WO2016064757A1

WO2016064757A1 - Modular microfluidic system for perfused cell culture

Info

Publication number: WO2016064757A1
Application number: PCT/US2015/056271
Authority: WO
Inventors: Thomas Neumann; Richard Carleton HULIT; Mark E. Fauver; Davis E. WEBB; Ken Fisher; Steve HULTENG
Original assignee: NORTIS Inc
Current assignee: NORTIS Inc
Priority date: 2014-10-19
Filing date: 2015-10-19
Publication date: 2016-04-28
Anticipated expiration: 2017-04-19

Abstract

A modular microfluidic cell culture system includes disposable, perfusable cell culture chips having one or more portable platforms that include detachable fluid reservoirs adapted to be connected to the perfusable cell culture chips and further adapted to be pneumatically-powered for fluid perfusion. One or more of the portable platforms are adapted to be disconnected from the cell incubator-based system. A cell incubator includes an organizational system to provide a pressurized, incubator- controlled air mixture to a number of portable platforms.

Description

MODULAR MICROFLUIDIC SYSTEM FOR PERFUSED CELL CULTURE

Cross- Reference to Related applications

This application is related to and claims priority from co-pending US Provisional Application No. 62/065,713 to Neumann et al. filed 19 October 2014 and entitled "MODULAR MICROFLUIDIC SYSTEM FOR PERFUSED CELL CULTURE," the disclosure of which is incorporated by reference.

Field of the Invention

The present invention relates to microfluidic devices for cell culture, and, more particularly, to a modular microfluidic cell culture system.

Background of the Invention

Microfluidic devices for cell culture have traditionally used individual tube connections for fluid connections driven by syringe pumps, or are open-systems using gravity-fed perfusate. These legacy systems are either cumbersome to set up, are tethered to tubing, or do not provide controlled steady flow rates. The tethering of the experiment to a fixed location makes it difficult to transport the culture to a microscope or other analytical equipment. Existing pneumatically-driven cell culture systems (CellASIC) are designed to be powered from gas cylinders; these systems are intolerant of air leaks, and do not scale up to large numbers well because total leak rate goes up proportionally. Other systems that employ electrical motors to power the perfusion, even if they maintain some level of portability, do not scale to large numbers well because of the proportional increase in hardware needed, hence complexity and cost.

Summary of the Disclosure

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

In one aspect a modular microfluidic cell culture system is disclosed having: one or more disposable, perfusable cell culture chips;

one or more portable platforms that include detachable fluid reservoirs adapted to be connected to the perfusable cell culture chips and further adapted to be pneumatically-powered for fluid perfusion;

one or more of said portable platforms adapted to be disconnected from the cell incubator-based system; and

a cell incubator including an organizational system to provide a pressurized, incubator-controlled air mixture to a plurality of said portable platforms.

In another aspect said platforms have self-contained pressurized air storage to allow maintenance of perfusion for occasions when the platform is used outside incubator.

In another aspect said platforms contain air valves that are actuated in the presence of a fluid reservoir, to selectively pressurize fluid reservoirs without user attention.

In another aspect said platform uses a lever with a cam to provide a visual indicator that the platform is properly closed and pneumatic seals are established.

In another aspect said fluid reservoirs contain a flow restrictor made from fused silica capillary tubing.

In another aspect said fluid reservoirs are optically clear.

In another aspect said waste reservoirs have a sterile filter to vent to the external environment.

In another aspect said waste containers contain a flow restrictor to provide a controlled backpressure.

In another aspect said waste containers can accommodate the insertion of a small Eppendorf or other collection tube.

In another aspect said platform has a base that is the size of a microplate or well plate.

In another aspect said organizational system allows visualization of fluid levels in reservoirs.

In another aspect said air mixture consists of carbon dioxide, nitrogen, and oxygen. In another aspect said reservoirs can be user-configured to enable different perfusion paths.

In another aspect, a cell-incubator based pump system for perfusing cell culture media to a plurality of independent cell cultures, is disclosed including:

a cell incubator including a manifold;

a pump having a first line connected to the cell incubator to draw incubator- controlled air mixture from the incubator and pressurize it;

the first line also connected to a moisture filter to remove water vapor;

a pressure regulator coupled to the moisture filter to route the pressurized air back into the cell incubator through the manifold; and

fluid-reservoirs couples to the manifold to drive cell culture media from the fluid-reservoirs through flow restrictors using the pressurized air to provide a controlled rate of perfusion.

In another aspect, the pump comprises a diaphragm pump to draw and pressurize air.

In another aspect the pump has sterile filters on both inputs and outputs.

In another aspect the flow restrictors are fused silica capillary tubing with < ±1 urn tolerance on the inner diameter.

In another aspect the pressure regulator maintains 0-10 PSI.

In another aspect an air storage reservoir is included to reduce pump duty cycle and increase pump lifetime.

In another aspect, a microfluidic channel structure is disclosed having a plurality of exterior channel sides relieved such that outer channel wall can deform perpendicularly to the direction of applied actuator force; and

a circular-segment channel cross-section, such that the seal pressure is increased relative to the applied actuator force.

In another aspect, the microfluidic channel has sides relieved to allow deformation of channel elastomer material.

In another aspect, microfluidic channel has only two acute angle corners which require less material deformation to achieve a fluidic seal.

In another aspect, said microfluidic channel has no discontinuities above the mating plane where the seal is formed. In another aspect, said channel structure contains one or more independent flow paths.

Brief Description of the Drawings

While the novel features of the invention are set forth with particularity in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings, in which:

FIG. 1 schematically shows an example of a reservoir to chip connection.

FIG. 2A - FIG. 2D schematically illustrate the difference between the present invention's new microfluidic channel structure (2A and 2B) and typical microfluidic valve designs (FIG. 2C and 2D).

FIG. 3 schematically shows an example of a CAD model showing construction in a cross-sectional view.

FIG. 4 schematically shows an example of a CAD model of actuator and valve on chip.

FIG. 5 schematically shows an example of platform reservoir indexing.

FIG. 6 schematically shows an example of a valve for air pressure.

FIG. 7 schematically shows an example of a modular perfusion platform.

FIG. 8 schematically shows an example of a gas pump and docking station in an incubator.

FIG. 9 schematically shows an example of a system schematic of a docking station with a manifold assembly connected to a gas pump.

FIG. 10A-FIG. 10C schematically show more detailed views of an upper chip shell.

FIG. 1 1 shows a more detailed schematic of a shut off valve actuator.

FIG. 12 shows a detail of an example of a biological chamber coupled to a plurality of fluid channels.

FIG. 13A-FIG. 13C schematically show more detailed views of an upper chip shell. FIG. 14 schematically shows an example of a gas pump.

FIG. 15 schematically shows an example of the major functional blocks of a gas pump as used for perfusion of an incubator.

FIG. 16 schematically shows a cut -away side view of an example of a media reservoir as used in the chip perfusion platform.

In the drawings, identical reference numbers identify similar elements or components. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements and angles are not drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Further, the particular shapes of the elements as drawn, are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the drawings.

Detailed Description of the Preferred Embodiments

The examples presented herein are for the purpose of furthering an understanding of the invention. The examples are illustrative and the invention is not limited to the example embodiments.

Unless the context requires otherwise, throughout the specification and claims which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising" are to be construed in an open, inclusive sense that is as "including, but not limited to."

Reference throughout this specification to "one example" or "an example embodiment," "one embodiment," "an embodiment" or combinations and/or variations of these terms means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Definitions Generally, as used herein, the following terms have the following meanings unless the context suggests otherwise:

As used herein, "CAD" is understood to mean computer aided design.

As used herein, "plurality" is understood to mean more than one. For example, a plurality refers to at least 3, 4, 5, 70, 1 ,000, 10,000 or more.

As used herein "well plate" includes standard microplates or micro well plates or multiwells, which are flat plates with multiple "wells" used as small test tubes.

Example Embodiments

The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by specifically different equipment, and devices and reconstruction algorithms, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.

Described herein is a modular microfluidic cell culture system. The features of the system include a pneumatically-powered, perfused microfluidic cell culture system that is modular on many levels (chip-to-chip, chip-to-fluid reservoir, reservoir-to- platform (aka portable unit), and platform-to-system). In operation, the following features are enabled:

• Individual cell culture chips can be grown and easily connected later on, minimizing human touch time and resultant loss of sterility, and without significant fluidic disturbance to biological-matrix constructs and cells within the chip.

• Multiple compartment chips (for example, lumenal, extracellular compartments and the like) can be configured at the user level to provide perfusion as desired.

• Various ports on the multiple compartment chip can be open or closed with on- chip valves as desired by the end user. This facilitates injection of biological matrix, and subsequent re-sealing of the chip to form a closed system. • A modular system where the chip and its fluid reservoirs are contained in a portable unit that is pneumatically-powered, allowing independent perfusion of hundreds of cell cultures to be performed without significant additional complexity or cost. The portable cell culture unit (aka platform) is a closed system, and remains sterile when removed from the cell incubator. This is made possible because the disconnection of the portable cell culture unit from the system is at the pneumatic line, not the fluid line.

• The system at the highest organizational level consists of a recirculatory gas pump, and shelf racks with air manifolds to deliver pressurized C02-adjusted air.

Level 1 : Chip-to-Reservoir and Chip-to-Chip

Referring now to FIG. 1 an example of a reservoir to chip connection is schematically shown. A chip 16 includes a microfluidic circuit 18. The microfluidic circuit 1 8 includes a biological chamber 17 coupled to a plurality of fluid channels 26 (shown in more detail in FIG. 12) onto which are mounted a series of shut-off valves 1 1 , where each shut off valve 1 1 includes a valve actuator 10. A reservoir 14 includes a fluidic connector 12 sized to couple to one of an array of connectors 13. In one example, the silicone chip 16 consists of a thin, flat bottom side and an upper side with a high-aspect ratio thin walled channel that is depressed by the actuator.

In one example, the chip 16 contains a cylindrical channel 20 with defined dimensions and a protruding ring 22 sized to fit into a cylindrical hole 23 in an upper chip shell 24. This feature creates a compression seal over a straight-shaft connector of a defined outer diameter (OD). The chip 1 6 contains a linear array of the connectors 13 at defined intervals to allow connection to a syringe, a pipette tip, a medial reservoir, a collection reservoir or to another chip through a jumper tube (not shown). The flow path is set by the placement of media (source) and collection reservoirs to given channels on the chip and by user actuated shut-off valves 1 1 located on the chip, allowing multiple flow path possibilities. The number of channels 26 is scalable to allow the design to work with chips with different flow configurations. It also creates a more stable system due to the elimination of tubing runs and connections between the chip 16 and reservoir 14. Using this design minimizes swept volume and fluid disturbance and improves chip-to-chip connectivity with a manifold style design. It also has the advantage of minimizing swept volume and fluid disturbance and improves chip-to-chip connectivity with a manifold style design.

Referring now simultaneously to FIG. 10A-FIG. 10C, more detailed views of the upper chip shell are schematically shown. FIG. 10A shows a perspective view of an upper chip shell 24. The upper chip shell 24 includes the cylindrical hole 23 for accepting the protruding ring 22 from the chip. The upper chip shell also includes a plurality of actuator locations 27 through which the actuators are inserted.

Referring now particularly to FIG. 10B, a side view of an upper chip shell is schematically shown. Openings 29 for the connectors 13 are included and configured at predetermined intervals to be aligned with the chip connectors. FIG. 10C shows a cross sectional cut detail of the upper chip shell.

Referring now simultaneously to FIG. 13A-FIG. 13C, more detailed views of the lower chip shell are schematically shown. FIG. 13A shows a perspective view of a lower chip shell 25. The lower chip shell is sized to mate with the upper chip shell to hold the chip 16. FIG. 13B shows a cross sectional cut detail of the lower chip shell.

Referring now particularly to FIG. 13C, a side view of a lower chip shell is schematically shown. A plurality of openings 29A aligned to accept the connectors 13 are included and configured at predetermined intervals to be aligned with the chip connectors.

Referring now particularly to FIG. 2A - FIG. 2D, these figures schematically illustrate the difference between the present invention's new microfluidic channel structure (2A and 2B) and typical microfluidic valve designs (FIG. 2C and 2D). Each fluid channel on the chip contains a manually actuated shut-off valve 1 1 to facilitate the system modularity. The valves enable connections and disconnections to be made without creating fluidic disturbances and allow chips to be reconfigured for different flow patterns or hooked up in series mid-experiment.

Referring now specifically to FIG. 2B, there shown is a shut off valve in a closed mode. The valve workings illustrated are as follows: A quarter-turn manual actuator 10 pinches a silicone channel 30 against a rigid surface 32 to close the channel to fluid flow. An upper chip layer 33 may comprise an elastomer applied to the substrate 32. In one useful example, the system consists of a rigid chip enclosure including an upper chip shell 24 mounted to a lower chip shell 25 (as shown in FIG. 13A), two silicone chip layers 32, 33 and a rigid plastic actuator 10. The lower chip shell 25 provides a flat hard surface 31 for the actuator to press against. The upper chip shell 24 is mechanically connected to the lower shell and may advantageously include (not shown) indexing pins for the valve actuator to form a tightly controlled rigid anchor for the actuator. The actuator uses a barrel-cam track indexing on the upper shell that allows it to be positioned in either fluid path open or fluid path closed positions, by turning the handle of the actuator 90 degrees of rotation. This design creates a reliable seal while displacing a minimum volume of fluid.

Now specifically referring to FIG. 2D, in contrast to the present design there shown is how this interior corner creates a leak path 42. The primary advantage to both the exterior sides of the channel being relieved, and the circular-segment channel cross-section is a significant reduction of the applied actuation force to achieve an acceptable seal pressure to withstand leaks. This is especially important in cell culture disposables where cost and the dimensional limitations of performing short working distance optical microscopy limit the amount of material that can be used to stiffen the structure and hence allow on-chip valves or pumps to be used. Note that these are non-direct contact valves or pumps— an important consideration for maintenance of sterility and avoiding other potential contamination in a cell culture system.

Now referring jointly to FIG. 3 and FIG. 4, the microfluidic channel structure shown can be used as a valve but is equally applicable for use in a peristaltic pump and has two notable features. The outer portion of each channel has material relieved around the exterior, which allows for deformation of the channel structure with less applied downward force than the typical microfluidic valve design. This is because the much of deformation can be accomplished in a flexural mode, rather than mostly compression of a large volume of elastomer. Secondly, the inner portion of the channel cross-section is not rectangular, but a circular segment 35 that can be flattened more easily than an interior molded corner. Micromachining of these channel features allows the circular cross-section to be fabricated much more easily than with lithographic techniques which create sharp corners. See, for example, channel 37 having sharp corners 39 as shown in FIG. 2C. Referring specifically to FIG. 3, channel 30A is shown in an open mode with the actuator 10A in a first position. Channel 30B is shown in a closed mode where actuator 10B has been engaged to press against the channel.

Referring now briefly to FIG. 1 1 a more detailed schematic of a shut off valve actuator is shown. A valve actuator 1 0 includes a head portion 202 including a slot 204 for accepting a slotted screw driver or the like. Of course, any configuration of screw head could be substituted as desired. The bottom portion 208 preferably comprises a barrel-cam track 210, where the track is sized to index on a mating bump, tab or similar protrusion on the upper shell. When turned, the actuator operates in connection with the upper shell to be positioned in either fluid path open or fluid path closed positions, by rotating the actuator 90 degrees.

Level 2: Reservoir to Platform

Now referring to FIG. 5 an example of platform reservoir indexing is schematically shown. A platform reservoir housing 55 contains a plurality of indexed slots 50 for reservoirs 14 that align with the connectors 13 on the chip 1 6 (as shown in FIG. 1 ) and fit within a well-plate format. The bottom plate 54 of the housing contains indexing features and supports the chip, while the top plate 56 contains an air manifold 60 (as best shown in FIG. 9). The top and bottom plates 56, 54 respectively contain a latch mechanism 58 to allow them to capture and seal the reservoirs 14. Each slot 50 can be populated with a media (source) reservoir, a collection reservoir or left empty.

Now referring to FIG. 6, an example of a valve for air pressure is schematically shown. When a media reservoir 14 is placed in a housing slot, it actuates a normally closed air valve 70 to allow the reservoir to be pressurized because the top 1 93 of a plate 163 will compress the actuator 62 opening the valve. Collection reservoirs 15 do not actuate the valves and remain at atmospheric pressure. Each media reservoir contains a micro-capillary tube that restricts fluid flow when the media is pressurized resulting in a controlled rate profusion from the media reservoir to the waste reservoir. When the reservoirs are in place on the platform, the connections to the chip are made simultaneously with a single motion parallel to the axis of all the connectors. Referring now to FIG. 7 schematically shows an example of a modular perfusion platform. In this view the connection between the chip 16 and the reservoir housing 55 is illustrated. Each chip may be connected to a reservoir housing 55 holding one or more media reservoirs 14 and collection or waste reservoirs 1 5. The media reservoirs pump media into the chip module and the media is then circulated out to a waste reservoir. Each such platform is portable and may be connected with a plurality of other platforms in an incubator as described hereinbelow.

Level 3: Platform to System

Referring now to FIG. 8 an example of a gas pump and docking station in an Incubator is schematically shown. In the example shown, a pneumatic system 100 consists of a recirculatory air pump 104 that provides 2 user set pressure lines 1 10, 1 12 and an incubator organization and manifold assembly 106, also called the "docking station," that allows for easy access to any chip/platform 5 and provides air to each platform on an individual basis. This assembly allows platforms 5 to be removed individually or in groups of 4 on a tray. Valved quick disconnects are used for all pneumatic connections to allow components to be disconnected and reconnected while maintaining system pressure. The fluid flow rate through the chips is set using the input air pressure, so all chips connected to a single air-line will have the same flow rate. This system also allows a single platform to be hooked directly to the air pump for priming or when a second flow rate is required.

In one example, the system includes a gas pump, pressurized air distribution manifold, and an organizational shelf ("docking station") for stacking platforms. It is designed to work with pre-existing cell incubators, and make use of their ability to adjust air to a desired carbon dioxide level (typically 5 %). Additionally, the recirculatory gas pump supports incubators which control other gas concentrations, such as oxygen to create hypoxic environments. The environment inside the incubator is mimicked inside the closed system of the microfluidic cell culture media reservoirs. The pump system, by drawing in the air from inside the cell incubator, provides an appropriate range of carbon dioxide gas to the cell culture media (within the fluid reservoirs) and hence maintains the pH of the cell culture media. Referring now to FIG. 9, an example of a system schematic of a docking station with a manifold assembly connected to a gas pump is schematically shown. The gas pump 104 includes a precision pressure regulator 122 coupled to a filtration device 124. The pressurized gas is transmitted through a first line 1 10 to a docking station manifold 901 . The docking station manifold, in turn routes pressurized gas to a plurality of secondary manifolds 903. Some or all of the secondary manifolds 903 may be coupled to one or more platform assemblies 5 through their normally closed air valves 70. An incubator holding a plurality of platform assemblies 5 is shown and described above with respect to FIG. 7.

In operation, the recirculatory gas pump 104 provides a driver for perfusing cell culture media to a multitude of independent cell cultures. The gas pump system operates to 1 ) draw carbon dioxide-adjusted air from a cell incubator 1 02, 2) pressurize it and remove water vapor, 3) pressure-regulate and route the pressurized air back into the cell incubator through a manifold, 4) via fluid-reservoirs dedicated to each independent cell culture, the pressurized air will forces cell culture media through a flow restrictor to provide a controlled rate of perfusion. The gas pump is considered recirculatory because it draws the carbon dioxide adjusted air from the incubator, increases the air pressure via a pump, and then routes it back inside the incubator to the multitude of platforms. Any air leaks on the platforms do not deplete the cell incubator of carbon dioxide-adjusted air. Given the potential for very large (1 0s-1000s) of pneumatic connections made, the total air leakage of the system can be »100 mL/min. At these rates, gas cylinders may last from only a few days to less than 1 month.

Referring now to FIG. 14, an example of a gas pump is schematically shown. The gas pump 104 includes a pump 401 , a set of pressure filters 41 0, an optional buzzer 41 1 , a digital pressure switch 412, a vacuum generator 413, a power supply 414, a DC transformer 432, a drain valve 415, and an air reservoir 416. In one useful embodiment the set of pressure filters 41 0 may include two pressure filters with a Schrader drain and one vacuum filter with a pulse drain. A precision pressure regulator 122 is also incorporated into the gas pump as are other standard components such as displays. In one embodiment the precision regulator may comprise a precision regulator as manufactured by the Fairchild Company, US. Construction and operation of the gas pump 104 will be readily understood by one skilled in the art who has the benefit of this disclosure.

Referring now to FIG. 15, an example of the major functional blocks of a gas pump as used for perfusion of an incubator is schematically shown. The gas pump 104 includes the pump 401 , such as a diaphragm pump connected to the pressure/transducer switch 412. A pressure reservoir, such as air reservoir 41 6 may be coupled to a micro-mist separator 152, which, in turn is coupled to the pressure regulator 122. In one example the gas input may be 37° C at 100% humidity while the output is specified at .25 LJmin, 0-70 +/- 2% KPa (0-10 psi). A digital gas pressure gage 1 53 may be coupled to provide output readings.

FIG. 16 schematically shows a cut -away side view of an example of a media reservoir as used in the chip perfusion platform. Each media reservoir contains a flow restrictor comprising, for example, a micro-capillary tube that restricts fluid flow when the media is pressurized resulting in a controlled rate profusion from the media reservoir to the waste reservoir. The media reservoir 14 includes a pair of parallel plates 163 which are sized to allow media to flow in between the plates from both left and right cavities 165, 166 of the reservoir 14. A micro-capillary tube 162 is coupled to the outlet 12 at a first end and held in place by collar 1 67 to draw media from the reservoir. In one example, the flow restrictors may comprise fused silica capillary tubing with < ±1 urn tolerance on the inner diameter.

Described hereinabove is a modular microfluidic cell culture system which in one example includes one or more disposable, perfusable cell culture chips; one or more portable platforms that include detachable fluid reservoirs adapted to be connected to the perfusable cell culture chips and further adapted to be pneumatically- powered for fluid perfusion; one or more of said portable platforms adapted to be disconnected from the cell incubator-based system; and a cell incubator including an organizational system to provide a pressurized, incubator-controlled air mixture to a plurality of said portable platforms.

In another example said platforms have self-contained pressurized air storage to allow maintenance of perfusion for occasions when the platform is used outside incubator. In another example said platforms contain air valves that are actuated in the presence of a fluid reservoir, to selectively pressurize fluid reservoirs without user attention. In another example said platform uses a lever with a cam to provide a visual indicator that the platform is properly closed and pneumatic seals are established. In another example said fluid reservoirs contain a flow restrictor made from fused silica capillary tubing. In another example said fluid reservoirs are optically clear. In another example said waste reservoirs have a sterile filter to vent to the external environment. In another example said waste containers contain a flow restrictor to provide a controlled backpressure. In another example said waste containers can accommodate the insertion of a small Eppendorf or other collection tube. In another example said platform has a base that is the size of a microplate or well plate. In another example said organizational system allows visualization of fluid levels in reservoirs. In another example said air mixture consists of carbon dioxide, nitrogen, and oxygen. In another example said reservoirs can be user- configured to enable different perfusion paths.

In another example, a cell-incubator based pump system for perfusing cell culture media to a plurality of independent cell cultures, is disclosed including:

a cell incubator including a manifold;

the first line also connected to a moisture filter to remove water vapor;

fluid-reservoirs couples to the manifold to drive cell culture media from the fluid-reservoirs through flow restrictors using the pressurized air to provide a controlled rate of perfusion. In another example, the pump comprises a diaphragm pump to draw and pressurize air. In another example the pump has sterile filters on both inputs and outputs. In another example the flow restrictors are fused silica capillary tubing with < ±1 um tolerance on the inner diameter. In another example the pressure regulator maintains 0-1 0 PSI. In another example an air storage reservoir is included to reduce pump duty cycle and increase pump lifetime. In another example, a microfluidic channel structure is disclosed having a plurality of exterior channel sides relieved such that outer channel wall can deform perpendicularly to the direction of applied actuator force; and a circular-segment channel cross-section, such that the seal pressure is increased relative to the applied actuator force.

In another example, the microfluidic channel has sides relieved to allow deformation of channel elastomer material. In another example, microfluidic channel has only two acute angle corners which require less material deformation to achieve a fluidic seal. In another example, said microfluidic channel has no discontinuities above the mating plane where the seal is formed. In another example, said channel structure contains one or more independent flow paths.

The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by specifically different equipment, and devises, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.

Claims

Claims What is claimed is:

1 . A modular microfluidic cell culture system comprising:

one or more disposable, perfusable cell culture chips;

2. The system of claim 1 where said platforms have self-contained pressurized air storage to allow maintenance of perfusion for occasions when the platform is used outside incubator.

3. The system of claim 1 wherein said platforms contain air valves that are actuated in the presence of a fluid reservoir, to selectively pressurize fluid reservoirs without user attention.

4. The system of claim 1 wherein said platform uses a lever with a cam to provide a visual indicator that the platform is properly closed and pneumatic seals are established.

5. The system of claim 1 wherein said fluid reservoirs contain a flow restrictor made from fused silica capillary tubing.

6. The system of claim 1 wherein said fluid reservoirs are optically clear.

7. The system of claim 1 wherein said waste reservoirs have a sterile filter to vent to the external environment.

8. The system of clam 1 wherein said waste containers contain a flow restrictor to provide a controlled backpressure.

9. The system of claim 1 wherein said waste containers can accommodate the insertion of a small Eppendorf or other collection tube.

10. The system of claim 1 wherein said platform has a base that is the size of a microplate or well plate.

1 1 . The system of claim 1 wherein said organizational system allows visualization of fluid levels in reservoirs.

12. The system of claim 1 wherein said air mixture consists of carbon dioxide, nitrogen, and oxygen.

13. The system of claim 1 wherein said reservoirs can be user-configured to enable different perfusion paths.

14. A cell-incubator based pump system for perfusing cell culture media to a plurality of independent cell cultures, comprising:

a cell incubator including a manifold;

the first line also connected to a moisture filter to remove water vapor;

15. The system of claim 14 wherein the pump comprises a diaphragm pump to draw and pressurize air.

16. The system of claim 14 wherein the pump has sterile filters on both inputs and outputs.

17. The system of claim 14 wherein the flow restrictors are fused silica capillary tubing with < ±1 urn tolerance on the inner diameter.

18. The system of claim 14 wherein the pressure regulator maintains 0-1 0 PSI.

19. The system of claim 14 further including an air storage reservoir to reduce pump duty cycle and increase pump lifetime.

20. A microfluidic channel structure comprising:

A plurality of exterior channel sides relieved such that outer channel wall can deform perpendicularly to the direction of applied actuator force; and

circular-segment channel cross-section, such that the seal pressure is increased relative to the applied actuator force.

21 . The structure of claim 20 wherein the microfluidic channel has sides relieved to allow deformation of channel elastomer material.

22. The structure of claim 21 wherein said microfluidic channel has only two acute angle corners which require less material deformation to achieve a fluidic seal.

23. The structure of claim 20 wherein said microfluidic channel has no discontinuities above the mating plane where the seal is formed.

24. The structure of claim 20 wherein said channel structure contains one or more independent flow paths