium, BMMY growth medium, Bird growth medium, Delft growth medium, LSM growth medium, or FM22 growth medium, or other liquid growth medium sufficient to support growth of cells.
  • the batch culture operation comprises culturing the cells for at least about 1 hour (h), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, or more.
  • the batch culture operation comprises culturing the cells for at most about 24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h, 16 h, 15 h, 14 h, 13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, or less.
  • the batch culture operation comprises culturing the cells for a period of time that ranges between any two of the preceding values.
  • the approach 1500 includes a feed culture operation 1540.
  • the feed culture operation comprises culturing the cells in induction medium without RTDP.
  • the feed culture operation comprises culturing the cells for at least about 1 hour (h), 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, or more.
  • the feed culture operation comprises culturing the cells for at most about 24 h, 23 h, 22 h, 21 h, 20 h, 19 h, 18 h, 17 h, 16 h, 15 h, 14 h, 13 h, 12 h, 11 h, 10 h, 9 h, 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 2 h, 1 h, or less.
  • the feed culture operation comprises culturing the cells for a period of time that ranges between any two of the preceding values.
  • the approach 1500 includes an assay operation 1550.
  • the assay operation includes a dye equilibration operation 1552, a DO assay operation 1556, and a post-assay rinse operation 1554.
  • the dye equilibration operation comprises culturing the cells in induction medium with RTDP for a first period of time.
  • the first period of time is at least about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 2 h, 3 h, 4 h, or more.
  • the first period of time is at most about 4 h, 3 h, 2 h, 1 h, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the first period of time ranges between any two of the preceding values.
  • the DO assay operation comprises culturing the cells in induction medium with RTDP for a second period of time, obtaining fluorescence images of the microfluidic device or chip, as described herein, and determining a DO level across the microfluidic device or chip from the fluorescence images, as described herein.
  • the second period of time is at least about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 2 h, 3 h, 4 h, or more.
  • the second period of time is at most about 4 h, 3 h, 2 h, 1 h, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the second period of time ranges between any two of the preceding values.
  • the post-assay rinse operation comprises rinsing the cells for a third period of time.
  • the third period of time is at least about 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 2 h, 3 h, 4 h, or more.
  • the third period of time is at most about 4 h, 3 h, 2 h, 1 h, 55 min, 50 min, 45 min, 40 min, 35 min, 30 min, 25 min, 20 min, 15 min, 10 min, 9 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, 1 min, or less.
  • the third period of time ranges between any two of the preceding values.
  • the approach 1500 includes repeating any of operations 1550, 1552, 1554, and 1556.
  • the approach comprises repeating any of operations 1550, 1552, 1554, and 1556 at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more times.
  • the approach comprises repeating any of operations 1550, 1552, 1554, and 1556 at most about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 times.
  • the approach comprises repeating any of operations 1550, 1552, 1554, and 1556 a number of times that ranges between any two of the preceding values.
  • the microfluidic device used to culture the biological micro-objects is permeable to gas flow.
  • oxygen may permeate from an environment surrounding the microfluidic device, or vice versa.
  • oxygen may permeate from one area of the microfluidic device to another area of the microfluidic device.
  • this “DO edge effect” may lead to non-uniform DO supply across the microfluidic device.
  • the DO edge effect may be mitigated using a variety of approaches.
  • the DO edge effect is mitigated by coating exterior surfaces of the microfluidic device with an oxygen-impermeable film.
  • the microfluidic device comprises a plurality of exterior surfaces and at least a portion of one or more surfaces of the plurality are coated with an oxygen- impermeable film. In accordance with various embodiments, a portion of at least about 1, 2, 3, 4, 5, 6, or more exterior surfaces of the microfluidic device are coated with the oxygen- impermeable film. In accordance with various embodiments, a portion of at most about 6, 5, 4, 3, 2, or 1 exterior surfaces of the microfluidic device are coated with the oxygen-impermeable film.
  • a portion of a number of exterior surfaces of the microfluidic device that ranges between any two of the preceding values are coated with the oxygen-impermeable film.
  • the portion of the surface is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the surface.
  • the portion of the surface is at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of the surface.
  • the portion of the surface ranges between any two of the preceding values.
  • the portion of the surface comprises substantially all of the surface.
  • the portion of the one or more surfaces comprises those portions of the one or more surfaces which are permeable to oxygen if the oxygen-impermeable film is omitted.
  • the oxygen-impermeable film has an oxygen permeability at 25°C of at least about 1 cm 3 mm-m’ 2 day 1 atm 1 , 2 cm 3 mm-m’ 2 day 1 atm’ x , 3 cm 3 mm-m’ 2 day 1 atm 1 , 4 cm 3 mm-m’ 2 day 1 atm 1 , 5 cm 3 mm-m’ 2 day 1 atm 1 , 6 cm 3 mm-m’ 2 day’ ⁇ tm 1 , 7 cm 3 mm-m’ 2 day 1 atm 1 , 8 cm 3 mm-m’ 2 day 1 atm 1 , 9 cm 3 mm-m’ 2 day 1 atm 1 , 10 cm 3 mm-m’ 2 day 1 atm 1 , 20 cm 3 mm-m’ 2 day 1 atm 1 , or more.
  • the oxygen-impermeable film has an oxygen permeability at 25°C of at most about 20 cm 3 mm-m’ 2 day 1 atm 1 , 10 cm 3 mm-m’ 2 day 1 atm 1 , 9 cm 3 mm-m’ 2 day 1 atm 1 , 8 2 day 1 atm 1 , or less. In accordance with various embodiments, the oxygen-impermeable film has an oxygen permeability at 25 °C that ranges between any two of the preceding values.
  • the oxygen-impermeable film has a thickness of at least about 0.01 micrometers (pm), 0.1 pm, 0.2 pm, 0.3 pm, 0.4 pm, 0.5 pm, 0.6 pm, 0.7 pm, 0.8 pm, 0.9 pm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm, 100 pm, 200 pm, 300 pm, 400 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1,000 pm, or more.
  • pm micrometers
  • the oxygen-impermeable film has a thickness of at most about 1,000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 400 pm, 300 pm, 200 pm, 100 pm, 90 pm, 80 pm, 70 pm, 60 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 9 pm, 8 pm, 7 pm, 6 pm, 5 pm, 4 pm, 3 pm, 2 pm, 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, 0.1 pm, or less.
  • the oxygen-impermeable film has a thickness that ranges between any two of the preceding values.
  • the oxygen-impermeable film comprises any suitable material that can be applied to the one or more exterior surfaces of the microfluidic device and that reduces oxygen transfer from outside the microfluidic device to inside the microfluidic device.
  • the oxygen-impermeable film comprises Parylene N (poly(p-xylene)), Parylene C (poly(2-chloro-l,4-dimethylbenzene), Parylene D (poly(2,5-dichloro-l,4-dimethylbenzene)), Parylene HT® (poly(l,4- Bis(difluoromethyl)benzene)), epoxy, Torr Seal® epoxy, or any combination thereof.
  • the oxygen-impermeable film is applied to a microfluidic device (such as microfluidic device 200 described herein with respect to FIG. 2A) using chemical vapor deposition, a conformal coating process, physical application of the film material such as by brushing, spraying, dipping, or dispensing from an applicator, or any other application process that is effective to provide the oxygen-impermeable film on the microfluidic device.
  • a microfluidic device such as microfluidic device 200 described herein with respect to FIG. 2A
  • chemical vapor deposition such as microfluidic device 200 described herein with respect to FIG. 2A
  • a conformal coating process such as by brushing, spraying, dipping, or dispensing from an applicator, or any other application process that is effective to provide the oxygen-impermeable film on the microfluidic device.
  • portions of the exterior surfaces of the microfluidic device are masked off with a masking material before the oxygen- impermeable film is applied to the microfluidic device, which is followed by removal of the masking material to provide microfluidic device having portions of the exterior surfaces that are not covered by the oxygen-impermeable film and portions of the exterior surfaces that are covered by the oxygen-impermeable film.
  • the portions of the exterior surfaces that are not covered by the oxygen-impermeable film can include portions of the microfluidic device that provide a functional interface to the microfluidic device, such as a port, inlet, outlet, electrical contact, or optical interface (for example, an optically transparent cover, or the like, for imaging chambers or flow regions or areas of interest (AOIs) or for projecting structured light onto a surface of the device to activate DEP forces within a DEP substrate), or can include portions of the microfluidic device that are constructed of materials which are oxygen impermeable.
  • the portions of the exterior surfaces that are covered by the oxygen-impermeable film can include portions of the microfluidic device that are constructed of materials that are not oxygen-impermeable or are oxygen permeable, or can include portions of the exterior surfaces of the microfluidic device that, in the absence of the oxygen-impermeable film, would permit diffusion of oxygen from the exterior of the device into a flow region or chamber of the microfluidic device, or can include other portions of the exterior surfaces.
  • the DO edge effect is mitigated using an oxygen delivery system (such as oxygen delivery system 1600 described herein with respect to FIG. 16) to deliver a supplied partial pressure of oxygen to at least a portion of one or more exterior surfaces of the plurality.
  • the oxygen delivery module is configured to couple to any microfluidic device described herein (such as microfluidic device 200 described herein with respect to FIG. 2A).
  • the DO edge effect is mitigated by delivering a supplied partial pressure of oxygen to at least a portion of one or more exterior surfaces of the plurality.
  • the supplied partial pressure of oxygen is at least about 0.01 bar, 0.02 bar, 0.03 bar, 0.04 bar, 0.05 bar, 0.06 bar, 0.07 bar, 0.08 bar, 0.09 bar, 0.1 bar, 0.11 bar, 0.12 bar, 0.13 bar, 0.14 bar, 0.15 bar, 0.16 bar, 0.17 bar, 0.18 bar, 0.19 bar, 0.2 bar, 0.21 bar, 0.22 bar, 0.23 bar, 0.24 bar, 0.25 bar, 0.3 bar, 0.4 bar, 0.5 bar, 0.6 bar, 0.7 bar, 0.8 bar, 0.9 bar, 1 bar, or more.
  • the supplied partial pressure of oxygen is at most about 1 bar, 0.9 bar, 0.8 bar, 0.7 bar, 0.6 bar, 0.5 bar, 0.4 bar, 0.3 bar, 0.25 bar, 0.24 bar, 0.23 bar, 0.22 bar, 0.21 bar, 0.2 bar, 0.19 bar, 0.18 bar, 0.17 bar, 0.16 bar, 0.15 bar, 0.14 bar, 0.13 bar, 0.12 bar, 0.11 bar, 0.1 bar, 0.09 bar, 0.08 bar, 0.07 bar, 0.06 bar, 0.05 bar, 0.04 bar, 0.03 bar, 0.02 bar, 0.01 bar, or less.
  • the supplied partial pressure of oxygen ranges between any two of the preceding values.
  • the supplied partial pressure of oxygen is delivered to at least the portion of the one or more exterior surfaces by one or more tubes placed in proximity to the one or more exterior surface.
  • FIG. 16 shows an oxygen delivery system 1600 comprising one or more tubes 1602 with one or more holes (lumens) 1604.
  • the one or more tubes are configured to be connected to a gas source for receiving a gaseous medium comprising the supplied partial pressure of oxygen.
  • the one or more tubes comprise one or more holes configured to allow the supplied partial pressure of oxygen to flow therethrough.
  • the one or more tubes comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more holes.
  • the one or more tubes comprise at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 holes. In accordance with various embodiments, the one or more tubes comprise a range of tubes defined by any two of the preceding values.
  • the DO edge effect is mitigated by surrounding the microfluidic device in an oxygen bath.
  • the supplied partial pressure of oxygen is delivered to the at least the portion of the one or more exterior surfaces by an oxygen bath surrounding the microfluidic device.
  • the supplied partial pressure of oxygen is at least about 0.01 bar, 0.02 bar, 0.03 bar, 0.04 bar, 0.05 bar, 0.06 bar, 0.07 bar, 0.08 bar, 0.09 bar, 0.1 bar, 0.11 bar, 0.12 bar, 0.13 bar, 0.14 bar, 0.15 bar, 0.16 bar, 0.17 bar, 0.18 bar, 0.19 bar, 0.2 bar, 0.21 bar, 0.22 bar, 0.23 bar, 0.24 bar, 0.25 bar, 0.3 bar, 0.4 bar, 0.5 bar, 0.6 bar, 0.7 bar, 0.8 bar, 0.9 bar, 1 bar, or more.
  • the supplied partial pressure of oxygen is at most about 1 bar, 0.9 bar, 0.8 bar, 0.7 bar, 0.6 bar, 0.5 bar, 0.4 bar, 0.3 bar, 0.25 bar, 0.24 bar, 0.23 bar, 0.22 bar, 0.21 bar, 0.2 bar, 0.19 bar, 0.18 bar, 0.17 bar, 0.16 bar, 0.15 bar, 0.14 bar, 0.13 bar, 0.12 bar, 0.11 bar, 0.1 bar, 0.09 bar, 0.08 bar, 0.07 bar, 0.06 bar, 0.05 bar, 0.04 bar, 0.03 bar, 0.02 bar, 0.01 bar, or less.
  • the supplied partial pressure of oxygen ranges between any two of the preceding values.
  • FIG. 22 shows a system 2200 configured to implement the methods described herein.
  • the system comprises a microfluidic device 200.
  • the microfluidic device is similar to any microfluidic device described herein, such as microfluidic device 200 described herein with respect to FIG. 2A.
  • the microfluidic device comprises a plurality of exterior surfaces 2210. In accordance with various embodiments, at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film 2220.
  • the oxygen-permeable film is similar to any oxygen- impermeable film described herein.
  • the oxygen-impermeable film has an oxygen permeability of at most 20 cm 3 mm-m’ 2 day 1 atm 1 .
  • the oxygen-impermeable film comprises Parylene N, Parylene C, Parylene D, Parylene HT®, epoxy, Torr Seal® epoxy, or any combination thereof.
  • the oxygen-impermeable film has a thickness of at least 1 nanometer (nm). In accordance with various embodiments, the oxygen-impermeable film has a thickness of at most 10 micrometers (pm).
  • the system further comprises an oxygen delivery module (not shown in FIG. 22) configured to deliver a supplied partial pressure of oxygen to at least a portion of one or more exterior surfaces of the plurality.
  • the oxygen delivery module comprises one or more tubes placed in proximity to the one or more exterior surfaces, the one or more tubes comprising one or more holes (lumens) configured to allow the supplied partial pressure of oxygen to flow therethrough.
  • the one or more tubes are similar to system 1600 described herein with respect to FIG. 16.
  • the oxygen delivery module comprises an oxygen bath surrounding the microfluidic device.
  • the kit comprises: any microfluidic device described herein (such as microfluidic device 200 described herein with respect to FIG. 2A, or such as the microfluidic device wherein at least a portion of one or more exterior surfaces are coated with an oxygen-impermeable film as described herein with respect to FIG. 22) and a buffer.
  • the kit further comprises a fluidic medium containing a dye.
  • the dye comprises a soluble and diffusible dye.
  • the dye comprises a ruthenium complex.
  • the dye comprises any dye described herein.
  • the kit comprises: any microfluidic device described herein (such as microfluidic device 200 described herein with respect to FIG. 2A, or such as the microfluidic device wherein at least a portion of one or more exterior surfaces are coated with an oxygen-impermeable film as described herein with respect to FIG. 22); and a fluidic medium containing a dye.
  • the dye comprises a soluble and diffusible dye.
  • the dye comprises a ruthenium complex.
  • the dye comprises any dye described herein.
  • the kit further comprises a buffer.
  • Citric acid stock solution (0.1 M) and sodium citrate solution (0.1 M) were prepared to prepare citrate buffer solutions of target pH of 3.0, 3.4, 3.6, 3.8, 4.0, 4.4, 4.6, 4.8, 5.0, 5.4, 5.6, and 6, respectively.
  • a pH-sensitive dye solution (LysoSensorTM Yellow/Blue DND-160, 5 uM) was prepared for this experiment.
  • a pH standard curve plate (96 wells) was prepared by using MantisTM liquid handler, and the citrate buffer solutions of various pH value prepared were introduced into respective wells (200 uL per well). Specifically in this experiment, Wells Al-Gl were introduced with solutions of pH 3.0; Wells A2-G2 were introduced with solutions of pH 3.4; Wells A3-G3 were introduced with solutions of pH 3.6; Wells A4-G4 were introduced with solutions of pH 3.8; Wells A5-G5 were introduced with solutions of pH 4; Wells A6-G6 were introduced with solutions of pH 4.4; Wells A7-G7 were introduced with solutions of pH 4.6; Wells A8-G8 were introduced with solutions of pH 4.8; Wells A9-G9 were introduced with solutions of pH 5; Wells A10-G10 were introduced with solutions of pH 5.4; Wells Al 1-G11 were introduced with solutions of pH 5.6; Wells A12-G12 were introduced with solutions of pH 6. Row H was used
  • the OptoSelectTM device included a substrate configured with OptoElectroPositioning (OEPTM) technology, which provides a phototransistor-activated dielectrophoresis (DEP) force.
  • the device also included a plurality of microfluidic channels, each having a plurality of NanoPenTM chambers (or sequestration pens) fluidically connected thereto. The volume of each sequestration pen was around IxlO 6 cubic microns.
  • the microfluidic device included conditioned interior surfaces, which are described in U.S. Patent Application Publication No.
  • the mixtures of the citrate buffer and the pH-sensitive dye in Wells A2-G2 were used in another chip, and so on so that the fluorescent signals determined from respective chips were collected.
  • the area of interest for determining the fluorescent signal in this experiment was the entire sequestration pen.
  • FIG. 25A illustrates the microfluidic chip (i.e. microfluidic device) used in this experiment and a top view showing the configuration of the microfluidic channels and the sequestration pens.
  • FIG. 25B shows the fluorescent images of each chip containing the citrate buffer solutions of various pH values. The darker the color, the stronger the intensity of the fluorescent signal is, and hence, lower pH value. The images show that, by detecting the fluorescent signals of the pH-sensitive dye, the pH of the medium disposed in the microfluidic device can be clearly observed.
  • FIG. 25C shows that the fluorescent signals representing each tested pH value can be clearly distinguished from each other.
  • the fluorescent signals obtained from the well plate and microfluidic chip corresponding to various pH values were normalized and plotted into curves (FIG. 26). The curve can be further calculated into a standard curve matching the measurement on chip with true pH value.
  • System and Microfluidic device The following experiments were performed using an OptoSelectTM microfluidic (or nanofluidic) device manufactured by Berkeley Eights, Inc. and controlled by an optical instrument which was also manufactured by Berkeley Lights, Inc.
  • the OptoSelectTM device included a substrate configured with OptoElectroPositioning (OEPTM) technology, which provides a phototransistor-activated dielectrophoresis (DEP) force.
  • the device also included a plurality of microfluidic channels, each having a plurality of NanoPenTM chambers (or sequestration pens) fluidically connected thereto. The volume of each sequestration pen was around IxlO 6 cubic microns.
  • the microfluidic device included conditioned interior surfaces, which are described in U.S. Patent Application Publication No.
  • On-chip pH acidification assay BIRD media without supplement of lysine was introduced to replace the culture media above to induce the production of the cells. Then, the pH-sensitive dye solution was introduced into the microfluidic channel and allowed to diffuse into the sequestration pen until equilibration (non-buffered; 0.01 uL/s). Then, the perfusion was stopped and the 3MTM NovecTM 7500 Engineered Fluid was introduced into the microfluidic channel to seal the sequestration pens. The series of fluorescent images were taken over 15 minutes to determine the signals of the pH-sensitive dye. The area of interest for determining the fluorescent signal in this experiment was the entire sequestration pen.
  • FIG. 27A and FIG. 27B show the fluorescent images of the same view taken at different time point.
  • FIG. 27A was taken at Min. 0 when the pH-sensitive dye was just introduced and reached equilibration within the microfluidic device, and
  • FIG. 27B was taken at Min. 14.
  • the sequestration pens in the image had different intensities of fluorescent signals.
  • sequestration pens such as those indicated by white arrows
  • exhibiting stronger fluorescent signals indicates the culture medium within the pens was more acidic and the cells therewithin were more likely to be better producers (have better productivities).
  • FIG. 28 shows the time lapse records of the fluorescent signals of each pen over 15 minutes.
  • Each line in the figure represents the data of a single sequestration pen.
  • the figure indicates the dynamics of the change of pH value (acidification) of the medium disposed in each sequestration pen over time.
  • the traces of some pens exhibited quick acidification and then reached an early plateau level.
  • the traces of other sequestration pens exhibited more gradual acidification and eventually reached a plateau level, at a later time point.
  • the traces of some of the sequestration pens exhibited a flat change of pH value, suggesting the cells in those pens were not producers.
  • the methods are able to monitor the pH change within each sequestration pen and the information from such monitoring can be used for selecting cell clones that are likely to be better producers.
  • Citric acid stock solution (0.1 M) and sodium citrate solution (0.1 M) were prepared to prepare citrate buffer solutions of target pH of 3.0, 3.4, 3.6, 3.8, 4.0, 4.4, 4.6, 4.8, 5.0, 5.6, 5.8, and 6, respectively.
  • a pH-sensitive dye solution (LysoSensorTM Yellow/Blue DND-160, 5 uM, pH 5.6) was prepared for this experiment.
  • Cells (Saccharomyces cerevisiae) were obtained from storage and inoculated in batch media (BMGY medium) in a deep well plate at 30 degrees Celsius on a shaker plate until OD 0.6. Cells were then collected and kept on ice until import.
  • FIG. 30 shows a standard curve calculated from the measurement of this example.
  • Hydrogel composition and polymerization A hydrogel polymer solution comprising polyethylene glycol acrylamide (8-arm acrylamide-terminated PEG), MEHQ, and LAP was prepared and introduced into the flow region of the microfluidic device. The polymer solution was allowed to diffuse into the chamber for 10 minutes. Then, the flow region was flushed with fresh PBS to remove the polymer solution remaining in the flow region. The solidification of the polymer solution was activated in a selected area within the sequestration pen thereby forming an in situ-generated barrier as shown in FIG. 31A and FIG. 3 IB.
  • FIG. 31A illustrates a configuration of a microfluidic device used in this example.
  • the micro fluidic device comprises a microfluidic channel 3101 and a plurality of sequestration pen 3102 (only one of the sequestration pens is shown in this figure).
  • the sequestration pen 3102 has a proximal opening 3103 to the microfluidic channel 3101.
  • An in situ-generated barrier 3110 as formed within the sequestration pen 3102 to divide it into a culture area 3104 and an assay area 3105.
  • the assay area 3105 is an area close to a distal end of the chamber
  • the culture area 3104 is an area close to a proximal end of the chamber.
  • FIG. 3 IB shows a photo taken before cells were disposed.
  • An area of interest for the detection of the pH acidification assay can be within the assay area 3105, for example, the area 3131.
  • the area of interest can be within the culture area 3104 and within an area close to the proximal opening 3103, for example the area 3132.
  • area 3131 was chosen to be the area of interest.
  • Example 4 Assay For Detection of a Level of Dissolved Oxygen in a Fluid Located Within a Microfluidic Device
  • System and Microfluidic device The foregoing experiments were performed using an OptoSelectTM microfluidic (or nanofluidic) device manufactured by Berkeley Lights, Inc. and controlled by an optical instrument which was also manufactured by Berkeley Lights, Inc.
  • the instrument included: a mounting stage for the microfluidic device coupled to a temperature controller; a pump and fluid medium conditioning component; an optical train including a camera and a structured light source suitable for activating phototransistors within the microfluidic device; and software for controlling the instrument, including performing image analysis and automated detection and repositioning of micro -objects.
  • the OptoSelectTM device included a substrate configured with OptoElectroPositioning (OEPTM) technology, which provides a phototransistor-activated dielectrophoresis (DEP) force.
  • the device also included a plurality of microfluidic channels, each having a plurality of NanoPenTM chambers (or sequestration pens) fluidically connected thereto. The volume of each sequestration pen was around IxlO 6 cubic microns.
  • the microfluidic device included conditioned interior surfaces, which are described in U.S. Patent Application Publication No.
  • the reference curve was generated using at least five different oxygen concentration levels, ranging from 21% O2, e.g., Clean Dry Air (CD A) to 2.02% O2, using custom pre-mixed gas (Praxair). The five concentrations used were: CDA, 14.14%, 7.98%, 5.19%, and 2.02%. In this experiment, a sixth gas, N2 (e.g., 0% O2), was also used. Generally, however, the five-point reference curve from 21% O2 to 2.02% O2 provided sufficiently reproducible and representative reference curves for the Dissolved Oxygen assay. In some variations, a reference curve may be obtained using four, three or two O2 reference points and still provide robust detection of dissolved oxygen levels. In other variations, a reference curve may be obtained using at least six, seven, eight, nine, ten, or more, reference points.
  • the cycle of perfusion provides periods of perfusion punctuated by intervals of no flow. Images were obtained at one minute intervals during both portions of the perfusion cycle over a period of at least 5 min, with excitation at 455 nm. Images centered on an Area of Interest (AOI) centered mid-pen within the sequestration pens as well as a set of images centered within the channel were obtained at 625 nm with 50 msec illumination (15% power) using a custom bandpass filter combination.
  • AOI Area of Interest
  • a useful AOI for the reference curve generation and for the DO assay may be any region within the sequestration pen where medium transference is dominated by diffusion.
  • the values obtained were normalized against the data obtained from the 21% O2 (CD A) images.
  • the average normalized intensity across the microfluidic device in the channel region for each O2 concentration was plotted and is shown in FIG. 9A (flow) and FIG. 9B (no flow).
  • the respective average normalized intensity across all of the sequestration pens were plotted against each O2 concentration and is shown in FIG. 9C (flow) and FIG. 9D (no flow).
  • the sequestration pens were examined to identify each sequestration pen having no cells within the AOI.
  • the optical density (OD) of each identified sequestration pen was obtained under brightfield, and quantified by comparison to the OD of that sequestration pen when empty under brightfield.
  • Fluorescence images at 625nm were obtained across the microfluidic device, in the channel region and within each identified sequestration pen at the mid-pen AOI, either at a single timepoint or over a ten minute period, using 50 msec illumination, 15% power as above.
  • the raw fluorescence values were averaged if more than one image per AOI was taken. The raw fluorescence was normalized against the fluorescence observed in that pen prior to cell importation at 21% O2 concentration to remove pen to pen aberrations.
  • the normalized (optionally averaged) fluorescence value was finally correlated to the dissolved O2 level from the reference curve generated above, which can be represented as O2 saturation percentage.
  • O2 saturation percentage When each correlated dissolved O2 level of the respective sequestration pen was plotted against the OD obtained for that pen (correlating with Biomass of the sequestration pen), the relationship was observed as shown in FIG. 8, where values of biomass along the x-axis are binned for easier review. The relationship is roughly linear, but there was clonal variation observable. For a given biomass point, a range of dissolved oxygen concentrations for a set of sequestration pens is shown in FIG. 8, extending in the y-space.
  • Colonies in the sequestration pens having a higher concentration of O2 are consuming less oxygen than the sequestration pens having an equivalent biomass (OD) where lower concentration of O2 was imaged, e.g., more fluorescence signal).
  • the more fluorescent, less O2 saturated, faster O2 consuming colonies can be selected for further analysis, as the sequestration pen identification was maintained throughout the assay.
  • the cells from preferred sequestration pens may be either the colonies having the highest biomass or may be pens where the cells have the highest O2 consumption per mass unit where the colony may not have the most number of cells but the individual cells are consuming oxygen at the highest rate per cell.
  • FIG. 13 illustrates exemplary DO standard curves generated by the process 1200. As shown in FIG. 13, the dynamic range included air saturation values from 9.5% air saturation to 100% air saturation. The normalized RTDP was converted to a DO value and achieved a coefficient of variation (CV) of 1.4%.
  • CV coefficient of variation
  • RTDP concentration testing The RTDP concentration was decreased from a standard value of 2 mg/mL to determine whether RTDP concentration affected DO assay performance.
  • a RTDP concentration of 0.4 mg/mL was prepared and testing was conducted at nominal O2 concentrations of 21%, 14%, and 2%.
  • the fluorescence exposure was set to 150 ms with 15% illumination.
  • FIG. 17 shows the variability of the normalized fluorescence intensity at a 0.4 mg/mL RTDP concentration. As shown in FIG. 17, the variability of the normalized intensity for the 0.4 mg/mL RTDP concentration was within tolerable limits and within the same range as a 2 mg/mL RTDP concentration.
  • Torr Seal® chip sealing Mitigation of the DO edge effect was tested using Torr Seal® epoxy sealing, as described herein. The results of an unsealed microfluidic device and a microfluidic device sealed using Torr Seal® epoxy were compared.
  • the RTDP concentration was 0.4 mg/mL and a fluorescence exposure of 150 ms with 15% illumination was utilized.
  • the nominal O2 concentration was varied between 21% and 2%.
  • the sparging and gas flush time was varied between 15 minutes and 60 minutes.
  • a first perfusion of RTDP was performed at 3 uL/s and the time of the first perfusion was varied between 5 minutes and 25 minutes.
  • a second perfusion of RTDP was performed at 4 uL/s and the time of the second perfusion was 5 minutes.
  • Example 8 Parylene sealing of microfluidic devices
  • Parylene chip sealing Mitigation of the DO edge effect was tested using Parylene sealing, as described herein. The results of an unsealed microfluidic device surrounded by an O2 supply and a microfluidic device sealed using Parylene were compared.
  • the RTDP concentration was 0.4 mg/mL and a fluorescence exposure of 150 ms with 15% illumination was utilized.
  • the nominal O2 concentration was varied between 21% and 2%.
  • the sparging and gas flush time was varied between 15 minutes and 60 minutes.
  • a first perfusion of RTDP was performed at 3 uL/s and the time of the first perfusion was varied between 15 minutes and 25 minutes.
  • a second perfusion of RTDP was performed at 4 uL/s and the time of the second perfusion was 10 minutes.
  • FIGs. 18A-18B shows the improvement in dissolved oxygen uniformity achieved by sealing the microfluidic chip from external gas exchange using Parylene.
  • the x-axis shows distance along the channel (from inlet to outlet) expressed as a 100% of the total length of the channel from inlet to outlet.
  • the y-axis is an average normalized intensity of fluorescence signal that correlates with dissolved oxygen level. Decreased signal correlates with increased oxygen.
  • Along the top axis is the uniformity of signal for 21% oxygen (ambient air) and 2% oxygen in both control chips (no sealing) and Parylene- sealed chips, with FIGs. 18A and 18B being the measurement in the pen (Assay Area, FIG. 18 A) and channel (ChannelArea, FIG.
  • FIGs. 19A-19B shows the different performance levels of various sealing techniques in limiting external gas exchange to improve dissolved oxygen uniformity.
  • the x-axis shows distance along the channel (from inlet to outlet) expressed as a 100% of the total length of the channel from inlet to outlet.
  • the y-axis is an average normalized intensity of fluorescence signal that correlates with dissolved oxygen level. Decreased signal correlates with increased oxygen.
  • Along the top axis is the uniformity of signal for 21% oxygen (ambient air) and 2% oxygen in 3 different sealing conditions: A-sealed (sealed using Torr Seal®), gas bath, and P- sealed (sealed using Parylene), with FIGs.
  • FIGs. 20A-20B show an example of how the above-described non-uniformities in external gas exchange impact the dissolved oxygen signal as observed over the whole chip.
  • the lower legends provide an intensity map from white to black, where white is the lowest average normalized intensity.
  • decreased signal correlates with increased oxygen (i.e. the whiter bands are gas exchange with the external channels).
  • A-Sealed chips have stark white bands associated with the channels that face the external surface when turning, pick up oxygen and decrease the assay signal. Gas-bath non-uniformities were less severe and absent from the P-sealed chip.
  • Pen-level DO measurements Growth medium with oxygen- sensitive dye RTDP was flushed through the chip at a high flow rate while fluorescence images were captured. The constant flow allowed the channel area to act as an oxygen source. Live cell colonies in the pen bottoms consumed oxygen at a rate determined by the number of cells and the cells’ biological characteristics, thereby acting as an oxygen sink. The difference in oxygen consumption between sink and source creates a steady-state fluorescence gradient in the oxygen-sensitive dye between the cell colony and the top of the pen meeting the channel.
  • FIG. 21A shows an exemplary brightfield image of cells in sequestration pens.
  • the boundary of the cell colony was determined by automated image analysis of the brightfield image.
  • the fluorescence intensity of the RTDP signal in the DO consumption assay was quantified in a region of interest dynamically defined by the boundary of the cell colony, allowing quantification of the differential fluorescence between the cell colony and the top of the pen.
  • the magnitude of this differential fluorescence signal was normalized against the spatial variation of illumination intensity in the brightfield image, the size of the cell colony, and the autofluorescence signal from the colony, giving a measure of the characteristic oxygen consumption of the strain in that pen.
  • the RTDP was dissolved in the same media that was used for the induction culture period of the workflow described herein. Because the DO assay can be repeated, the cells were altematingly cultured in the regular induction media and the RTDP media for the assay. These cells had been culturing for approximately 30 hours total on chip since they were loaded.
  • FIG. 2 IB shows an exemplary fluorescence image of dissolved oxygen in the sequestration pens. As shown in FIG. 21B, sequestration pens containing cells produced fluorescence signals in proportion to the number of cells contained therein, while sequestration pens containing no cells did not produce fluorescence signals.
  • the embodiments described herein can be practiced with other computer system configurations including hand-held devices, microprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers and the like.
  • the embodiments can also be practiced in distributing computing environments where tasks are performed by remote processing devices that are linked through a network.
  • any of the operations that form part of the embodiments described herein are useful machine operations.
  • the embodiments, described herein also relate to a device or an apparatus for performing these operations.
  • the systems and methods described herein can be specially constructed for the required purposes or it may be a general purpose computer selectively activated or configured by a computer program stored in the computer.
  • various general purpose machines may be used with computer programs written in accordance with the teachings herein, or it may be more convenient to construct a more specialized apparatus to perform the required operations.
  • Certain embodiments can also be embodied as computer readable code on a computer readable medium.
  • the computer readable medium is any data storage device that can store data, which can thereafter be read by a computer system. Examples of the computer readable medium include hard drives, network attached storage (NAS), read-only memory, randomaccess memory, CD-ROMs, CD-Rs, CD-RWs, magnetic tapes, and other optical, FLASH memory and non-optical data storage devices.
  • the computer readable medium can also be distributed over a network coupled computer systems so that the computer readable code is stored and executed in a distributed fashion.
  • Embodiment 1 A method of determining a level of oxygen in a medium disposed within a microfluidic device comprising a flow region and one or more chambers fluidically coupled to the flowing region, the method comprising: flowing a fluidic medium containing a dye and a supplied partial pressure of oxygen into the microfluidic device for a period of time, wherein fluorescence emitted by the dye changes based on availability of oxygen proximate to the dye; taking a fluorescence image of an area of interest (AOI) within the flow region or one or more of the chambers; and correlating fluorescence detected in the fluorescence image of the AOI with a reference to determine an observed level (e.g., a partial pressure) of oxygen in the AOI.
  • AOI area of interest
  • Embodiment 2 The method of embodiment 1 , further comprising : determining a level of oxygen consumption by a biological micro-object or a population of biological microobjects (e.g., a clonal population) disposed within one of the one or more chambers.
  • a biological micro-object or a population of biological microobjects e.g., a clonal population
  • Embodiment 3 The method of embodiment 2, further comprising: comparing the determined level of oxygen consumption with a threshold value; and selecting the biological micro-object or the population of biological micro-objects (e.g., a clonal population) if the determined level of oxygen consumption is above the threshold value.
  • Embodiment 4 The method of embodiment 2 or 3, further comprising: forecasting a level of productivity of an expanded population of biological micro-objects expanded from the biological micro-object or the population of biological micro-objects based at least in part upon the determined level of oxygen consumption.
  • Embodiment 5 The method of embodiment 4, further comprising: determining a number of biological micro-objects present in the chamber, wherein the forecast level of productivity is based at least in part on the determined number of biological micro-objects in the chamber.
  • Embodiment 6 The method of embodiment 4 or 5, further comprising: comparing the forecast level of productivity with a threshold value; and selecting the biological micro-object or the population of biological micro-objects (e.g., a clonal population) if the forecast level of productivity is above the threshold value.
  • Embodiment 7 The method of embodiment 3 or 6, wherein the selected biological micro-object or the population of biological micro-objects is removed from the microfluidic device (e.g., exported) and, optionally, cultured so as to produce an expanded population of biological micro-objects.
  • the microfluidic device e.g., exported
  • Embodiment 8 The method of embodiment 7, wherein the expanded population of biological micro-objects is expanded at least partially following export from the microfluidic device (e.g., in a macro-scale culture device, which can be any culture device having a volume that can be used for cell culture of at least 1 mL).
  • a macro-scale culture device which can be any culture device having a volume that can be used for cell culture of at least 1 mL.
  • Embodiment 9 A method of determining a level of oxygen consumption by a biological micro-object or a population of biological micro-objects, the method comprising: optionally disposing the biological micro-object or the clonal population of biological microobjects in a chamber of a microfluidic device comprising a flow region, wherein the chamber is fluidically connected to the flow region; flowing a fluidic medium containing a dye and a supplied partial pressure of oxygen into the microfluidic device for a period of time, wherein fluorescence emitted by the dye changes based on availability of oxygen proximate to the dye; taking a fluorescence image of an area of interest within the flow region and/or the chamber; correlating fluorescence detected in the fluorescence image of the area of interest with a reference to determine an observed level (e.g., partial pressure) of oxygen in the area of interest; and determining the level of oxygen consumption by the biological micro-object or the population of biological micro-objects disposed within the chamber.
  • Embodiment 10 A method of selecting a biological micro-object or a population of biological micro-objects, the method comprising: optionally disposing the biological microobject or the clonal population of biological micro-objects in a chamber of a microfluidic device comprising a flow region, wherein the chamber is fluidically connected to the flow region; flowing a fluidic medium containing a dye and a supplied partial pressure of oxygen into the microfluidic device for a period of time, wherein fluorescence emitted by the dye changes based on availability of oxygen proximate to the dye; taking a fluorescence image of an area of interest within the flow region and/or the chamber; correlating fluorescence detected in the fluorescence image of the area of interest with a reference to determine an observed level (e.g., partial pressure) of oxygen in the area of interest; determining the level of oxygen consumption by the biological micro-object or the population of biological micro-objects disposed within the chamber; and selecting the biological micro-object or the population of biological micro-object
  • Embodiment 11 A method of forecasting a level of productivity of a population of biological micro-objects expanded from a biological micro-object or a clonal population of biological micro-objects, the method comprising: optionally disposing a biological microobject or a clonal population of biological micro-objects in a chamber of a microfluidic device comprising a flow region, wherein the chamber is fluidically connected to the flow region; flowing a fluidic medium containing a dye and a supplied partial pressure of oxygen into the microfluidic device for a period of time, wherein fluorescence emitted by the dye changes based on availability of oxygen proximate to the dye; taking a fluorescence image of an area of interest within the flow region and/or the chamber; correlating fluorescence detected in the fluorescence image of the area of interest with a reference to determine an observed level (e.g., partial pressure) of oxygen in the area of interest; determining a level of oxygen consumption by the biological micro-object or the population of biological
  • Embodiment 12 The method of embodiment 10 or 11, wherein the microfluidic device comprises a plurality of chambers, each fluidically connected to the flow region, wherein there is a plurality of biological micro-objects and/or populations of biological microobjects, each one of the biological micro-objects and/or populations of biological micro-objects disposed within a corresponding chamber of the plurality of chambers, and wherein selecting the biological micro-object or the population of biological micro-objects comprises selecting one or more of the plurality of biological micro-objects and/or populations of biological microobjects.
  • Embodiment 13 The method of any one of embodiments 1-12, wherein the dye comprises a soluble and diffusible dye.
  • Embodiment 14 The method of any one of embodiments 1-13, wherein the dye comprises a ruthenium complex.
  • Embodiment 15 The method of any one of embodiments 1-14, wherein the fluorescence emitted by the dye is quenched when the dye is in proximity to oxygen and fluoresces when the dye is not in proximity to oxygen.
  • Embodiment 16 The method of any one of embodiments 1-15, wherein the level of oxygen consumption corresponds to a decrease in the observed partial pressure of the oxygen compared to the supplied partial pressure.
  • Embodiment 17 The method of any one of embodiments 1-16, wherein the fluidic medium is flowed at a flow rate ranging between 0.1 microliter/s and 10 microliters/s.
  • Embodiment 18 The method of any one of embodiments 1-17, wherein the biological micro-object or population of biological micro-objects in the chamber of the microfluidic device is cultured under aerobic conditions comprising flowing a fluidic medium through the fluidic region, wherein the fluidic medium comprises at least a minimum supplied partial pressure of oxygen of 0.04 bar.
  • Embodiment 19 The method of any one of embodiments 1-18, wherein the AOI is disposed within the chamber at a location wherein transference of components of the fluidic medium flowing in the flow region (e.g., a channel to which the chamber is fluidically connected) is dominated by diffusion.
  • the AOI is disposed within the chamber at a location wherein transference of components of the fluidic medium flowing in the flow region (e.g., a channel to which the chamber is fluidically connected) is dominated by diffusion.
  • Embodiment 20 The method of any one of embodiments 1-19, wherein the AOI is disposed in the flow region (e.g., a channel), at a position proximal to an opening from the chamber to the flow region.
  • the flow region e.g., a channel
  • Embodiment 21 The method of any one of embodiments 1-20, wherein the AOI contains no biological micro-objects.
  • Embodiment 22 The method of any one of embodiments 1-21, wherein the fluidic medium comprises a liquid medium, a gaseous medium, or a mixture thereof.
  • Embodiment 23 The method of any one of embodiments 1-22, wherein the flowing the fluidic medium containing the dye and the supplied partial pressure of oxygen into the microfluidic device comprises alternately flowing a liquid medium into the microfluidic device and flowing a gaseous medium comprising the supplied partial pressure of oxygen into the microfluidic device.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the medium comprises a liquid medium saturated with the supplied partial pressure of the oxygen.
  • Embodiment 25 The method of any one of embodiments 1-24, wherein the correlating the fluorescence of the fluorescence image of the AOI to a reference to determine the observed partial pressure of the oxygen at the AOI comprises identifying an intensity of the fluorescence on a reference curve correlating fluorescence intensities of the dye with supplied partial pressures of the oxygen.
  • Embodiment 26 The method of any one of embodiments 1-25, wherein the method further comprises constructing a reference curve correlating an observed intensity of fluorescence of the dye with a supplied partial pressure of oxygen.
  • Embodiment 27 The method of embodiment 26, wherein the constructing the reference curve comprises: flowing a liquid medium through the channel of the microfluidic device for a first reference period of time, wherein the liquid medium comprises the dye and a first supplied partial pressure of the oxygen; detecting a first fluorescence intensity of the dye within a selected region of the microfluidic device; flowing a second liquid medium through the channel of the microfluidic device for a second reference period of time, wherein the second liquid medium comprises the dye and a second supplied partial pressure of the oxygen; detecting a second fluorescence intensity of the dye within the selected region of the microfluidic device; and correlating each of the first and the second fluorescence intensities with the first and second supplied partial pressures of the oxygen, respectively.
  • Embodiment 28 The method of embodiment 26, wherein the constructing the reference curve further comprises flowing liquid media comprising the dye and successive supplied partial pressures of the oxygen at successive points in time; detecting successive fluorescence intensities of the dye at the successive points in time; and correlating each of the fluorescence intensities with a supplied partial pressure of the oxygen.
  • Embodiment 29 The method of any one of embodiments 26-28, wherein the microfluidic device does not contain any biological micro-objects while constructing the reference curve.
  • Embodiment 30 The method of any one of embodiments 26-29, wherein the selected supplied partial pressure of oxygen is from about 0.02 bar to about 0.21 bar.
  • Embodiment 31 The method of any one of embodiments 26-30, wherein the method further comprises detecting fluorescence intensities associated with at least three, four, five, or more supplied partial pressures of the oxygen.
  • Embodiment 32 The method of any one of embodiments 1- 31, wherein the fluorescence image is taken under a perfusion condition.
  • Embodiment 33 The method of embodiment 32, wherein the perfusion condition comprises a constant flow, a steady state flow, a pre-programmed flow with one or more defined flow rates, or zero flow after equilibrium.
  • Embodiment 34 The method of any one of embodiments 1-33, wherein the microfluidic device comprises a plurality of chambers, and wherein the method further comprises: introducing the population of biological micro-objects into the plurality of chambers.
  • Embodiment 35 The method of embodiment 34, wherein the flow region of the microfluidic device comprises a plurality of channels, and wherein the method further comprises: introducing the population of biological micro-objects into the plurality of channels.
  • Embodiment 36 The method of any one of embodiments 1-35, wherein the flowing the fluidic medium and the taking the fluorescence image are performed at a selected temperature.
  • Embodiment 37 The method of embodiment 36, wherein the temperature is from about 20°C to about 40°C.
  • Embodiment 38 The method of embodiment 36 or 37, wherein the temperature is from about 28°C to about 30°C.
  • Embodiment 39 The method of any one of embodiments 1-38, wherein the flowing the fluidic medium and the taking the fluorescence image is performed at a selected pH.
  • Embodiment 40 The method of embodiment 39, wherein the pH is from about 3.0 to about 9.0.
  • Embodiment 41 The method of any one of embodiments 1-40, wherein the method further comprises taking a plurality of fluorescence images at a plurality of timestamps and correlating a respective fluorescence of each fluorescence image to determine a respective observed partial pressure of the oxygen in the AOI at the respective timestamp.
  • Embodiment 42 The method of any one of embodiments 1-41, wherein the method further comprises taking a plurality of fluorescence images at a plurality of points within the AOI.
  • Embodiment 43 The method of any one of embodiments 1-42, wherein the chamber comprises a sequestration pen, wherein the sequestration comprises an isolation region and a connection region, and wherein the connection region has a proximal opening to the channel and a distal opening to the isolation region.
  • Embodiment 44 The method of embodiment 43, wherein the isolation region comprises a single opening to the connection region.
  • Embodiment 45 The method of embodiment 43 or 44, wherein the population of biological micro-objects is disposed within the isolation region of the sequestration pen.
  • Embodiment 46 The method of any one of embodiments 43-45, wherein the method further comprises taking a plurality of fluorescence images at a plurality of points extending from the isolation region of the sequestration pen along an axis of diffusion towards the channel.
  • Embodiment 47 The method of any one of embodiments 43-46, wherein the AOI comprises at least part of the connection region.
  • Embodiment 48 The method of any one of embodiments 1-47, wherein the microfluidic device comprises a plurality of exterior surfaces, wherein each of the plurality of exterior surface is oxygen-impermeable.
  • Embodiment 49 The method of any one of embodiments 1-48, wherein the microfluidic device comprises a plurality of exterior surfaces and wherein at least a portion of one or more exterior surfaces of the plurality is coated with an oxygen-impermeable film.
  • Embodiment 50 The method of embodiment 49, wherein the oxygen-impermeable film has an oxygen permeability of 20 cm 3 mm-m’ 2 day’ 1 atm’ 1 or less.
  • Embodiment 51 The method of embodiment 49 or 50, wherein the oxygen- impermeable film has an oxygen permeability of between 1 cm 3 mm-m’ 2 day’ 1 atm’ 1 and 20 cm 3 mnr m’ 2 day ’ 1 atm’ 1 .
  • Embodiment 52 The method of any one of embodiments 49-51 , wherein the oxygen- impermeable film comprises Parylene N, Parylene C, Parylene D, Parylene HT, epoxy, Torr- Seal epoxy, or any combination thereof.
  • Embodiment 53 The method of any one of embodiments 49-52, wherein the oxygen- impermeable film has a thickness of at least 1 nanometer (nm).
  • Embodiment 54 The method of any one of embodiments 49-53, wherein the oxygen- impermeable film has a thickness of at most 10 micrometers (pm).
  • Embodiment 55 The method of any one of embodiments 1-54, wherein the microfluidic device comprises a plurality of exterior surfaces and wherein the method further comprises delivering a supplied partial pressure of oxygen to at least a portion of one or more exterior surfaces of the plurality.
  • Embodiment 56 The method of embodiment 55, wherein the supplied partial pressure of oxygen is delivered to at least the portion of the one or more exterior surfaces by one or more tubes placed in proximity to the one or more exterior surfaces, the one or more tubes comprising one or more holes (or lumens) configured to allow the supplied partial pressure of oxygen to flow therethrough.
  • Embodiment 57 The method of embodiment 55 or embodiment 56, wherein the supplied partial pressure of oxygen is delivered to the at least the portion of the one or more exterior surfaces by an oxygen bath surrounding the microfluidic device.
  • Embodiment 58 A system comprising: a microfluidic device comprising: a flow region (e.g. comprising a channel); a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region (or channel); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen.
  • a flow region e.g. comprising a channel
  • a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region (or channel); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen.
  • Embodiment 59 A system comprising: a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region; and a plurality of exterior surfaces, wherein at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film.
  • a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region; and a plurality of exterior surfaces, wherein at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film.
  • Embodiment 60 The system of embodiment 59, wherein the oxygen-impermeable film has an oxygen permeability of at least 1 cm 3 mm-m’ 2 day 1 atm 1 .
  • Embodiment 61 The system of embodiment 59 or 60, wherein the oxygen- impermeable film has an oxygen permeability of at most 20 cm 3 mm-m’ 2 day 1 atm 1 .
  • Embodiment 62 The system of any one of embodiments 59-61, wherein the oxygen- impermeable film comprises Parylene N, Parylene C, Parylene D, Parylene HT, epoxy, Torr- Seal epoxy, or any combination thereof.
  • Embodiment 63 The system of any one of embodiments 59-62, wherein the oxygen- impermeable film has a thickness of at least 1 nanometer (nm).
  • Embodiment 64 The system of any one of embodiments 59-63, wherein the oxygen- impermeable film has a thickness of at most 10 micrometers (pm).
  • Embodiment 65 The system of any one of embodiments 59-64, wherein the microfluidic device comprises a plurality of chambers, each chamber of the plurality configured to receive a population of biological micro-objects therein.
  • Embodiment 66 The system of any one of embodiments 59-65, wherein the flow region of the microfluidic device comprises a plurality of channels.
  • Embodiment 67 The system of any one of embodiments 59-66, wherein the chamber comprises a sequestration pen, wherein the sequestration pen comprises an isolation region and a connection region, and wherein the connection region has a proximal opening to the flow region (or a channel of the flow region) and a distal opening to the isolation region.
  • Embodiment 68 The system of embodiment 67, wherein the isolation region comprises a single opening to the connection region.
  • Embodiment 69 The system of embodiment 67 or 68, wherein the isolation region of the sequestration pen is configured to receive the population of biological micro-objects therein.
  • Embodiment 70 A system comprising: an oxygen delivery module; a nest comprising a support structure configured to support a microfluidic device in proximity to the oxygen delivery module; a gas source in fluidic communication with the oxygen delivery module; and a controller configured to control a flow of gas from the gas source to the oxygen delivery module.
  • Embodiment 71 The system of embodiment 70, wherein the oxygen delivery module comprises one or more tubes, the one or more tubes comprising one or more holes configured to allow a supplied partial pressure of oxygen to flow therethrough.
  • Embodiment 72 The system of embodiment 70 or 71, wherein the oxygen delivery module comprises an oxygen bath surrounding the microfluidic device.
  • Embodiment 73 The system of any one of embodiments 70-72, wherein the nest is configured to provide a fluidic connection between the system and said microfluidic device.
  • Embodiment 74 The system of any one of embodiments 70-73, wherein the nest further comprises a socket configured to provide an electrical interface between the system and said microfluidic device.
  • Embodiment 75 The system of any one of embodiments 70-74, further comprising a fluidic medium source comprising a sparging component in fluidic communication with the gas source.
  • Embodiment 76 The system of any one of embodiments 70-75, wherein the system further comprises a structured light source positioned to direct structured light to said microfluidic device and configured to thereby activate phototransistors within said microfluidic device.
  • Embodiment 77 The system of any one of embodiments 70-76, further comprising a microfluidic device disposed on the support structure, the microfluidic device comprising a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region.
  • a microfluidic device disposed on the support structure, the microfluidic device comprising a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological micro-objects therein, wherein the chamber opens to the flow region.
  • Embodiment 78 The system of any one of embodiments 70-77, wherein the microfluidic device comprises a plurality of chambers, each chamber of the plurality configured to receive a population of biological micro-objects therein.
  • Embodiment 79 The system of any one of embodiments 70-78, wherein the flow region of the microfluidic device comprises a plurality of channels.
  • Embodiment 80 The system of any one of embodiments 70-79, wherein the chamber comprises a sequestration pen, wherein the sequestration comprises an isolation region and a connection region, and wherein the connection region has a proximal opening to the flow region (or a channel of the flow region) and a distal opening to the isolation region.
  • Embodiment 81 The system of embodiment 80, wherein the isolation region comprises a single opening to the connection region.
  • Embodiment 82 A kit comprising: a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological microobjects therein, wherein the chamber opens to the flow region (or a channel of the flow region); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen or wherein at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film; and a buffer.
  • a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological microobjects therein, wherein the chamber opens to the flow region (or a channel of the flow region); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen or wherein at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film; and
  • Embodiment 83 The kit of embodiment 82, further comprising a fluidic medium containing a dye configured to emit a fluorescence signal that changes based on availability of oxygen proximate to the dye.
  • Embodiment 84 The kit of embodiment 83, wherein the dye comprises a soluble and diffusible dye.
  • Embodiment 85 The kit of embodiment 83 or 84, wherein the dye comprises a ruthenium complex.
  • Embodiment 86 A kit comprising: a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological microobjects therein, wherein the chamber opens to the flow region (or a channel of the flow region); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen or wherein at least a portion of one or more exterior surfaces of the plurality are coated with an oxygen-impermeable film; and a fluidic medium containing a dye configured to emit a fluorescence signal that changes based on availability of oxygen proximate to the dye.
  • a microfluidic device comprising: a flow region (e.g., comprising a channel); a chamber configured to receive a population of biological microobjects therein, wherein the chamber opens to the flow region (or a channel of the flow region); and a plurality of exterior surfaces, wherein each exterior surface of the plurality is impermeable to oxygen or
  • Embodiment 87 The kit of embodiment 86, wherein the dye comprises a soluble and diffusible dye.
  • Embodiment 88 The kit of embodiment 86 or 87, wherein the dye comprises a ruthenium complex.
  • Embodiment 89 The kit of any one of embodiments 86-88, further comprising a buffer.
  • Embodiment 90 A method of monitoring a local pH of a medium spatially distributed within a microfluidic device comprising an enclosure, wherein the enclosure comprises a flow region and a chamber having a proximal opening fluidically connecting the chamber to the flow region, the method comprising: introducing a pH-sensitive molecule into the microfluidic device; and detecting a signal associated with the pH-sensitive molecule in an area of interest within the enclosure.
  • Embodiment 91 The method of embodiment 90, wherein the area of interest is within the chamber.
  • Embodiment 92 The method of embodiment 90 or embodiment 91, wherein the area of interest comprises about 5% to 100% of the cross-sectional area of the chamber.
  • Embodiment 93 The method of any one of embodiments 90 to 92, the area of interest does not comprise a wall of the chamber.
  • Embodiment 94 The method of any one of embodiments 90 to 93, further comprising disposing a micro-object into the chamber.
  • Embodiment 95 The method of embodiment 94, wherein the micro-object is a biological micro-object.
  • Embodiment 96 The method of embodiment 94 or embodiment 95, wherein the area of interest is substantially free of the micro-object.
  • Embodiment 97 The method of embodiment 94 or embodiment 95, wherein the area of interest has the micro-object disposed therewithin.
  • Embodiment 98 The method of any one of embodiments 90 to 97, further comprising introducing an in situ-generated barrier in the chamber, wherein the in situ-generated barrier defines an assay area and a culture area within the chamber.
  • Embodiment 99 The method of embodiment 98, wherein the area of interest is within the assay area.
  • Embodiment 100 The method of embodiment 99, wherein the area of interest comprises about 30% to 100% of the assay area.
  • Embodiment 101 The method of any one of embodiments 98 to 100, wherein the area of interest does not comprise the in situ-generated barrier.
  • Embodiment 102 The method of any one of embodiments 98 to 101, wherein the micro-object is within the culture area.
  • Embodiment 103 The method of any one of embodiments 98 to 102, wherein the in situ-generated barrier is formed in a central portion of the chamber.
  • Embodiment 104 The method of any one of embodiments 98 to 103, wherein the assay area is located proximal to one side of the in situ-generated barrier, between the in-situ generated barrier and a distal end of the chamber, and the culture area is located proximal to another side of the in situ-generated barrier, between the in-situ generated barrier and the proximal opening of the chamber.
  • Embodiment 105 The method of embodiment 104, wherein the in situ-generated barrier separates the culture area from the distal end of the chamber.
  • Embodiment 106 The method of any one of embodiments 98 to 105, further comprising introducing a polymer solution into the flow region of the microfluidic device; allowing the polymer solution to diffuse into the chamber; and solidifying the polymer solution thereby forming the in situ-generated barrier within the chamber.
  • Embodiment 107 The method of embodiment 106, wherein polymer solution is soluble in a fluidic medium within the microfluidic device.
  • Embodiment 108 The method of any one of embodiments 98 to 107, wherein the in situ-generated barrier has a porosity restricting passage of the micro-object.
  • Embodiment 109 The method of any one of embodiments 98 to 108, wherein the enclosure is configured to receive a fluidic medium, and the in situ-generated barrier is porous to the fluidic medium.
  • Embodiment 110 The method of any one of embodiments 90 to 109, wherein the microfluidic device comprises a three-layer structure comprising a cover, a microfluidic circuit structure, and a substrate, and the chamber comprises a height defined as a distance from a bottom of the cover to a surface of the substrate.
  • Embodiment 111 The method of embodiment 110, wherein the area of interest is set at a depth of about 30% to 70% of the height of the chamber.
  • Embodiment 112. The method of any one of embodiments 90 to 111, further comprising: introducing a water immiscible fluidic medium into the flow region to fill a portion of the flow region adjacent to the chamber.
  • Embodiment 113 The method of embodiment 112, wherein the portion of the flow region adjacent to the chamber is adjacent to the proximal opening of the chamber.
  • Embodiment 114 The method of embodiment 112 or embodiment 113, wherein the water immiscible fluidic medium seals the proximal opening of the chamber.
  • Embodiment 115 The method of any one of embodiments 90 to 114, further comprising: introducing a gaseous fluid into the microfluidic circuit to fill a portion of the flow region adjacent to the chamber with the gaseous fluid.
  • Embodiment 116 The method of embodiment 115, wherein the portion of the flow region adjacent to the chamber is adjacent to the proximal opening of the chamber.
  • Embodiment 117 The method of embodiment 115 or embodiment 116, wherein the gaseous fluid seals the proximal opening of the chamber.
  • Embodiment 118 The method of any one of embodiments 90 to 117, wherein detecting the signal comprises: obtaining an image of the area of the interest and determining an intensity of the signal associated with the pH-sensitive molecule in the area of interest from the image.
  • Embodiment 119 The method of any one of embodiments 90 to 118, further comprising normalizing the detected signal associated with the pH-sensitive molecule with a reference signal.
  • Embodiment 120 The method of any one of embodiments 90 to 119, wherein the pH-sensitive molecule is a fluorescent dye.
  • Embodiment 121 The method of any one of embodiments 90 to 120, wherein the chamber comprises an isolation region and a connection region fluidically connecting the isolation region to the flow region; and wherein the connection region comprises the proximal opening to the flow region and a distal opening to the isolation region.
  • Embodiment 122 The method of embodiment 121, wherein the area of interest is within the isolation region.
  • Embodiment 123 The method of embodiment 121, wherein the area of interest is within the connection region.
  • Embodiment 124 The method of any one of embodiments 90 to 123, wherein the pH-sensitive molecule is an acidotropic dye.
  • Embodiment 125 The method of any one of embodiments 90 to 124, wherein introducing the pH-sensitive molecule into microfluidic device comprises: introducing the pH- sensitive molecule into the flow region and allowing the pH-sensitive molecule to diffuse into the chamber.
  • Embodiment 126 The method of any one of embodiments 90 to 125, wherein introducing the pH-sensitive molecule into microfluidic device comprises: introducing a fluidic medium comprising the pH-sensitive molecule into the microfluidic device.
  • Embodiment 127 The method of embodiment 126, wherein the fluidic medium comprising the pH-sensitive molecule is non-buffered.
  • Embodiment 128 The method of embodiment 126 or embodiment 127, wherein introducing a fluidic medium comprising the pH-sensitive molecule comprises perfusing the fluidic medium comprising the pH-sensitive molecule and allowing the pH-sensitive molecule to diffuse into the chamber.
  • Embodiment 129 The method of embodiment 128, further comprising stopping the perfusion of fluidic medium comprising the pH-sensitive molecule while detecting the signal associated with the pH-sensitive molecule.
  • Embodiment 130 The method of any one of embodiments 90 to 129, wherein detecting a signal comprises: detecting a first signal associated with the pH-sensitive molecule in the area of interest at a first time point; and detecting a second signal associated with the pH- sensitive molecule in the area of interest at a second time point.
  • Embodiment 131 The method of embodiment 130, further comprising comparing the first signal with the second signal.
  • Embodiment 132 The method of any one of embodiments 94 to 131, wherein the chamber is a first chamber, and the microfluidic device further comprises a second chamber fluidically connected to the flow region, and introducing a pH-sensitive molecule into the microfluidic device comprises allowing the pH-sensitive molecule to diffuse into both the first chamber and the second chamber.
  • Embodiment 133 The method of embodiment 132, wherein detecting the signal associated with the pH-sensitive molecule in the area of interest comprises detecting a first signal associated with the pH-sensitive molecule within the first chamber and a second signal associated with the pH-sensitive molecule within the second chamber.
  • Embodiment 134 The method of embodiment 132, wherein the micro-object is a first micro-object, and wherein disposing the micro-object in the chamber comprises disposing the first micro-object in the first chamber; wherein the method further comprises disposing a second micro-object into the second chamber; and wherein detecting the signal associated with the pH-sensitive molecule in the area of interest comprises detecting a first signal associated with the pH-sensitive molecule within the first chamber and detecting a second signal associated with the pH-sensitive molecule within the second chamber.
  • Embodiment 135. The method of embodiment 134, further comprising selecting a desired micro-object from the first micro-object, the second micro-object or both by comparing the first signal with the second signal or by comparing the first signal and/or the second signal with a pre-determined threshold value.
  • Embodiment 136 The method of embodiment 135, further comprising exporting the desired micro-object out from the microfluidic device.
  • Embodiment 137 The method of any one of embodiments 90 to 136, further comprising determining a level of oxygen in the microfluidic device.
  • Embodiment 138 The method of embodiment 137, wherein determining the level of oxygen comprises: introducing an oxygen-sensitive molecule into the micro fluidic device, and detecting a signal associated with the oxygen- sensitive molecule in a second area of interest.
  • Embodiment 139 The method of embodiment 138, wherein introducing the oxygensensitive molecule comprises introducing a fluidic medium comprising the oxygen- sensitive molecule into the flow region and allowing the oxygen-sensitive molecule to diffuse into the chamber.
  • Embodiment 140 The method of embodiment 139, wherein the fluidic medium comprising the oxygen-sensitive molecule comprises a supplied partial pressure of oxygen.
  • Embodiment 141 The method of any one of embodiments 138 to 140, wherein detecting the signal associated the oxygen- sensitive molecule comprises obtaining an image of the second area of the interest, and determining an intensity of the signal associated with the oxygen-sensitive molecule.
  • Embodiment 142 The method of any one of embodiments 138 to 141, further comprising normalizing the detected signal associated with the oxygen- sensitive molecule with a reference signal.
  • Embodiment 143 The method of any one of embodiments 138 to 142, wherein the signal associated with the of the pH-sensitive molecule and the signal associated with the oxygen-sensitive molecule are detected in different areas of the chamber.
  • Embodiment 144 The method of any one of embodiments 138 to 143, wherein the oxygen-sensitive molecule is introduced together with the pH-sensitive molecule.
  • Embodiment 145 The method of any one of embodiments 138 to 143, wherein the oxygen-sensitive molecule is introduced separately with the pH-sensitive molecule.
  • Embodiment 146 The method of any one of embodiments 90 to 145, wherein the pH-sensitive molecule is responsive to a pH range from 1 to 9.
  • Embodiment 147 The method of embodiment 146, wherein the pH-sensitive molecule is responsive to a pH range from 3 to 6.
  • Embodiment 148 The method of any one of embodiments 90 to 147, wherein the microfluidic device does not comprise an integrated pH sensor.
  • Embodiment 149 A non-transitory computer-readable medium in which a program is stored for causing a system comprising a computer to perform a method of any one of embodiments 90 to 148.
  • Embodiment 150 A method of ranking a micro-object population within a microfluidic device, comprising: introducing the micro-object population into the microfluidic device, wherein the microfluidic device comprises an enclosure comprising a flow region and a plurality of chambers, wherein each of the plurality of the chamber is fluidically connected through a proximal opening to the flow region; disposing individual micro-object of the microobject population into a respective chamber of the plurality of chamber resulting in disposed micro-objects within respective chambers; allowing the disposed micro-objects to produce a molecule of interest; introducing a pH-sensitive molecule into the microfluidic device; detecting a first signal associated with the pH-sensitive molecule in a first area of interest within respective chamber; and ranking the disposed micro-objects based on the first signals detected in the respective chambers.
  • Embodiment 151 The method of embodiment 150, further comprising culturing the disposed micro-objects in the respective chambers.
  • Embodiment 152 The method of embodiment 150 or embodiment 151, wherein the first area of interest comprises about 5% to 100% of the respective chamber.
  • Embodiment 153 The method of any one of embodiments 150 to 152, the first area of interest does not comprise a wall of the respective chamber.
  • Embodiment 154 The method of any one of embodiments 150 to 153, further comprising measuring a biomass of the disposed micro-objects in the respective chambers.
  • Embodiment 155 The method of embodiment 154, wherein ranking the disposed micro-objects is further based on the biomass measured in the respective chambers.
  • Embodiment 156 The method of any one of embodiments 150 to 155, further comprising introducing an oxygen-sensitive molecule into the microfluidic device, and detecting a second signal associated with the oxygen-sensitive molecule in a second area of interest within respective chamber.
  • Embodiment 157 The method of embodiment 156, wherein ranking the plurality of micro-objects is further based on the second signal detected in the respective chambers.
  • Embodiment 158 The method of embodiment 156 or embodiment 157, wherein the second area of interest is within a micro-object-free area within the respective chamber.
  • Embodiment 159 The method of embodiment 158, wherein the first area of interest and the second area of interest are the same area.
  • Embodiment 160 The method of embodiment 158 or embodiment 159, wherein second area of interest comprises about 5% to 100% of the micro-object-free area.
  • Embodiment 161 The method of any one of embodiments 150 to 160, further comprising introducing a water immiscible fluidic medium into the flow region to fill a portion of the flow region adjacent to the plurality of chambers.
  • Embodiment 162 The method of embodiment 161, wherein the portion of the flow region adjacent to the plurality of chambers is adjacent to the proximal openings of each of the plurality of chambers.
  • Embodiment 163 The method of embodiment 161 or embodiment 162, wherein the water immiscible fluidic medium seals the proximal opening of the chamber.
  • Embodiment 164 The method of any one of embodiments 150 to 163, further comprising selecting a desired micro-object based on the ranking and exporting the desired micro-object out of the micro-fluidic device.
  • Embodiment 165 The method of any one of embodiments 150 to 164, wherein each chamber of the plurality of chambers comprises an isolation region and a connection region fluidically connecting the isolation region to the flow region; and wherein the connection region comprises the proximal opening to the flow region and a distal opening to the isolation region.
  • Embodiment 166 The method of embodiment 165, wherein the first area of interest is within the isolation region.
  • Embodiment 167 The method of embodiment 165, wherein the first area of interest is within the connection region.
  • Embodiment 168 The method of any one of embodiments 150 to 167, wherein introducing a pH-sensitive molecule into the microfluidic device comprises introducing the pH-sensitive molecule into the flow region and allowing the pH-sensitive molecule to diffuse into the plurality of chambers.
  • Embodiment 169 A non-transitory computer-readable medium in which a program is stored for causing a system comprising a computer to perform a method of any one of embodiments 150 to 168.

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Abstract

L'invention concerne des procédés et des kits pour surveiller et déterminer un pH pour une sélection de micro-objets biologiques à l'intérieur d'un dispositif microfluidique. Les procédés et les kits répondent à l'écoulement d'un milieu fluidique comprenant une molécule sensible au pH et la détection d'un signal associé à la molécule sensible au pH pour déterminer un pH local d'un milieu distribué spatialement à l'intérieur du dispositif microfluidique. Le signal détecté peut être utilisé pour évaluer une acidification de milieu à l'intérieur d'une zone sélectionnée à l'intérieur du dispositif microfluidique et fournit par conséquent des informations de valeur pour sélectionner un micro-objet biologique souhaité à partir d'une population cultivée.
PCT/US2023/063233 2022-02-25 2023-02-24 Appareils, procédés et kits pour dosages microfluidiques Ceased WO2023164623A2 (fr)

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WO2025071355A1 (fr) * 2023-09-27 2025-04-03 포항공과대학교 산학협력단 Dispositif de réaction microfluidique et procédé de synthèse d'arnm l'utilisant

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US20050266582A1 (en) * 2002-12-16 2005-12-01 Modlin Douglas N Microfluidic system with integrated permeable membrane
US8398922B2 (en) * 2009-10-08 2013-03-19 The United States of America as represented by the Secretary of Commerce, the National Institute of Standards and Technology Highly sensitive oxygen sensor for cell culture
WO2017091601A1 (fr) * 2015-11-23 2017-06-01 Berkeley Lights, Inc. Structures d'isolation microfluidiques produites in situ, kits et procédés d'utilisation de celles-ci
US11724260B2 (en) * 2019-04-08 2023-08-15 The Regents Of The University Of Michigan Microfluidic sensor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025071355A1 (fr) * 2023-09-27 2025-04-03 포항공과대학교 산학협력단 Dispositif de réaction microfluidique et procédé de synthèse d'arnm l'utilisant

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