HK1249131B - Pens for biological micro-objects - Google Patents

Description

Fences for biological micro-targets

The application is a divisional application of a PCT application entering the national phase with an application date of October 30, 2013, application number 201380061831.3, an earliest priority date of October 31, 2012, and an invention title of “Fence for Biological Micro-Targets”.

Background Art

In the field of biological science, the activity of biological micro-targets, such as cells, is often studied and analyzed. For example, cells that produce at least a minimum number of clones or secrete a desired substance can be used in the production of pharmaceuticals or in the study of diseases. Therefore, it can be advantageous to determine cells that produce clones or secrete a specific substance at or above a minimum rate. Embodiments of the present invention relate to improved microfluidic devices and processes for placing selected biological micro-targets into a holding enclosure, regulating micro-targets in the enclosure, monitoring the biological activity of micro-targets in the enclosure, and/or removing micro-targets whose biological activity reaches a predetermined threshold from the enclosure for further use or processing.

Summary of the Invention

In certain embodiments, a method for processing biological microtargets may include: actively placing individual biological microtargets into an interior space of a holding enclosure in a microfluidic device; and providing a flow of a first liquid medium to the enclosure for a period of time. The method may also include: while providing the flow, preventing the first medium from the flow from directly flowing into the interior space of the holding enclosure.

In certain embodiments, a microfluidic device may include a housing and a retaining fence. The housing may be disposed on a base and may include a flow path for a first liquid medium. The retaining fences may be disposed within the housing and each fence may include a perimeter surrounding an interior space. The perimeter may be configured to retain biological micro-targets suspended in a second liquid medium and prevent the first medium from flowing directly into the second medium within the interior space.

A method for processing biological microtargets may include forming a virtual retention fence in a microfluidic device by illuminating a light pattern in the form of a retention fence into the microfluidic device, thereby activating a dielectrophoresis (DEP) electrode. The method may also include placing individual biological microtargets within the retention fences, wherein each of the retention fences isolates any one or more of the individual microtargets within the retention fence from all microtargets outside the retention fence. The method may also include providing a common flow of a liquid medium to the microtargets within the retention fences for a period of time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an example of a microfluidic device according to some embodiments of the present invention.

1B is a side cross-sectional view of the device of FIG. 1A .

1C is a top cross-sectional view of the device of FIG. 1A .

2 shows a cross-sectional side view of the device of FIG. 1A , illustrating an example of a device configured with an optoelectronic tweezers (OET) device according to some embodiments of the present invention.

3 is a top, partial cross-sectional view of the device of FIG. 1A constructed with the OET device of FIG. 2 and a virtual fence according to certain embodiments of the present invention.

4 is a top, partial cross-sectional view of the device of FIG. 1A configured with the OET device of FIG. 2 and a fence, which may be physical and/or virtual, according to certain embodiments of the present invention.

5A illustrates an example of a microfluidic structure disposed on a base defining fluid channels and enclosures according to some embodiments of the present invention.

5B is a top cross-sectional view of the microfluidic structure and base of FIG. 5A .

6A illustrates an example of a microfluidic structure disposed on a base defining fluid channels and enclosures according to some embodiments of the present invention.

6B is a top cross-sectional view of the microfluidic structure and base of FIG. 6A .

FIG. 7 illustrates an example of a variation of the fence shown in FIG. 6B , according to some embodiments of the present invention.

8A, 8B, 9 and 10 illustrate examples of alternative configurations of fences according to some embodiments of the present invention.

11A and 11B illustrate the use of an optical trap to select and move a micro-target according to some embodiments of the present invention.

12A and 12B illustrate the use of virtual barriers to select and move a micro-target according to some embodiments of the present invention.

13A illustrates an example of a microfluidic structure disposed on a base defining fluid channels and enclosures according to certain embodiments of the present invention.

13B is a top cross-sectional view of the microfluidic structure and base of FIG. 13A .

14 and 15 illustrate examples of alternative configurations for the fence array of FIG. 13B , according to embodiments of the present invention.

16 illustrates an example of a process including placing a biological microtarget into an enclosure in a microfluidic device, according to certain embodiments of the present invention.

FIG. 17 illustrates a process according to certain embodiments of the present invention, showing an example of the operation of the apparatus of FIG. 1A configured with the OET apparatus of FIG. 2 .

18A-18C illustrate examples of processing cells according to the steps of FIG. 17 .

FIG. 19 illustrates an example of an enclosure expanding as clones multiply within the enclosure according to the steps of FIG. 17 .

FIG. 20 illustrates an example of removing pens in which clones reproduce too slowly and draining the clones in these pens according to the steps of FIG. 6 .

FIG. 21 illustrates an example of placing a daughter clone into a new enclosure according to the steps of FIG. 17 .

FIG. 22 illustrates a process according to certain embodiments of the present invention, showing another example of the operation of the apparatus of FIG. 1A configured with the OET apparatus of FIG. 2 .

DETAILED DESCRIPTION

This specification describes example embodiments and applications of the present invention. However, the present invention is not limited to these example embodiments and applications, or to the manner in which the example embodiments and applications operate or are described herein. Furthermore, the accompanying drawings may show simplified or partial views, and the dimensions of elements in the accompanying drawings may not be enlarged or reduced to scale for clarity. In addition, when the terms "on," "attached to," or "coupled to" are used herein, an element (e.g., a material, layer, substrate, etc.) can be "on," "attached to," or "coupled to" another element, regardless of whether the element is directly on, attached to, or coupled to the other element, or whether there are one or more intervening elements between the element and the other element. In addition, if provided, directions (e.g., above, below, top, bottom, side, up, down, below, above, below, horizontal, vertical, "x," "y," "z," etc.) are relative and are provided merely as examples, for ease of illustration and discussion, and not as limitations. Furthermore, where reference numbers are given to a list of elements (eg, elements a, b, c), these reference numbers are intended to include any one of the listed elements by itself, any combination of less than all of the listed elements, and/or combinations of all of the listed elements.

The words "substantially" and "generally" mean sufficient to achieve the intended purpose. The term "plurality" means more than one.

The term "cell" refers to a biological cell. With respect to cells, the term "clone" refers to identical cells, each derived from the same parent cell. Clones are therefore all "daughter cells" of the same parent cell.

As used herein, the term "biological microtarget" includes biological cells and complexes such as proteins, embryos, plasmids, oocytes, sperm, genetic material (e.g., DNA), transfection vectors, hybridomas, transfected cells, and the like, as well as combinations thereof.

As used herein, a dielectrophoresis (DEP) electrode refers to a terminal on the inner surface of a chamber for containing a liquid medium or an area on the inner surface of a chamber for containing a liquid medium, at which terminal or area, a DEP force in the medium sufficient to attract or repel a micro-target in the medium can be selectively activated or deactivated.

As used herein with respect to a liquid or gas, the term "flow" includes, but is not limited to, continuous, pulsed, periodic, random, intermittent, or reciprocating flow of a liquid or gas. "Convection flow" is a flow of a liquid or gas driven by pressure. "Diffusive flow" or "diffusion" is a flow of a liquid or gas driven by random thermal motion. As used herein with respect to two or more liquid or gaseous media, the term "diffusive mixing" refers to the mixing of the media due to spontaneous mixing of the media caused by random thermal motion. As used herein with respect to "convection flow," "diffusive flow," "diffusion," or "diffusive mixing," the term "substantially" means greater than 50%.

The term "deterministic," when used to describe selecting or placing a micro-target, refers to selecting or placing a specifically determined or desired micro-target from a group of micro-targets. Deterministically selecting or placing a micro-target therefore does not include any one of the micro-targets being merely randomly selected or placed from a group or subgroup of micro-targets.

As used herein, the term "processing" a microtarget means any one or more of the following: moving (e.g., in a liquid medium flow, with the aid of an OET device, etc.), sorting and/or selecting one or more of the microtargets; changing one or more of the microtargets, where examples of such changes include multiplying a population of microtargets of cells or other living organisms, fusing two or more such microtargets together, and transfecting one or more microtargets; monitoring the microtargets; monitoring the proliferation, secretion, etc. of microtargets of cells or other living organisms; and/or regulating the microtargets of cells or other living organisms.

Embodiments of the present invention include deterministically placing individual biological microtargets within a holding enclosure within a microfluidic device. A first liquid medium flow can be provided to the enclosure, but the enclosure can be configured to prevent the first medium from flowing directly into a second medium within the enclosure, while allowing diffusional mixing of the first medium within the flow and the second medium within the enclosure.

1A-1C illustrate an example of a microfluidic device 100 according to certain embodiments of the present invention. As shown, the microfluidic device 100 may include a housing 102, an electrode mechanism 108, and a monitoring mechanism 118. Also as shown, the housing 102 may include an interior chamber 110 that holds one or more liquid media 114, in which a plurality of microtargets 116 may be suspended. The media 114 may be disposed on an interior surface 120 of the chamber 110. A plurality of retaining fences 112 for the microtargets 116 may be disposed within the chamber 110. As will be appreciated, each fence 112 may be a virtual fence, a physical fence, and/or a combination virtual/physical fence.

The media 114 in the device 100 may include, for example, a first media 122 and a second media 124. The first media 122 may be the media 114 in the flow path 126, while the second media 124 may be the media 114 inside the retaining fence 112. The first media 122 may be the same type of media as the second media 124. Alternatively, the first media 122 may be a different type of media than the second media 124.

The housing 102 may include a perimeter defining a chamber 110. As shown, the housing 102 may also include one or more inlets 104 through which a medium 114 and a microtarget 116 may be introduced into the chamber 110. One or more flow paths 126 for the medium 114 may exist within the channel 110. For example, as shown in FIG1C , the channel 110 may include a flow path 126 for the medium 114 from the inlet 104 to the outlet 106.

Inlet 104 may be, for example, an input port, an opening, a valve, a channel, etc. Housing 102 may also include one or more outlets through which medium 114 and microtarget 116 may be removed. Microtarget 116 may alternatively be removed from housing 102 in other ways. For example, as described below, a needle-like aspirator (not shown) may pierce housing 102, and one or more microtargets 116 may be removed by the aspirator. Outlet 106 may be, for example, an input port, an opening, a valve, a channel, etc. As another example, outlet 106 may include a droplet output mechanism such as any of the output mechanisms disclosed in U.S. patent application Ser. No. 13/856,781 (Attorney Docket No. BL1-US), filed April 4, 2013. All or part of housing 102 may be gas permeable to allow gas (e.g., ambient air) to enter and exit chamber 110, e.g., to maintain microtarget 116 in chamber 110. For example, a gas stream may be applied to a gas permeable portion of housing 102. For example, pulsed, modulated, or otherwise controlled gas flow may be applied as needed (eg, when experiments indicate that micro-targets (eg, cells) within housing 102 require gas).

Although not shown, the device 100 may include sensors or similar components that detect relevant conditions of the medium 114 or chamber 110, such as temperature, chemical composition of the medium 114 (e.g., the level of dissolved oxygen, carbon dioxide, etc. in the medium 114), pH of the medium 114, osmotic pressure of the medium 114, etc. The housing 102 may, for example, include such sensors or components, which may be configured with a controller (not shown) to control the input of the medium 114 through the inlet 104 to maintain certain conditions of the medium 114 constant or to controllably adjust certain conditions of the medium 114 (such as the conditions identified above).

The electrode mechanism 108 (shown in FIG. 1B ) can be configured to generate selective electrokinetic forces on microtargets 116 in a medium 114. For example, the electrode mechanism 108 can be configured to selectively activate (e.g., turn on) and deactivate (e.g., turn off) dielectrophoresis (DEP) electrodes at an inner surface 120 of a chamber 110 on which the medium 114 is disposed. The DEP electrodes can generate forces in the medium 114 that attract or repel the microtargets 116, and the electrode mechanism 108 can thereby select and/or move one or more microtargets 116 in the medium 114. For example, in some embodiments, the electrode mechanism 108 can be configured such that a hard-wired electrical connection to the DEP electrodes at the inner surface 120 can activate and deactivate the respective DEP electrodes. In other embodiments, the respective DEP electrodes at the inner surface 120 can be optically controlled. Examples including an optoelectronic tweezers mechanism are shown in FIG. 2 and described below.

For example, the electrode mechanism 10 may include one or more optical (e.g., laser) tweezers devices and/or one or more electro-optical tweezers (OET) devices (e.g., as disclosed in U.S. Patent No. 7,612,355, the entire contents of which are incorporated herein by reference). As another example, the electrode mechanism 108 may include one or more devices (not shown) for moving one or more droplets of the medium 114 suspended therein in the micro-target 116. Such devices (not shown) may include electrowetting devices, such as electro-electro-wetting (OEW) devices (e.g., as disclosed in U.S. Patent No. 6,958,132). The electrode mechanism 108 may therefore be characterized as a DEP device in certain embodiments.

Monitoring mechanism 118 may include any mechanism for observing, identifying, or detecting individual micro-targets in medium 114. In certain embodiments, monitoring mechanism 118 may also include a mechanism for monitoring the biological activity or biological state of micro-targets 116 in enclosure 112.

As shown in FIG2 , the monitoring mechanism 118 may include an imaging device 220. For example, the imaging device 220 may include a camera or similar device for capturing images of the micro-targets 116 in the chamber 110, including in the enclosure 112. Also as shown, the controller 218 may control the imaging device 220 and process the images captured by the imaging device 220. Although shown in FIG2 as being disposed below the apparatus 102, the imaging device 220 may be disposed in other locations, such as above or to the side of the apparatus 102.

As also shown in FIG2 , the electrode mechanism 108 may comprise an OET device. For example, as shown, the electrode mechanism 108 may comprise a first electrode 204, a second electrode 210, an electrode activation substrate 208, and a power source 212. As shown, the medium 114 and the electrode activation substrate 208 within the chamber 110 may separate the electrodes 204 and 210. A pattern of light 216 from a light source 214 may selectively activate a desired pattern of individual DEP electrodes on the interior surface 120 of the chamber 110. In other words, the light in the light pattern 216 may reduce the electrical impedance of the electrode activation substrate 208 at the pattern of small "electrode" areas on the interior surface 120 of the chamber 110 to less than the impedance of the medium 114. This creates an electric field gradient in the medium 114 from the electrode areas on the surface 120 to the first electrode 204, which in turn creates a localized DEP force that attracts or repels nearby micro-targets 116. Different patterns of individual DEP electrodes that attract or repel microtargets 116 in the medium 114 can thus be selectively activated and deactivated at many different such electrode regions on the interior surface 120 of the chamber 110 by projecting different light patterns 216 from a light source 214 (e.g., a laser source or other type of light source) into the microfluidic device 100.

In some embodiments, the electrode activation base 208 can be a photoconductive material, and the inner surface 120 can be featureless. In these embodiments, the DEP electrode can be formed anywhere and in any pattern on the inner surface 120 of the chamber 110. Several examples are described in the aforementioned U.S. Patent No. 7,612,355, where the undoped amorphous silicon material 24 shown in the drawings of the aforementioned patent can be an example of a photoconductive material that can constitute the electrode activation base 208.

In other embodiments, electrode activation substrate 208 may comprise a circuit substrate such as a semiconductor material including multiple doped layers, electrically insulating layers, and conductive layers that form semiconductor integrated circuits, such as those known in the semiconductor art. In these embodiments, circuit elements may form electrical connections between electrode regions at inner surface 120 of chamber 110 and second electrode 210, which can be selectively activated and deactivated by varying the pattern of light pattern 216. When deactivated, each electrical connection may have a high impedance, such that the voltage drop from the corresponding electrode region at inner surface 120 of chamber 110 to second electrode 210 is greater than the voltage drop from first electrode 204 through dielectric 114 to the corresponding electrode region. However, when activated by light in light pattern 216, each electrical connection may have a low impedance, such that the voltage drop from the corresponding electrode region at inner surface 120 of chamber 110 to second electrode 210 is less than the voltage drop from first electrode 204 through dielectric 114 to the corresponding electrode region, thereby activating the DEP electrode at the corresponding electrode region as described above. Thus, the DEP electrodes that attract or repel micro-targets in the medium 114 can be selectively activated and deactivated at many different "electrode" regions on the interior surface 120 of the chamber 110 by the light pattern 216. Non-limiting examples of such configurations of electrode activation substrates 208 include the phototransistor-based OET device 200 shown in Figures 21 and 22 of U.S. Patent No. 7,956,339.

In some embodiments, the first electrode 204 may be part of the upper wall 202 of the housing 102, and the electrode activation base 208 and the second electrode 210 may be part of the lower wall 206 of the housing 102, as generally illustrated in FIG2 . As shown, the upper wall 202 and the lower wall 206 may define the chamber 110, and the dielectric 114 may be disposed on the inner surface 120 of the chamber 110. However, this is merely an example. In other embodiments, the first electrode 204 may be part of the lower wall 206, while one or both of the electrode activation base 208 and/or the second electrode 210 may be part of the upper wall 202 of the housing 102. As another example, the first electrode 204 may be part of the same wall 202 or 206 as the electrode activation base 208 and the second electrode 210. For example, the electrode activation base 208 may include the first electrode 204 and/or the second electrode 210. Furthermore, the light source 214 may alternatively be located below the housing 102 , and/or the imaging device 220 and the light source 214 may alternatively be located on the same side of the housing 102 .

As mentioned, in certain embodiments of the present invention, portion or all of each fence 112 may be “virtual,” as used herein, meaning that portion or all of the fence 12 comprises DEP forces from activated DEP electrodes at electrode regions on the interior surface 120 of the chamber 110 (as described above) rather than a physical barrier.

FIG3 (which shows a partial top cross-sectional view of a portion of the housing 102) illustrates an example of the device 100 of FIG1A-1C, wherein the fence 112 (which is labeled 302 in FIG3) is a virtual fence 302 according to certain embodiments of the present invention. The virtual fence 302 in FIG3 can be formed in the chamber 110 by, for example, the electrode mechanism 108 configured as the OET device of FIG2. That is, the virtual fence 302 can include a pattern of activated DEP electrodes at the inner surface 120 of the chamber 110. Although one microtarget 116 is shown in each fence 302, more than one microtarget 116 may alternatively be present in each fence.

3 , a flow 314 of medium 114 can be provided through chamber 110 in flow path 126. As shown in FIG3 , each enclosure 302 can isolate the microtarget(s) 116 in that enclosure 302 from the microtargets 116 in other enclosures 302. However, flow 314 of medium 114 can be a common flow 314 provided to some or all of the enclosures 302, and thus the microtargets 116 in the enclosures 302. Constructed as shown in Figure 3, each fence 302 can thereby isolate the microtarget(s) 116 on the inside of the fence 302 from the microtargets 116 on the outside of the fence 302, including the microtargets 116 in other fences 302, and thereby prevent the microtargets 116 from the outside of a particular fence 302 from mixing with the microtarget(s) on the inside of that particular fence 302, while allowing a common flow 314 of the medium 114 to flow into (by convection) and out of the multiple fences 116, and thereby, for example, supply nutrients and transport waste away from the microtargets 116 in the multiple fences 116.

Virtual fences 302 may include light boundaries in light patterns 216 projected into housing 102 of microfluidic device 100 by light source 214, as shown in FIG2 . Power source 212 of the OET device of FIG2 may be configured with a frequency that causes the light boundaries defining each fence 302 to repel microtarget 116, thereby retaining microtarget 116 within each fence 302. Furthermore, by varying light patterns 216 projected into housing 102, one or more of virtual fences 302 may be moved, expanded or contracted, removed, etc.

As shown in FIG3 , the OET device depicted in FIG2 can also form a light trap 304 (e.g., a cage) that captures microtargets 116 to select and move microtargets 116. Light trap 304 can be, for example, a light cage that captures microtargets 116. The frequency of power source 212 in FIG2 can be such that light trap 304 repels selected microtargets 116. Microtargets 116 can thus be moved within chamber 110 by moving light trap 304 on electrode-activated substrate 208. Detector 220 can capture images of microtargets 116 in channel 110 (e.g., flow path 126), which can be an example of a common space. Specific desired individual microtargets among microtargets 116 can thus be identified and selected by selector 118 (e.g., configured as the OET device of FIG2 ), for example, via light traps 302, 412, as discussed below with reference to FIG3 and FIG4 . Detector 220 and selector 118 (eg, configured as the OET device of FIG. 2 ) may therefore be examples of devices that deterministically select or place one or more of micro-targets 116 .

Although shown as a square in FIG3 , fence 302 may alternatively have other shapes. For example, fence 302 may be circular, oval, rectangular, triangular, etc. Furthermore, fence 320 need not be completely enclosed. For example, any fence in fence 302 may have opening 308 , as illustrated by fence 302 in FIG3 . Although shown as a circle, light trap 304 may have other shapes, such as square, oval, rectangular, triangular, etc. Furthermore, fence 302 may have different sizes and may be arranged in different orientations.

FIG4 (which shows a partial top cross-sectional view of a portion of the housing 102) shows another example of a configuration for the apparatus 100 of FIG1A-1C. In the configuration shown in FIG4, the enclosures 112 (labeled 402 in FIG4) can be entirely physical or both physical and virtual. For example, as shown, each enclosure 402 can include a physical barrier 404 (e.g., as part of the housing 102) that can define or be a portion of a perimeter 406 having an opening 408 in fluid communication with (e.g., in contact with) the flow 314 of the medium 114 passing through the chamber 110.

3 , a flow 314 of medium 114 can be provided through chamber 110 in flow path 126. Each fence 402 can isolate microtarget(s) 116 in that fence 402 from microtargets 116 in other fences 302. For example, each fence 302 can prevent any microtarget 116 outside that fence 302 from mixing with any microtarget 116 inside that fence 302. However, flow 314 of medium 114 can be a common flow 314 provided to all fences 402 and, therefore, all microtargets 116 within those fences 402. However, fences 402 can be configured such that a first medium 122 from flow 314 does not flow directly into any of the fences 402, but the structure of fences 402 can allow for diffuse mixing of the first medium 122 from flow 314 with the second medium 124 of the fences 402.

For example, the barrier 404 of each fence 402 can be shaped and oriented to prevent the first medium 122 from the flow 314 in the flow path 126 from flowing directly into the fence 402. For example, each fence 402 can be shaped and oriented such that a portion of the physical barrier 404 directly faces the direction of the flow 314, while no openings (e.g., openings 408) directly face the direction of the flow 314. In the example shown in FIG3 , each of the fences 402 thus prevents the first medium 122 from the flow 314 in the flow path 126 from flowing directly into the fence 402.

As another example, barrier 404 may be shaped and oriented to prevent convective flow of first medium 122 from flow 314 in flow path 126 into fence 402. However, each fence 402 may be shaped and oriented to allow substantially only diffuse mixing of first medium 122 from flow 314 in flow path 126 with second medium 424 inside fence 402. For example, each fence 402 may include openings shaped and oriented to allow such diffuse mixing.

However, in some embodiments, the openings 402 can be oriented so that the openings 408 point in any direction relative to the flow 314 of the medium 114. Also as shown, any of the fences 402 can include both physical barriers and virtual portions. For example, in some embodiments, a virtual gate 410, including adjacent activated DEP electrodes on the inner surface 120 of the chamber 110, can be formed or removed at the openings 408 of one or more of the physical barriers 404 to selectively fully enclose the fence 402, as generally illustrated in FIG4 . The virtual gate 410 can correspond to light in the light pattern 214 projected onto the electrode-activated substrate 208. (See FIG2 .)

As also shown in FIG4 , one or more of the fences 402 include one or more such virtual doors 410. For example, as shown, fence 402a includes one or more openings 408a, 408b for entering fence 402a, and a virtual door 410a, 410b may be present at each of these openings 408a, 408b. In operation, micro-object 116 may move into fence 402a through first opening 408a when first virtual door 410a is closed, and micro-object 116 may later move out of fence 402a through second opening 408b when second virtual door 410b is closed.

A light trap 412 (which may be similar to or identical to light trap 304) may be formed on surface 120 of chamber 110 by electrode mechanism 108 configured as the OET device of FIG2 . Light trap 412 may be formed to capture microtarget 116 to select microtarget 116. The frequency of power source 212 in FIG2 may be such that light trap 412 repels selected microtarget 116. Thus, microtarget 116 may be moved within chamber 110 by moving light trap 412 across photoconductive layer 308. For example, microtarget 116 may be selected and moved into and/or out of enclosure 402 by forming light trap 412 that captures microtarget 116 and then moving light trap 412 across interior surface 102.

4, the fence 402 may alternatively be of other shapes. For example, the fence 402 may be of a partial circle, an ellipse, a rectangle, a triangle, etc. The light trap 412 may similarly have other shapes besides the circular shape shown.

Similar to fences 302 and 402, fence 112 can also be configured in some embodiments to prevent the first medium 122 from the common flow in the flow path 126 from directly (e.g., convective flow) into the fence 112, while allowing essentially only diffusion mixing of the first medium 122 from the common flow in the flow path 126 with the second medium inside the fence 112.

However, the housing 102 need not be configured with a single common space for the medium 114. Rather, the housing 102 may include one or more interconnected chambers, channels, etc. that contain the medium 114 and through which the medium 114 may flow. Examples are shown in Figures 5A-7.

As shown in Figures 5A and 5B, the housing 102 of the device 100 (see Figures 1A-1C) can include a base (e.g., substrate) 502 on which one or more microfluidic structures 500 are disposed. The base 502 can include, for example, a lower wall 206 as described above with reference to Figure 2, and all or part of the top surface of the microfluidic structure 500 can include an upper wall 202 including any of the variations described above.

As shown, the microfluidic structure 500 can include channels 504 and fences 506, each of which can include a perimeter 510 and an opening 508 to the channel 504. As shown, the fences 506 and the channels 504 can be at the same height or at different heights from the base 502. The channels 504 and fences 506 can correspond to the chamber 110 of Figures 1A-1C and 2, while the surface 522 of the base 502 can correspond to the interior surface 120 of the chamber 110 of Figures 1A-1C and 2. Therefore, in embodiments of the present invention in which the housing 102 includes the base 502 and the OET device of Figure 2, the DEP electrodes can be activated and deactivated according to the light pattern 216 at the surface 522 of the base 502 rather than at the interior surface 120 of the chamber 110.

One or more micro-targets 116 can be deterministically selected (e.g., using the detector 220 and selector 118 described above) and moved from the channel 504 (which can be an example of a common space and/or flow path) through the opening 508 into the perimeter 510 of the enclosure 506. The micro-target 116 can then retain the micro-target(s) within the enclosure 506 for a period of time. The opening 508 and perimeter 510 of each enclosure can be sized and configured, and the flow rate of the flow 520 of the medium 114 in the channel 504 can be such that the flow 520 creates little to no significant convection inside the perimeter 510. Once placed within the enclosure 506, the micro-target(s) 116 thus tend to remain within the enclosure 506 until actively removed from the enclosure 506. Diffusion through the opening 508 between the medium 114 in the channel 504 and the enclosure 510 can provide an inflow of nutrients for the microtarget(s) 116 in the enclosure 506 from the channel 504 into the enclosure 510 and an outflow of waste from the microtarget(s) 116 from the enclosure 510 into the channel 504.

The fence 506 can be configured such that the first medium 122 in the flow 520 in the channel 504 does not flow directly into any fence therein, but the structure of the fence 506 allows the first medium 122 from the flow 520 to diffusely mix with the second medium 124 inside the fence 506 through the openings 508 in the fence 506, generally as discussed above.

The channel 504 and the fences 506 can be physical structures, as shown in Figures 5A and 5B. For example, the microfluidic structure 500 can include a flexible material (e.g., rubber, plastic, and elastomers, polydimethylsiloxane ("PDMS"), etc.), which can also be breathable in certain embodiments. Alternatively, the microfluidic structure 500 can include other materials including rigid materials. Although one channel 504 and three fences 506 are shown, the microfluidic structure 500 can include more than one channel 504 and more or less than three fences 506. As shown in Figure 5B, virtual gates 512 can be optionally formed and removed to close and open the opening 508 of each of the fences 506. These virtual gates 512 can be formed by activating DEP electrodes at the surface 522 of the base 502, generally as described with respect to the inner surface 120.

5A and 5B , the channels 504 and the fences 506 may alternatively be virtual. For example, all or part of the channels 504 and/or the fences 506 may be formed by activating DEP electrodes at the upper surface 522 of the base 502, as generally described above.

In the example shown in Figures 6A and 6B, the housing 102 of the device 100 (see Figures 1A-1C) can include the base 502 of Figures 5A and 5B and a microfluidic structure 602 disposed on the surface 522 of the base 502. As can be seen in Figure 6B, the microfluidic structure 602 can include a fence structure 612, which can include fences 606. Each such fence 606 can include a boundary 610 within which a microtarget 116 can be placed and held for a period of time. Also as shown in Figure 6B, the microfluidic structure 602 can define channels 604, and the opening 608 of each fence 606 can be in fluid communication with (e.g., in contact with) the medium 114 in one of the channels 604.

One or more micro-targets 116 (as described above) can be deterministically selected and moved from one of the channels 604 (which can be examples of common spaces and/or flow paths) through opening 608 into a perimeter 610 of enclosure 606. The micro-target(s) 116 can then be held within enclosure 606 for a period of time. Thereafter, the micro-target(s) 116 can be moved from perimeter 610 into channel 604 through opening 608. The flow 620 of the medium 114 in channel 604 can cause the micro-target 116 to move within channel 604.

Because openings 608 of enclosure 606 are in fluid communication with channel 604, flow 620 of medium 114 in channel 604 can provide nutrients to microtargets 116 in enclosure 606 and allow waste products from microtargets 116 to flow out during the period that microtargets 116 are held in enclosure 606. Flow 620 in channel 604 can thus constitute a common flow of medium 114 to enclosure 606, which, like enclosure 506, can otherwise physically separate and isolate microtargets 116.

The fence 606 can be configured such that the first medium 122 in the flow 620 in the channel 604 does not flow directly into any of the fences 606, but the structure of the fence 606 allows the first medium 122 from the flow 620 to diffusely mix with the second medium 124 inside the fence 606 through the openings 608 in the fence 606. For example, the fence 606 can be physical (rather than virtual), and the openings 608 of the fence 606 can be oriented in any direction as long as no portion of the openings 608 directly faces the flow 620. Thus, the fence 606 can prevent the first medium 122 from flowing directly into the fence 606.

The enclosures 606 may be physical structures as shown in FIG6B . For example, the microfluidic structure 600 may include any of the materials described above with respect to the microfluidic structure 500 of FIG5A and FIG5B . Although two channels 604 and twelve enclosures 606 are shown in FIG6B , the microfluidic structure 602 may include more or less than two channels 604 and more or less than twelve enclosures 606. Although not shown, a virtual gate, similar to the gate 512 of FIG5B , may optionally be formed at the opening 608 of one or more of the enclosures 606.

While the microfluidic structure 602, including the fence structure 612, is shown as physical in Figures 6A and 6B, all portions of the structure 602 may alternatively be virtual and, therefore, generated by activating DEP electrodes at the surface 522 of the base 502, as described above with respect to the interior surface 120. For example, all or part of the fence structure 612 may be virtual rather than physical.

FIG. 7 is similar to FIG. 6B , except that channels 704 (which may be examples of flow paths 126) are disposed between enclosure structures 712, as shown. Alternatively, each enclosure 706 may be similar to each enclosure 606. For example, each enclosure 706 may include a perimeter 710 within which microtargets 116 may be placed and retained. Also as shown in FIG. 7 , an opening in each enclosure 706 may be in fluid communication (e.g., in contact) with the medium 114 in the channel 704. One or more microtargets 116 may be deterministically selected (as described above) and moved from the channel 704 (which may be an example of a common space) through the opening 708 into the perimeter 710 of the enclosure 706. The microtarget(s) 116 may be retained within the perimeter 710 for a period of time. Thereafter, the microtarget(s) 116 may be moved from the perimeter 710 through the opening 708 into the channel 704. The flow 720 of the medium 114 in the channel 704 can cause the microtarget 116 to move in the channel 704. Alternatively or in addition, the microtarget 116 can be moved by DEP forces, centrifugal forces, or the like.

Because openings 708 of enclosures 706 are in fluid communication with channels 704, flow 720 of medium 114 in the channels can also provide nutrients to micro-targets 116 in enclosures 706 and provide for the outflow of waste products from micro-targets 116 during the period that the micro-targets 116 are held in enclosures 706. Flow 720 in channels 704 can thus constitute a common flow of medium 114 to all of the enclosures 706.

The fence 706 can be configured such that the first medium 122 in the flow 720 in the channel 704 does not flow directly into any of the fences 706, but the structure of the fence 706 allows the first medium 122 in the channel 704 to diffusely mix with the second medium 124 in the fence 706 through the openings 706 in the fence 706. For example, the fence 706 can be physical and can be oriented such that no opening of the fence 706 directly faces the flow 720.

Although one channel 704 and twelve fences 706 are shown in FIG7 , there may be more or fewer. Although not shown, a virtual gate, similar to gate 512 of FIG5B , may optionally be formed at opening 706 of one or more of the fences 708. Although microfluidic structure 712 is shown as physical in FIG7 , all or part of the fence structure 702 may alternatively be virtual and thus formed by activating DEP electrodes at surface 522 of base 502, as described above with respect to interior surface 120.

The shapes and configurations of the fences 506, 606, 706 (or any fence disclosed herein) shown in Figures 5A-7 are merely examples, and these fences 506, 606, 706 (or any fence disclosed herein) can take other shapes and/or configurations. For example, any of the fences 506, 606, 706 (or any fence disclosed herein) can be circular, oval, triangular, etc., rather than square or rectangular. As another example, any of the fences 506, 606, 706 (or any fence disclosed herein) can be replaced by the fences 806, 826, 906, 926 shown in Figures 8A-10.

As shown in FIG8A , the fence may include an opening 812 (e.g., corresponding to openings 506, 606, 706) that is smaller than the full width of the perimeter 810 (e.g., corresponding to perimeters 510, 610, 710). Also as shown in FIG8A , the fence 806 may include one or more second openings 814 (one is shown, but more may be present). Opening 812 may be larger than microtarget 116 (not shown in FIG8A ), and second opening 814 may be smaller than microtarget 116. Second opening 814 may allow, for example, medium 114 (not shown in FIG8A ) to flow into or out of the fence 806. For example, medium 114 may flow into the fence 806 through opening 812 and out of the fence 806 through second opening 814. Also as shown in FIG8A , the walls of the fence do not need to have the same thickness.

As shown in FIG. 8B , the fence 826 may include an inner wall 834 extending from an opening 832 (eg, corresponding to openings 508 , 608 , 708 , 812 ) to form an inner receiving space 836 within a perimeter 840 (eg, corresponding to perimeter 510 , 610 , 710 ).

As shown in FIG9 , fence 906 may include one or more additional fences 916 (one shown, but more may be present). For example, one or more second inner fences 916 (one shown, but more may be present) including an opening 922 and a perimeter 920 may be disposed inside perimeter 910 of outer fence 906, which may include opening 912. One or more micro-targets 116 (not shown in FIG9 ) may be disposed within each inner fence 916 and the perimeter of outer fence 906.

As shown in FIG10 , fence 926 (including opening 932 and perimeter 930) may include a plurality of holding spaces 936 (although three are shown, more or fewer may exist) separated by interior walls 934. One or more micro-targets 114 (not shown in FIG10 ) may be disposed in each holding space 936. For example, different types of micro-targets 116 may be disposed in each holding space 936.

Any of the fences disclosed herein may be configured similarly to or have any of the features of the fences 806, 826, 906, 926 shown in Figures 8A-10.

Regardless of the configuration of the enclosure, a micro-target 116 can be deterministically selected and moved from a stream 520, 620, 720 in a channel 504, 604, 704 into an enclosure 506, 606, 706 in Figures 5A-7 (including variations of the enclosures 506, 6060, 706 shown in Figures 8A-10) by any of a variety of mechanisms. Figures 11A-12B illustrate an example in which the OET device of Figure 2 is used for this purpose. In Figures 11A-12B, channel 1104 can be any of channels 504, 604, 704; enclosure 1106 can be any of channels 506, 606, 706; and stream 1120 of medium 114 can be any of the streams 520, 620, 720 in Figures 5A-7.

As shown in FIG11A , micro-targets 116 can be deterministically selected in a stream 1120 in a channel 1104 by forming a light trap 1108 (e.g., similar to light trap 304) that captures the micro-targets 116, which can trap the micro-targets 116 in the light trap 1108. As shown in FIG11B , the light trap 1108 can then be moved from the channel 1104 into an enclosure 1106, where the micro-targets 116 can be released from the light trap 1108. The light trap 1108 can be similar to light traps 304, 412 and can be formed and moved on the surface 522 of the base 502 by the OET device of FIG2 in the same manner as the light traps 304, 412 are formed and moved on the interior surface 120 as described above.

As shown in FIG12A , by forming a virtual barrier 1208 in the path of the micro-target 116 in the channel 1104, the micro-target 116 can be deterministically selected in the flow 1120 in the channel 1104. As shown in FIG12B , the virtual barrier 1208 can deflect the micro-target 116 into the fence 1106. The virtual barrier 1208 can be formed by activating the DEP electrodes on the surface 522 of the base 502 using the OET device of FIG2 , as generally described above. Once the selected micro-target 116 is deflected into the fence 1106, the virtual barrier 1208 can be removed from the channel 1104.

As described above, micro-target 116 can be housed in any of the enclosures disclosed herein for a period of time, after which micro-target 116 can be removed from the enclosure. In some embodiments, micro-target 116 can be removed from the enclosure in any of the ways shown in Figures 11A-12B.

For example, a light trap 1108 can be formed in the fence 1106 to capture the micro-target 116, and the light trap 1108 can be moved out of the fence 1106 and into the channel 1104, which is the reverse of the process shown in Figures 11A and 11B. Once in the channel 1104, the light trap 1108 can be removed to release the micro-target 116 into the flow 1120 of the medium 114 in the channel 1104.

As another example, a virtual barrier similar to barrier 1208 shown in Figures 12A and 12B can be formed in fence 1106 to nudge microtarget 116 out of fence 1106 and into flow 1120 of medium 114 in channel 1104. This is the reverse of the process shown in Figures 12A and 12B.

As yet another example, any of the physical fences disclosed herein can be configured similarly to the output mechanism 800 disclosed in the aforementioned U.S. Patent Application Serial No. 13/856,781 (Attorney Docket No. BL1-US). In such a configuration, the fence can be configured similarly to the extrusion mechanism 804 in the aforementioned patent application, and a striking mechanism similar to the striking mechanism in the aforementioned patent can be provided to extrude the micro-target 116 from the fence.

13A and 13B illustrate a microfluidic device 1300, which may be an example of the device 100 of FIGS. 1A-1C , wherein the base 502 and the microfluidic structure 1302 are examples of the housing 102, the chamber 1308 is an example of the chamber 110, the inlet 1314 is an example of the inlet 104, the outlet 1316 is an example of the outlet 106, and the fence 1306 is an example of the fence 112. (Compare to FIGS. 1A-1C )

As shown in Figures 13A and 13B, device 1300 may include a microfluidic structure 1302 disposed on a base 502 (described above with reference to Figures 5A and 5B). As can be seen in Figure 13B, the microfluidic structure 1302 and base 502 may define a chamber 1308 for the medium 114 and the microtarget 116. The medium 114 with the microtarget 116 may be input into the chamber 1308 through an inlet 1314 and output from the chamber 1308 through an outlet 1316. Thus, a flow 1320 of the medium 114 may be provided in the chamber 1308 from the inlet 1314 to the outlet 1316. The inlet 1314 and the outlet 1316 may be the same or similar to the inlet 104 and the outlet 106 of Figures 1A-1C, as described above. The channel 1304 is an example of a flow path and/or common space for the medium 114.

13B , the aerator 1310 and the array 1312 of fences 1306 and the passage 1304 can be disposed in the chamber 1308 between the inlet 1314 and the outlet 1316 and, therefore, in the flow 1320 of the medium 114. The flow 1320 of the medium 114 can thus pass from the inlet 1314, through the aerator 1310, through the passage 1304 of the fence array 1312, and out the outlet 1316. Alternatively, the inlet 1314 can be located between the aerator 1310 and the fence 1304, and, therefore, the aerator 1310 can be located upstream of the inlet 1314.

Channel 1304 and fence 1306 can be similar to any of the channels and fences described herein. For example, channel 1304 can be similar to any of channels 504, 604, 704, 1104, 1304, including any variations of those channels, and fence 1306 can be similar to any of fences 112, 302, 402, 506, 606, 706, 806, 806, 1106, 1206, including any variations of those fences.

The opening of enclosure 1306 can be in fluid communication with (e.g., in contact with) one of the channels in channel 1304. As microtargets 116 (not shown in Figures 13A and 13B) move with flow 1320 of medium 114, some of the microtargets 116 in channel 1304 can be selected and moved into enclosure 1306. Microtargets 116 can be deterministically selected in channel 1304 and moved into enclosure 1306 using any of the techniques or mechanisms described above (e.g., by light traps such as light traps 304, 412, 1108; by virtual barriers such as barrier 1208; or the like). Flow 1320 of medium 114 can also be a common flow that delivers nutrients to microtargets 116 in enclosure 1306 and provides an outflow of waste from microtargets 116 in enclosure 1306, which can additionally isolate microtargets 116 from each other. In addition, each fence 1306 can be configured such that the medium 114 in the flow 1320 in the channel 1304 (e.g., the first medium 122 shown in Figures 1B and 1C) does not flow directly into any of the fences 1306, but the structure of each fence 130 can allow the medium 114 from the flow 1320 in the channel 1304 to diffusely mix with the medium 114 in the fence 1306 (e.g., the second medium 124 shown in Figures 1B and 1C), as generally described above.

The configuration of the fence array 1312 in Figure 13B is merely an example. Figures 14 and 15 illustrate examples of alternative configurations.

14 , fence array 1400 may include multiple rows of fences 1402, and openings of fences 1402 may be in fluid communication with (e.g., in contact with) a single channel 1404. Fence array 1400 and channel 1404 may replace fence array 1312 and channel 1304 in FIG. 13B , and flow 1320 of medium 114 in FIG. 13B may pass through channel 1404.

The fence array 1500 and channels 1504 in FIG15 can also replace the fence array 1312 and channels 1304 in FIG13B. As shown in FIG15, the fence array 1500 may include multiple rows of fences 1502 having openings in direct fluid communication with channels 1504c. Multiple first branch channels 1504b can connect input channels 1504a to channels 1504c that flow directly through the fences 1502. Other (second) branch channels 1504d can connect channels 1504c to output channels 1504e. The flow 1320 of the medium 114 in FIG13B can enter the first channel 1504a, pass through the branch channels 1504b to the channel 1504c in direct fluid communication with the fences 1502, and pass through other branch channels 1504d to the second channel 1504e.

The channels 1404 and 1504 in Figures 14 and 15 can be similar to the channel 1304 described above. The fences 1402, 1502 can also be similar to the fence 1306 described above. The channels 1404, 1504 can be examples of common spaces and/or flow paths. Each fence 1402, 1502 can be configured so that the medium 114 in the flow in the channel 1404, 1504 (e.g., the first medium 122 shown in Figures 1B and 1C) does not flow directly into the fence 1402, 1502, but the structure of each fence 1402, 1502 can allow the medium 114 from the flow in the channel 1404, 1504 to diffusely mix with the medium in the fence 1402, 1502 (e.g., the second medium 124 shown in Figures 1B and 1C), as generally described above.

FIG16 shows an example of a process 1600 for processing biological microtargets in an enclosure. The process 1600 can be implemented using any of the microfluidic devices described above or similar devices. For example, the process 1600 can be implemented using microfluidic devices 100 and 1300, including any variations of those described above (e.g., as shown in FIG2-12B , FIG14 , and FIG15 ).

As shown in FIG16 , at step 1602, process 1600 may load a biological microtarget into a microfluidic device. For example, process 1600 may introduce microtarget 116 in medium 114 into chamber 110 of device 100 of FIG1A-1C via inlet 104. As another example, process 1600 may introduce microtarget 116 in medium 114 into chamber 1308 of device 1300 of FIG13A and 13B via inlet 1314.

At step 1604, the process may select individual micro-targets from the biological micro-targets loaded at step 1602. For example, process 1600 may select a subset of less than all of the micro-targets 116 in medium 114 that have a particular characteristic. The micro-targets 116 may be monitored, for example, using imaging device 220 of FIG. 2 . At step 1604, one micro-target 116 having a specific desired characteristic may be deterministically selected and loaded into one enclosure, such that step 1604 results in one and only one micro-target 116 in each of a plurality of enclosures. Alternatively, more than one micro-target 116 may be loaded into an enclosure.

At step 1606, process 1600 may place the microtarget 116 selected at step 1604 into an enclosure of the microfluidic device. For example, at step 1606, process 1600 may place the selected microtarget 116 into an enclosure 112, 302, 402, 506, 606, 706, 806, 906, 1106, 1206, 1306, 1402, 1502 using any of the techniques described above. As described above and shown throughout the figures, the enclosures described above may physically separate the microtargets 116 from one another. That is, each enclosure may physically separate the microtarget 116 or microtargets 116 in that enclosure from all other microtargets 116 in the microscopic device 100, 1300. After placing the selected microtarget 116 in the enclosure at step 1606, process 1600 may maintain the microtarget 116 in the enclosure for a period of time.

At step 1608, process 1600 can provide a flow of liquid medium 114 to the enclosure. Step 1608 can be accomplished by providing any of flows 314, 314, 520, 620, 720, 1120, 1320 in chambers 110, 1308 or channels 504, 604, 704, 1104 as described. Note that at step 1606, the various microtargets 116 can be physically isolated from one another by being placed in physically separate enclosures, but at step 1608, those microtargets 116 in the enclosures can be provided with the same flow of medium 114. As described above, the fence 112, 302, 404, 506, 606, 706, 806, 906, 1106, 1206, 1402, 1502 can be configured to prevent the medium 114 (e.g., the first medium 122 shown in Figures 1B and 1C) from flowing 314, 314, 520, 620, 720, 1120, 1320 in the chamber 110, 1308 or the channel 504, 604, 704, 1104 directly from the chamber 110, 1308. 1B and 1C ) is allowed to diffusely mix with the medium 114 on the inside of the fence (e.g., the second medium 124 shown in FIG. 1B and 1C ).

As mentioned, the microtargets 116 placed into the enclosure at step 1606 may be maintained in the enclosure for a period of time, during which period, step 1608 may provide a flow of medium 114 to the microtargets 116, which, through the aforementioned diffusion mixing, may provide nutrients to the microtargets 116 in the enclosure and provide for the outflow of waste products from the microtargets 116. At step 1610, process 1600 may monitor one or more biological activities of the microtargets 116 in the enclosure. Examples of such biological activities may include colony production, secretion of certain biological substances, etc. The monitoring at step 1610 may be continuous during the period of time, periodic during the period of time, at the end of the period of time, etc. The monitoring at step 1610 may be implemented in any manner suitable for analyzing the biological activities of the microtargets 116. For example, the monitoring at step 1610 may be implemented using the imaging device 220 of FIG. 2 , using sensors (not shown) in or adjacent to the enclosure, etc.

At step 1612, process 1600 may select microtargets 116 in the enclosure that meet a predetermined criterion, threshold, or condition associated with the biological activity or state monitored at step 1610. The microtargets 116 selected at step 1612 may be removed from the enclosure for further processing or use. For example, the selected microtargets 116 may be removed from the enclosure using any of the techniques or processes described above. As another example, one or more microtargets 116 may be removed from the enclosure by piercing the housing with a needle aspirator (not shown) and removing the microtargets 116 with the aspirator. A specific controlled number of microtargets 116 may be removed, for example, by selecting and removing that number of microtargets 116, or if the microtargets 116 are biological cells, removing all cells when the colony of cloned cells reaches a desired number.

At step 1614 , process 1600 may discard micro-targets 116 not selected at step 1612 , which are micro-targets 116 that do not meet a predetermined criterion, threshold, or condition associated with the biological activity or state monitored at step 1610 .

FIG17 illustrates an example process 1700 for propagating a population of clonal cells from a single mother cell, according to certain embodiments of the present invention. This process 1700 may be an instance of process 1600 of FIG16 . For example, process 1700 may begin after performing steps 1602 and 1604 of FIG16 ; steps 1702-1706 may be performed between steps 1606 and 1608; step 1708 may be an instance of step 1610; step 1710 may be an instance of step 1614; and step 1712 may be an instance of step 1612.

For ease of illustration and discussion, process 1700 is discussed below as being implemented by device 100 configured with the OET device of Figure 2 to form and manipulate virtual fence 302 of Figure 3. However, process 1700 may be implemented by other configurations of device 100 or device 1300 in which the fence is a virtual fence.

As shown in Figure 17, process 1700 may process the cells in the enclosure at step 1702. Such processing may include fusing two cells into one cell, transfecting the cells by injecting a biological vector into the cells, etc. Figures 18A-18C illustrate examples.

As shown in FIG18A , two different types of microtargets 116 and 1804 can be placed in a medium 114 in a chamber 110. The OET device of FIG2 can generate optical traps 1806, 1808 (e.g., like optical trap 304) to select one of the first cell type 116 and one of the second cell type 1804. The optical traps 1806 and 1808 can then be moved into contact, such that the first cell type 116 and the second cell type come into contact as shown in FIG18B . These paired cells 1810 can then be subjected to one or more treatments (e.g., a fusion chemical (e.g., polyethylene glycol (PEG), Sendai virus, a thin film that pierces one of the cells 116, 1804, an electric field, pressure, etc.) included in the stream 314) to fuse the paired cells 1810 together to form fused cells 1812 as shown in FIG18C . That is, each fused cell 1812 can include one of the first cell type 116 and one of the second cell type 1804 fused together. Light traps 1806 and 1808 may be similar and may be formed and operate similarly to the light traps 304 , 412 described above, including any variations thereof.

18C , each fused cell 1812 can be placed in a virtual enclosure 1814, 1816, 1818, 1820. Although four enclosures 1814, 1816, 1818, 1820 are shown, there may be more or fewer. Virtual enclosures 1814, 1816, 1818, 1820 can be the same as or similar to enclosure 302 of FIG. 3 , as described above.

Alternatively, component 1804 in Figure 18A can be a cell vector to be transfected into microtargets 116. In this case, each cell 1812 in Figure 18C can be one of the microtargets 116 transfected with vector 1804.

As yet another alternative, cells 1812 in Figure 18C (whether fused or transfected) can be processed in another device and then placed into enclosures 1814, 1816, 1818, 1820. In this alternative, step 1702 is not included in the process of Figure 17. As yet another alternative, cells 1812 can be single cells rather than fused or transfected cells.

Referring again to FIG. 17 , at step 1704, clones can be propagated in each enclosure 1814, 1816, 1818, 1820 from the cells 1812 within the enclosure. This can be facilitated by including culture medium in the flow 314 through the chamber 114. At step 1706, the enclosures 1814, 1816, 1818, 1820 can be expanded as the clones propagate within each enclosure. An example is shown in FIG. 19 . As shown in FIG. 19 , as the number of cells 1812 within each enclosure 1814, 1816, 1818, 1820 increases, the size of the enclosures 1814′, 1816′, 1818′, 1820′ can be expanded to accommodate the population of propagated clones within each enclosure.

At step 1708 of Figure 17, each enclosure 1814, 1816, 1818, 1820 can be inspected and analyzed for clonal proliferation within the enclosure. For example, a fluorescent tag (e.g., a bioluminescent compound that fluoresces when stimulated or otherwise) bound to the clones can be included in the flow 314 passing through the chamber 110. The level of fluorescence emitted by each enclosure 1814, 1816, 1818, 1820 can then be analyzed to determine the clonal proliferation within each enclosure.

At step 1710, clones in enclosures 1814, 1816, 1818, 1820 where clones 1812 have reproduced less than a minimum amount (or are otherwise undesirable) may be discarded. FIG20 illustrates an example. For the purposes of the example shown in FIG20 , assume that at step 1710, it is determined that clones 1812 in enclosures 1814′, 1820′ of FIG19 have reproduced less than a minimum threshold amount and are to be discarded. As shown in FIG20 , the enclosures may be removed to release the clones 1812 in these enclosures. This can be accomplished simply by removing the light corresponding to enclosures 1814′, 1820′ from the light pattern 216 incident on housing 102 of FIG2 . The now-released clones 1812 previously in enclosures 1814′, 1820′ may then be ejected from chamber 110 (e.g., via flow 314) and discarded.

As shown in FIG17 , steps 1704 through 1710 can be repeated to continue propagating clones 1812 in enclosures 1816′, 1818′. Alternatively, at step 1712, individual clones 1812 can be selected from enclosures 1814′, 1820′ and placed in new enclosures as child clones. Repeating steps 1704 through 1710 can propagate, test, and discard slowly propagating clones in the new enclosures. FIG21 illustrates an example in which individual child clones 1812 from enclosures 1816′, 1818′ in FIG20 are selected and placed in new enclosures 2102. New enclosures 2102 can be formed and manipulated in the same manner as enclosures 1814, 1816, as described above. Individual child clones 1812 can be selected and moved generally as described above (e.g., using light traps similar to light traps 304, 412 of FIG4 ).

FIG22 illustrates a process 2200 , which is a variation of process 1700 of FIG17 .

As shown in FIG22 , one or more cells can be held in and secreted into the enclosure. For example, as shown in FIG18C , a cell 1812 can be disposed in each of enclosures 1814, 1816, 1818, and 1820. Alternatively, more than one cell 1812 can be present in each of enclosures 1814, 1816, 1818, and 1820. Cells 1812 can be fused or transfected cells as described above with reference to FIG18A-18C . Alternatively, cells 1812 can be simple cells rather than fused or transfected cells.

22 , each enclosure 1814, 1816, 1818, 1820 can be inspected and the productivity of the cells 1812 in the enclosure can be analyzed. For example, one or more cells 1812 can be removed from each enclosure 1814, 1816, 1818, 1820 and observed, tested, etc. to determine the secretion productivity of the removed cells.

At step 2206, enclosures 1814, 1816, 1818, 1820 in which cells 1812 are secreted at less than a threshold level can be discarded. This can be accomplished generally as shown in FIG20 and described above. That is, enclosures 1814, 1816, 1818, 1820 containing slow-rate producing cells 1812 can be removed, and the slow-rate producing cells 1812 can be washed away generally according to the discussion of FIG20 above.

22 , steps 2202 through 2206 may be repeated to continue causing secretion from the cells 1812 in the remaining enclosures, testing the secretion productivity of the cells in each enclosure, and discarding the cells in the slow-producing enclosures 1812. Alternatively, at step 2208, individual ones of the fast-producing cells 1812 may be selected and placed as daughter cells in new enclosures (e.g., generally according to the example shown in FIG. 21 ), and steps 2202 through 2206 may be repeated to cause secretion from the daughter cells in their new enclosures, testing the secretion productivity of the daughter cells in each enclosure, and discarding the daughter cells in the slow-secreting enclosures.

While specific embodiments and applications of the present invention have been described in this specification, these embodiments and applications are merely exemplary and many variations are possible.

Claims (42)

1. A microfluidic device, comprising: A housing, comprising a substrate and a microfluidic structure disposed on a surface of the substrate, wherein the substrate and the microfluidic structure define an internal chamber for accommodating a first liquid medium and a biological microtarget suspended in the first liquid medium, and wherein the microfluidic structure comprises (i) a channel providing a flow path for the first liquid medium, and (ii) a plurality of physical retaining fences, wherein each of the plurality of retaining fences comprises a boundary and a single opening, wherein: The enclosure surrounds an interior space, which is configured to maintain biological micro-targets suspended in a second liquid medium. The single opening leads to the channel, and The single opening is oriented such that no part of the flow path directly faces the channel, thus preventing the first liquid medium from flowing directly into the second liquid medium in the interior space when the channel accommodates the flow of the first liquid medium and the retaining fence accommodates the second liquid medium, while allowing the first liquid medium to diffuse and mix with the second liquid medium in the interior space; and The surface of the substrate includes dielectrophoretic (DEP) electrodes configured to be selectively activated and deactivated. 2. The microfluidic device of claim 1, wherein at least one of the plurality of retaining fences includes an inner wall extending from the opening into the enclosure. 3. The microfluidic device of claim 2, wherein the inner wall of the at least one retaining fence includes a barrier between the opening of the retaining fence and the internal storage space within the enclosure of the retaining fence. 4. The microfluidic device according to claim 1, wherein at least one of the retaining fences further comprises an inner fence, the inner fence comprising a boundary and an opening, wherein the inner fence is disposed inside the boundary of the at least one retaining fence. 5. The microfluidic device according to claim 1, wherein: At least one of the plurality of retaining fences includes a retaining space, the retaining space being separated by an internal wall disposed inside the boundary of the at least one retaining fence, and Each of the holding spaces is configured to hold one of the biological micro-targets. 6. The microfluidic device of claim 1, further comprising means for selectively forming and removing a dielectrophoretic (DEP) gate at any of the openings of the plurality of retaining fences. 7. The microfluidic device according to claim 1, wherein: (1) The DEP electrode includes a hardwired electrical connection; (2) The DEP electrode includes a photoconductive layer; or (3) The DEP electrode includes a phototransistor. 8. The microfluidic device of claim 7, wherein the DEP electrode is optically controlled. 9. The microfluidic device according to claim 1, further comprising an electrode mechanism, the electrode mechanism comprising: First electrode; The second electrode; and Electrode activation substrate, The first electrode is a portion of a first wall defining the chamber, and the second electrode and the electrode activation substrate are portions of a second wall defining the chamber. 10. The microfluidic device of claim 9, wherein the electrode activation substrate is a photoconductive material. 11. The microfluidic device of claim 10, wherein the electrode activation substrate has a featureless surface. 12. The microfluidic device of claim 10, wherein the photoconductive material is undoped amorphous silicon. 13. The microfluidic device of claim 9, wherein the electrode activation substrate comprises a semiconductor material, the semiconductor material comprising a plurality of doped layers, an electrically insulating layer, and a conductive layer forming a semiconductor integrated circuit. 14. The microfluidic device of claim 9, wherein the electrode activation substrate comprises a phototransistor. 15. The microfluidic device according to any one of claims 1-14, further comprising an electrode mechanism including an electrowetting device for moving one or more droplets of liquid medium suspended therein in the biological microtarget. 16. The microfluidic device of claim 15, wherein the electrowetting device comprises an optoelectronic wetting (OEW) device. 17. The microfluidic device according to any one of claims 1-14, further comprising: The entrance into the housing to the chamber; and The outlet that exits the housing from the chamber. 18. The microfluidic device of claim 17, wherein the channel and the plurality of retaining barriers are disposed in the chamber between the inlet and the outlet. 19. The microfluidic device of claim 18, further comprising a ventilator disposed in the chamber between the inlet and the plurality of retaining fences. 20. The microfluidic device according to any one of claims 1-14, further comprising a ventilator disposed in the housing. 21. The microfluidic device according to any one of claims 1-14, wherein the microfluidic device comprises a plurality of the channels, wherein each of the plurality of channels is fluidly connected to a plurality of the retaining fences. 22. The microfluidic device according to any one of claims 1-14, wherein all or part of the housing is breathable. 23. The microfluidic device according to any one of claims 1-14, wherein the housing comprises a flexible material. 24. The microfluidic device according to any one of claims 1-14, further comprising a sensor for monitoring the bioactivity of micro-targets within the enclosure. 25. The microfluidic device of claim 24, wherein the sensor is further configured to detect the temperature, chemical composition, pH, or osmotic pressure of the medium within the chamber. 26. A method for processing biological micro-targets, the method comprising: Each biological micro-target is actively placed within the internal space of the retaining fence in the microfluidic device according to claim 1; Providing a flow of a first liquid medium to the retaining fence for a period of time; and When providing the flow, the first liquid medium is prevented from flowing directly into the interior space of the retaining fence. 27. The method of claim 26, wherein the blocking includes allowing the first liquid medium from the flow to diffuse and mix with the second liquid medium in the interior space of the retaining fence. 28. The method of claim 26, further comprising monitoring the characteristics of the micro-target within the retaining fence during the time period. 29. The method of claim 28, wherein the characteristic is the preservation of the bioactivity of the microtargets within the enclosure. 30. The method according to claim 29, wherein: The bioactivity includes clone production, and The monitoring includes monitoring the clone production of the microtargets within the retention fence. 31. The method according to claim 29, wherein: The biological activities include secreting secretions, and The monitoring includes monitoring the secretions of the micro-targets within the retaining fence. 32. The method of claim 28, wherein the characteristic is the biological state of the micro-target within the retaining enclosure. 33. The method according to any one of claims 26-32, wherein the first liquid medium is a medium of a different type from the second liquid medium. 34. The method according to any one of claims 26-32, wherein the first liquid medium is a medium of the same type as the second liquid medium. 35. The method according to any one of claims 26-32, further comprising selecting a subset of the microtargets having predetermined characteristics from a plurality of microtargets in the microfluidic device, wherein the active placement comprises placing only the selected subset of the microtargets in the retaining fence. 36. The method of claim 35, wherein: The plurality of micro-targets are located in a common position within the microfluidic device. The selection includes selecting each micro-target from the selected subset within the public location, and The active placement includes moving each of the micro-targets in the selected subset from the common location to one of the holding fences in the holding fence. 37. The method according to any one of claims 26-32, wherein the active placement comprises placing one and only one of the respective micro-targets in each of the retaining fences. 38. The method according to any one of claims 26-32, wherein the active placement includes activating a dielectrophoresis (DEP) electrode that generates a DEP force on the respective microtargets. 39. The method of claim 38, wherein activating the DEP electrode comprises activating the DEP electrode using a photoelectric tweezers device. 40. The method according to any one of claims 26-32, wherein the providing includes allowing the first liquid medium to flow through an opening in the retaining fence. 41. The method according to any one of claims 26-32, further comprising maintaining the respective micro-targets within the retaining fence during the time period. 42. The method of claim 26, wherein the placement comprises placing each of a plurality of different types of micro-targets in each of a plurality of different holding spaces in one of the holding fences.