Isolation of live cells following high-throughput long-term time-lapse microscopy

Abstract

The microfluidic device includes a cell flow layer and a control layer. The cell flow layer includes a proliferation channel, a collection channel, multiple bridge channels connecting the proliferation and collection channels, multiple bridge valve portions, and multiple cell growth trenches connected to the proliferation channel. The proliferation channel includes an inlet valve portion and an outlet valve portion that control flow into and out of the proliferation channel. The collection channel includes an inlet valve portion and an outlet valve that control flow into and out of the collection channel. The bridge valve portion controls flow between the proliferation channel and the collection channel. The control layer includes a first control channel that operates the bridge valve portion and a second control channel that operates the inlet valve portion and the outlet valve portion of the proliferation channel and the collection channel.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 864,091, filed June 20, 2019, and entitled "ISOLATING LIVE CELLS AFTER HIGH-THROUGHPUT, LONG-TERM, TIME-LAPSE MICROSCOPY," which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. HR0011-16-2-0049 awarded by the Defense Advanced Research Projects Agency, Grant No. 1615487 awarded by the National Science Foundation, and Grant No. DARPA-BAA-16-17 awarded by the Defense Advanced Research Projects Agency. The United States Government has certain rights in this invention.

[0003] The present disclosure relates to microfluidic devices, particularly those that enable long-term monitoring of cell populations and efficient extraction of cells of interest. [Background technology]

[0004] Genetic screens play a fundamental role in biology by identifying which genes or gene parts determine phenotypic traits. Their power depends on the breadth of mutant libraries that can be considered, the types of traits that can be measured, the ability to control growth conditions while ensuring spatiotemporal uniformity, and (since many mutations only alter the distribution of phenotypes) how reliably these distributions can be sampled for each mutant. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,691,164 [Patent Document 2] U.S. Patent No. 6,767,706 [Patent Document 3] U.S. Patent No. 8,075,748 [Patent Document 4] U.S. Pat. No. 9,149,806 [Patent Document 5] US Patent Application Publication No. 2019 / 0271634 Summary of the Invention [Problem to be solved by the invention]

[0006] Current techniques only provide low-resolution snapshots of end points, providing little information about proliferation, subcellular dynamics, and responses to environmental changes. Moreover, because each cell is probed only once, current techniques struggle to distinguish genetically stable characteristics from transient phenotypic heterogeneity. Thus, new devices and methods for imaging and analyzing cells are needed. [Means for solving the problem]

[0007] A microfluidic device for use in analyzing cells and extracting one or more cells of interest includes a substrate, a cell flow layer coupled to the substrate, and a control layer coupled to the cell flow layer. The cell flow layer includes a proliferation channel, a plurality of cell growth trenches, a collection channel, and a plurality of bridge channels. The proliferation channel has an inlet portion, an outlet portion, a main portion, an inlet valve portion, and an outlet valve portion. The proliferation channel inlet valve portion is configured to assist in selectively controlling flow between the proliferation channel inlet portion and the proliferation channel main portion. The proliferation channel outlet valve portion is configured to assist in selectively controlling flow between the proliferation channel main portion and the proliferation channel outlet portion. A plurality of cell growth trenches are fluidly coupled to the proliferation channel main portion. The collection channel has an inlet portion, an outlet portion, a main portion, an inlet valve portion, and an outlet valve portion. The collection channel inlet valve portion is configured to assist in selectively controlling flow between the collection channel inlet portion and the collection channel main portion. The collection channel outlet valve portion is configured to assist in selectively controlling flow between the collection channel main portion and the collection channel outlet portion. Each of the plurality of bridge channels connects the proliferation channel main portion and the collection channel main portion. Each of the plurality of bridge channels includes a bridge valve portion configured to assist in selectively controlling flow between the proliferation channel and the collection channel. The control layer is configured to assist in actuating (i) the bridge valve portion of each of the plurality of bridge channels, (ii) the proliferation channel inlet valve portion, (iii) the proliferation channel outlet valve portion, (iv) the collection channel inlet valve portion, and (v) the collection channel outlet valve portion.

[0008] A method for analyzing cells and extracting one or more cells of interest using a microfluidic device having a proliferation channel, a plurality of cell growth trenches fluidly connected to the proliferation channel, and a collection channel fluidly connected to the proliferation channel includes injecting one or more cells and growth medium into an inlet portion of the proliferation channel to cause the one or more cells and growth medium to flow into a main portion of the proliferation channel and fill at least one of the plurality of cell growth trenches, closing an inlet valve portion of the proliferation channel and an outlet valve portion of the proliferation channel, washing the inlet portion of the proliferation channel to remove contaminants from the inlet portion of the proliferation channel, and analyzing one or more cells in at least one of the plurality of cell growth trenches to extract one or more cells of interest. the plurality of bridge valve portions to permit fluid communication between the main portion of the proliferation channel and the main portion of the collection channel through the plurality of bridge channels; moving one or more target cells from at least one of the plurality of cell growth trenches through the main portion of the proliferation channel and one or more of the plurality of bridge channels to the main portion of the collection channel; closing the plurality of bridge valve portions; opening the collection channel inlet valve portion and the collection channel outlet valve portion; moving the one or more target cells from the main portion of the collection channel to the collection channel outlet portion; and collecting the one or more target cells from the collection channel outlet portion.

[0009] The above summary is not intended to represent each implementation or every aspect of the present disclosure. Additional features and advantages of the present disclosure will be apparent from the detailed description and drawings set forth below.

[0010] The present disclosure will be better understood from the following description of exemplary implementations with reference to the accompanying drawings. [Brief description of the drawings]

[0011] [Figure 1A] FIG. 1 is a perspective view of an exemplary microfluidic device for analyzing cells and extracting one or more cells of interest, according to an embodiment of the present disclosure. [Figure 1B] FIG. 1B is an exploded view of the exemplary microfluidic device of FIG. 1A according to an embodiment of the present disclosure. [Figure 2A] FIG. 1B is a top view of a channel layout of the exemplary microfluidic device of FIG. 1A, according to an embodiment of the present disclosure. [Figure 2B] FIG. 2B is an enlarged view of a portion of the channel layout of FIG. 2A according to an embodiment of the present disclosure. [Figure 3A] FIG. 1B is a cross-sectional view of two valve portions of the exemplary microfluidic device of FIG. 1A in an open state, according to an embodiment of the present disclosure. [Figure 3B] 3B is a cross-sectional view of the two valve portions of FIG. 3A when moving from an open state to a closed state, according to an embodiment of the present disclosure. [Figure 3C] FIG. 3B is a cross-sectional view of the two valve portions of FIG. 3A in a closed state, according to an embodiment of the present disclosure. [Figure 4A] FIG. 1B is a top view of an inlet portion of the exemplary microfluidic device of FIG. 1A during injection of cells and growth medium, according to an embodiment of the present disclosure. [Figure 4B] FIG. 4B is a top view of the inlet portion of FIG. 4A after injection of cells and growth medium, according to an embodiment of the present disclosure. [Figure 4C] FIG. 4B is a top view of the inlet portion of FIG. 4A during cleaning to remove residual contaminants, according to an embodiment of the present disclosure. [Figure 5A] FIG. 1B is a top view of a cell growth trench of the exemplary microfluidic device of FIG. 1A according to an embodiment of the present disclosure. [Figure 5B] FIG. 1B is a top view of a cell growth trench of the exemplary microfluidic device of FIG. 1A coupled to a backchannel, according to an embodiment of the present disclosure. [Figure 6] 1 is a kymograph showing proliferation of cells within cell proliferation trenches, according to an embodiment of the present disclosure. [Figure 7] 1 is a flow chart of a method for analyzing cells and extracting one or more cells of interest according to an embodiment of the present disclosure. [Figure 8A]FIG. 1B is a top view of the proliferation and harvest channels of the exemplary microfluidic device of FIG. 1A as cells and growth medium are injected into the proliferation channel, according to an embodiment of the present disclosure. [Figure 8B] FIG. 8B is a top view of the proliferation and collection channels of FIG. 8A as the inlet portion of the proliferation channel is cleaned, according to an embodiment of the present disclosure. [Figure 8C] FIG. 8B is a top view of the proliferation and collection channels of FIG. 8A as cells are transferred from the proliferation channel to the collection channel, according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] While the present disclosure is susceptible to various modifications and alternative forms, specific implementations have been shown by way of example in the drawings and are described in detail herein. It should be understood, however, that the present disclosure is not intended to be limited to the particular forms disclosed. Rather, the present disclosure should cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0013] While the present disclosure is susceptible of many different forms, it has been shown in the drawings and is described herein in detail in exemplary implementations of the disclosure, with the understanding that this disclosure should be considered as an example of the principles of the disclosure and is not intended to limit the broad aspects of the disclosure to the implementations shown.

[0014] FIG. 1A shows a perspective view of a microfluidic device 100 that can analyze cells and extract one or more cells of interest. FIG. 1B shows an exploded view of the microfluidic device 100. In some implementations, the device 100 is used for multi-generational time-lapse microscopy. In these implementations, an isogenic population of cells can be trapped and grown within the device 100, allowing cells to be imaged for multiple generations. One or more cells of interest can then be extracted from the device 100 for use in a variety of downstream applications.

[0015] The device 100 includes a cover slip 102, a cell flow layer 110 coupled to the cover slip, and a control layer 150 coupled to the cell flow layer 110. Generally, the cell flow layer 110 is mounted on the cover slip 102 and the control layer 150 is mounted on the cell flow layer 110. The cell flow layer 110 includes various channels through which cells (as well as other fluids such as growth medium and washing fluids or solutions) may flow during use. The control layer 150 includes various fluid-filled channels to actuate various valves of the cell flow layer 110. As discussed in more detail herein, the valves can be actuated to selectively control the flow of cells and other fluids through the various channels of the cell flow layer 110.

[0016] In some implementations, the cell flow layer 110 and the control layer 150 are both constructed from blocks of polydimethylsiloxane (PDMS) and cast from separate molds. In some implementations, the cover slip 102 is made from glass. The various channels of the cell flow layer 110 and the control layer 150 can be formed using any suitable manufacturing technique(s). In some implementations, the cell flow layer 110 and the control layer 150 are fabricated using multilayer soft lithography. In these implementations, a mold is first formed from a silicon wafer using UV lithography techniques. The PDMS layer is then cast by pouring liquid PDMS into the silicon mold, followed by curing to solidify the PDMS. The two PDMS layers can be bonded together (e.g., via curing or partial curing) and bonded to the cover slip 102 (e.g., via plasma bonding) before being further baked. Thus, the negative spaces of the channels in the cell flow layer 110 and control layer 150 are imprinted from the positive silicon wafer mold.

[0017] The end product of the fabrication method is a three-dimensional device 100. In some implementations, the cell flow layer 110 and the control layer 150 have a length of between about 20.0 mm and about 40.0 mm, or about 30.0 mm, the cell flow layer 110 and the control layer 150 have a width of between about 10.0 mm and about 20.00 mm, or about 17.0 mm, the cell flow layer 110 has a height of between about 40.0 μm and about 60.0 μm, or about 50.0 μm, and the control layer 150 has a height of between about 2.0 mm and about 10.0 mm, or about 6.0 mm.

[0018] The various channels of the cell flow layer 110 are defined on the lower surface of the cell flow layer 110. Thus, the cell flow layer 110 includes an upper wall 113A that forms the upper walls (e.g., ceiling) of the various channels defined in the cell flow layer 110. When the cell flow layer 110 is bonded to the cover slip 102, the cover slip 102 forms the lower walls (e.g., floor) of the various channels of the cell flow layer 110. Similarly, the various channels of the control layer 150 are defined on the lower surface of the control layer 150. An upper wall 113B of the control layer 150 forms the upper walls (e.g., ceiling) of the various channels of the control layer 150. An upper wall 113A of the cell flow layer 110 forms the lower walls (e.g., floor) of the various channels of the control layer 150. The channels of the cell flow layer 110 and the control layer 150 are each fluidly connected to the atmosphere via a plurality of vertical channels extending upwardly through the cell flow layer 110 and / or the control layer 150 and a plurality of openings defined in the upper wall of the control layer 150.

[0019] FIG. 2A is a top view of device 100 showing the two-dimensional layout of the various channels of cell flow layer 110 and control layer 150. FIG. 2B is an enlarged top view of device 100 showing a set of proliferation and collection channels. Cell flow layer 110 includes four pairs of analysis channels. Each pair of analysis channels includes a proliferation channel 112 and a collection channel 122. Cells are generally located in proliferation channel 112 during any monitoring and imaging processes during use of device 100. Once a cell of interest is identified, it can be moved to collection channel 122 from where it can be collected. In general, any one or more pairs of proliferation channel 112 and collection channel 122 can be used during operation of device 100. For example, all four pairs can be used simultaneously to analyze the same type of cells, all four pairs can be used simultaneously to analyze different types of cells, and fewer than all four pairs can be used simultaneously to analyze the same or different types of cells. Although device 100 having four pairs of proliferation channels 112 and collection channels 122 is shown in FIG. 2A, device 100 can have any number of proliferation channels 112 and collection channels 122, including one or more.

[0020] The proliferation channel 112 is formed from different portions, including an inlet portion 114A, an inlet valve portion 116A, a main portion 118, an outlet valve portion 116B, and an outlet portion 114B. The different portions of the proliferation channel 112 are located at different positions along the length of the cell flow layer 110. For example, the inlet portion 114A and the inlet valve portion 116A are located at a first end 111A of the cell flow layer 110, while the outlet valve portion 116B and the outlet portion 114B are located at a second end 111B of the cell flow layer 110. The main portion 118 is located between the first end 111A and the second end 111B. In general, each portion of the proliferation channel 112 is a channel through which cells and fluids may flow.

[0021] The cell flow layer 110 further includes a plurality of cell growth trenches 120 that are fluidly connected to at least a main portion 118 of the proliferation channel 112. The cell growth trenches 120 are disposed on a first side of the proliferation channel 112 and are configured to extend from the proliferation channel 112 in a direction perpendicular to the direction in which the proliferation channel 112 extends between a first end 111A of the device 100 and a second end 111B of the device 100.

[0022] The cell growth trenches 120 are configured to be filled with cells during use of the device 100. The cell growth trenches 120 have a width that is approximately equal to or slightly larger than the width of the cells to be analyzed during use of the device 100. Thus, the cells within any one of the cell growth trenches 120 are arranged together in a linear, one-dimensional group (e.g., the cells are geometrically constrained to a single line). As the cells that initially fill the cell growth trenches 120 begin to divide, they eventually fill the end of the cell growth trench 120 with an isogenic cell lineage. These cells generally have the same genetic makeup and are arranged in a single, one-dimensional row. The length of the cell growth trenches 120 (e.g., the distance the cell growth trenches 120 extend from the growth channel 112) can be between about 1.0 μm and about 100.0 μm. The width of the cell growth trenches 120 can be between about 0.1 μm and about 50.0 μm. The height of cell growth trenches 120 can be between about 0.1 μm and about 50.0 μm. In general, the dimensions of cell growth trenches 120 can be adjusted during the wafer fabrication process depending on the required microscopy sensitivity (e.g., closer spaced cell growth trenches will have greater trench-to-trench signal spillover than cell growth trenches spaced farther apart, e.g., due to the point spread function of light), the size of cells used in device 100, the required throughput, etc. The distance between adjacent pairs of cell growth trenches 120 can be between about 0.1 μm and about 10.0 μm.

[0023] The collection channel 122 is disposed on a second side of the proliferation channel 112 opposite the cell growth trench 120. Thus, the proliferation channel 112 is disposed between the cell growth trench 120 and the collection channel 122. The collection channel 122 also extends in a direction parallel to the proliferation channel 112 and perpendicular to the cell growth trench 120 between the first end 111A of the device 100 and the second end 111B of the device 100. Similar to the proliferation channel 112, the collection channel 122 includes an inlet portion 124A, an inlet valve portion 126A, a main portion 128, an outlet valve portion 126B, and an outlet portion 124B. The different portions of the collection channel 122 are located at different positions along the length of the cell flow layer 110. For example, inlet portion 124A and inlet valve portion 126A are located at a first end 111A of cell flow layer 110, while outlet valve portion 126B and outlet portion 124B are located at a second end 111B of cell flow layer 110. Main portion 128 is located between first end 111A and second end 111B. In general, each portion of collection channel 122 is a channel through which cells and fluids may flow.

[0024] In the illustrated implementation, the cell flow layer may also include a plurality of cell growth trenches 130 fluidly connected to the main portion 128 of the collection channel 122. The cell growth trenches 130 are disposed on a side of the collection channel 122 opposite the proliferation channel 112. The cell growth trenches 130 are configured to extend from the collection channel 122 in a direction perpendicular to the direction in which the collection channel 122 extends between the first end 111A of the device 100 and the second end 111B of the device 100. In general, the cell growth trenches 130 may have the same or similar dimensions as the cell growth trenches 120.

[0025] The cell flow layer 110 further includes a plurality of bridge channels 132 disposed between the proliferation channels 112 and the collection channels 122. The bridge channels 132 are voids defined between adjacent pairs of bridge protrusions 133 that generally extend the entire height of the cell flow layer 110 to the top wall of the cell flow layer 110. In some implementations, the bridge channels 132 are located only between the main portions 118 of the proliferation channels 112 and the main portions 128 of the collection channels 122. In other implementations, the bridge channels 132 may extend further such that the cell flow layer 110 includes bridge channels 132 located between other portions of the proliferation channels 112 and the collection channels 122. The bridge channels 132 are configured to allow cells and fluids to flow between the proliferation channels 112 and the collection channels 122.

[0026] As shown herein, proliferation channel 112 includes inlet valve portion 116A and outlet valve portion 116B, and harvest channel 122 includes inlet valve portion 126A and outlet valve portion 126B. These valve portions can be actuated to help selectively control the flow of cells and fluids through proliferation channel 112 and harvest channel 122. Inlet valve portion 116A of proliferation channel 112 controls flow between inlet portion 114A of proliferation channel 112 and main portion 118 of proliferation channel 112. Inlet valve portion 126A of harvest channel 122 controls flow between inlet portion 124A of harvest channel 122 and main portion 128 of harvest channel 122. Outlet valve portion 116B of proliferation channel 112 controls flow between main portion 118 of proliferation channel 112 and outlet portion 114B of proliferation channel 112. The outlet valve portion 126B of the collection channel 122 controls flow between the main portion 128 of the collection channel 122 and the outlet portion 124B of the collection channel 122.

[0027] In the illustrated implementation, valve portions 116A, 116B, 126A, 126B of proliferation channel 112 and collection channel 122 are at least partially formed by portions of the top wall of cell flow layer 110. Because the top wall of cell flow layer 110 forms the top walls of proliferation channel 112 and collection channel 122, valve portions 116A, 116B, 126A, 126B are likewise at least partially formed by portions of the top walls of proliferation channel 112 and collection channel 122. When valve portions 116A, 116B, 126A, 126B are actuated, the top walls of valve portions 116A, 116B, 126A, 126B are compressed downward toward coverslip 102, blocking proliferation channel 112 and collection channel 122 at valve portions 116A, 116B, 126A, 126B. Valve portions 116A, 116B, 126A, 126B are in an open state when valve portions 116A, 116B, 126A, 126B are compressed, blocking the channels in cell flow layer 110 and preventing fluid flow, and valve portions 116A, 116B, 126A, 126B are in a closed state when valve portions 116A, 116B, 126A, 126B are not compressed, opening the channels in cell flow layer 110 and allowing fluid flow.

[0028] The inlet valve portion 116A of the proliferation channel 112 is located between the inlet portion 114A and the main portion 118. Thus, when the upper wall of the inlet valve portion 116A is compressed downward, the portion of the upper wall of the proliferation channel 112 between the inlet portion 114A and the main portion 118 is compressed downward, preventing cells and fluids from flowing between the inlet portion 114A and the main portion 118 in the proliferation channel 112. The inlet valve portion 126A of the collection channel 122 is located between the inlet portion 124A and the main portion 128. Thus, when the upper wall of the inlet valve portion 126A is compressed downward, the portion of the upper wall of the collection channel 122 between the inlet portion 124A and the main portion 128 is compressed downward, preventing cells and fluids from flowing between the inlet portion 124A and the main portion 128 in the collection channel 122.

[0029] The outlet valve portion 116A of the proliferation channel 112 is located between the main portion 118 and the outlet portion 114B. Thus, when the upper wall of the outlet valve portion 116B is compressed downward, the portion of the upper wall of the proliferation channel 112 between the main portion 118 and the outlet portion 114B is compressed downward, preventing cells and fluids from flowing between the main portion 118 and the outlet portion 114B in the proliferation channel 112. The outlet valve portion 126A of the collection channel 122 is located between the main portion 128 and the outlet portion 124B. Thus, when the upper wall of the outlet valve portion 126B is compressed downward, the portion of the upper wall of the collection channel 122 between the main portion 128 and the outlet portion 124B is compressed downward, preventing cells and fluids from flowing between the main portion 128 and the outlet portion 124B in the collection channel 122.

[0030] The cell flow layer 110 also includes a plurality of bridge valve portions 134 configured to assist in selectively controlling flow through the bridge channel 132. Thus, the bridge valve portions 134 can be actuated to prevent cells and fluids from flowing between the proliferation channel 112 and the harvest channel 122. Similar to the valve portions 116A, 116B, 126A, and 126B, the bridge valve portions 134 are at least partially formed by a portion of the top wall of the cell flow layer 110. Because the top wall of the cell flow layer 110 forms the top wall of the bridge channel 132, the bridge valve portions 134 are likewise at least partially formed by a portion of the top wall of the bridge channel 132 itself. When the bridge valve portions 134 are actuated, at least a portion of the top wall of the bridge channel 132 is compressed downward toward the coverslip 102, blocking the bridge channel 132.

[0031] The bridge valve portion 134 at least partially overlaps with the bridge channel 132 because the bridge valve portion 134 is the portion of the top wall of the bridge channel 132 that can be compressed downward. To prevent cell and fluid flow through the bridge channel 132, the full width of the top wall of each bridge channel 132 (between adjacent bridge protrusions 133) forms a respective bridge valve portion 134. Thus, when the bridge valve portion 134 is actuated, the full width of the top wall of the bridge channel 132 is compressed downward to block the bridge channel 132.

[0032] In some implementations, the bridge valve portion 134 extends along the entire length of the bridge channel 132 between the proliferation channel 112 and the harvest channel 122. In these implementations, the entire top wall of each bridge channel 132 forms the bridge valve portion 134. In other implementations, the bridge valve portion 134 extends along only a portion of the length of the bridge channel 132 between the proliferation channel 112 and the harvest channel 122. In these implementations, only a portion of the top wall of each bridge channel 132 forms the bridge valve portion 134. However, because the bridge valve portion 134 still extends across the entire width of the bridge channel 132 between adjacent pairs of bridge protrusions 133, the bridge valve portion 134 can still control flow through the bridge channel 132.

[0033] Generally, each bridge valve portion 134 is configured to control the flow of cells and fluids through one or more bridge channels 132. In some implementations, each bridge valve portion 134 controls flow through a respective one of the bridge channels 132. In other implementations, at least one of the bridge valve portions 134 controls flow through two or more bridge channels 132.

[0034] The control layer 150 includes one or more control channels that can be pressurized to actuate the various valve portions of the cell flow layer 110. The control channels of the control layer 150 overlap portions of the top wall of the cell flow layer 110 that form the various valve portions of the cell flow layer 110. When a control channel is pressurized, the top wall of the valve portions compresses downward to move them to their closed state, and the control channel expands. The control channel can then be depressurized to return the valve portions to their open state, and the control channel contracts.

[0035] In the illustrated implementation, the control layer 150 defines a first control channel 152A and a second control channel 152B. The first control channel 152A is configured to control all of the inlet valve portions 116A and 126A and all of the outlet valve portions 116B and 126B. The second control channel 152B is configured to control all of the bridge valve portions 134. Thus, the first control channel 152A extends into the control layer 150 such that the first control channel 152A overlaps a portion of the upper wall of the cell flow layer 110 that forms the upper wall of all of the inlet valve portions 116A and 126A and all of the outlet valve portions 116B and 126B. In the illustrated embodiment, the first control channel 152A may have a U-shape. A base 154A of the U-shape extends between a first end 111A and a second end 111B of the cell flow layer 110. One leg of the U-shape 154B extends across all of the inlet portion 114A of the proliferation channel 112 and all of the inlet portion 124A of the collection channel 122. Similarly, the other leg of the U-shape 154C extends across all of the outlet portion 114B of the proliferation channel 112 and all of the outlet portion 124B of the collection channel 122. In other implementations, the first control channel 152A can have other shapes and / or configurations, so long as the first control channel 152A overlaps all of the required valve portions of the cell growth layer 110.

[0036] The second control channel 152B also extends into the control layer 150 such that the second control channel 152B overlaps a portion of the top wall of the cell flow layer 110 that forms the top wall of all of the bridge valve portions 134. In the illustrated implementation, the second control channel 152B includes a first portion 158A, a second portion 158B, and a third portion 158C. Portion 158A overlaps all of the bridge valve portions 134 of the upper pair of proliferation and collection channels 112, 122. Portion 158B overlaps all of the bridge valve portions 134 of the middle pair of proliferation and collection channels 112, 122. Portion 158C overlaps all of the bridge valve portions 134 of the lower pair of proliferation and collection channels 112, 122. Second control channel 152B also includes a first intervening portion 160A connecting first portion 158A and second portion 158B and a second intervening portion 160B connecting second portion 158B and third portion 158C. In other implementations, second control channel 152B can have other shapes and / or configurations, so long as second control channel 152B overlaps all of the required valve portions of cell growth layer 110.

[0037] 3A-3C show cross-sectional views of an exemplary control channel 302 of the control layer 150 as pressurization actuates two exemplary valve portions 304A and 304B of the cell flow layer 110 and dams the underlying channel. The two exemplary valve portions 304A, 304B can be inlet valve portions 116A and 126A, two outlet valve portions 116B and 126B, or two adjacent bridge valve portions 134. In FIG. 3A, the control channel 302 is filled with an incompressible or substantially incompressible fluid, such as distilled water. In FIG. 3A, the control channel 302 is filled with fluid but is not yet pressurized. Thus, the upper walls 306A and 306B of the valve portions 304A and 304B are not compressed and the valve portions 304A and 304B are in their open state.

[0038] In FIG. 3B, the fluid in the control channel 302 begins to be pressurized (e.g., pressure is applied to the control channel 302). Thus, the upper walls 306A and 306B of the valve portions 304A and 304B begin to compress and move toward the coverslip 102. Because the valve portions 304A and 304B are not completely closed, fluid can still flow through the valve portions 304A and 304B. In FIG. 3C, a higher pressure is applied to the control channel 302, forcing the upper walls 306A and 306B of the valve portions 304A and 304B up to the coverslip 102. With this compression, the valve portions 304A and 304B move to their closed state and fluid cannot flow through the valve portions 304A and 304B. To return the valve portions 304A and 304B to their open state, pressure is removed from the control channel 302. The material forming top walls 306A and 306B (which in some implementations may be PDMS) is generally elastic, so that when pressure is removed from control channel 302, top walls 306A and 306B return to their uncompressed state (FIG. 3A).

[0039] As shown in FIG. 3A, the valve portions 304A and 304B generally have a dome-shaped cross section, and the thickness of the top wall of the cell flow layer 110 at the valve portions 304A and 304B is thinner than the thickness of the top wall of the cell flow layer 110 at different locations. Because the thickness of the top wall at the valve portions 304A and 304B is thinner, the top wall is flexible and can be compressed as shown in FIG. 3B and FIG. 3C. Furthermore, the dome-shaped cross section helps ensure that the top walls of the valve portions 304A and 304B do not compress and resist pressure from the control channel 302. Thus, the cross sections of the inlet valve portions 116A, 126A, the outlet valve portions 116B, 126B, and the bridge valve portion 134 are all dome-shaped. These locations also have thinner top walls than other locations in the proliferation channel 112, the harvest channel 122, and the bridge channel 132, which helps to allow compression of the top walls of the valve portions. At locations within proliferation channel 112, collection channel 122, and bridge channel 132 that do not have valve portions, the top wall is relatively thick and the channels have a generally square or generally rectangular cross-section.

[0040] 2A and 2B, to actuate the valve portions 116A, 116B, 126A, and 126B, pressurizing the first control channel 152A causes the upper walls of the valve portions 116A, 116B, 126A, and 126B to compress to the coverslip 102, and fluid cannot flow through the valve portions 116A, 116B, 126A, and 126B. When pressure is removed from the first control channel 152A, the valve portions 116A, 116B, 126A, and 126B return to their open state, and fluid can again flow through the valve portions 116A, 116B, 126A, and 126B. Similarly, to actuate the bridge valve portion 134, pressurizing the second control channel 152B causes the upper wall of the bridge valve portion 134 to compress to the coverslip 102, and fluid cannot flow through the bridge valve portion 134. Removing pressure from the second control channel 152B causes the bridge valve portions 134 to return to their open state, allowing fluid to flow through the bridge channel 132 again. In some implementations, the first control channels 152A and 152B are always filled with incompressible fluid during use (regardless of whether the valve portions remain open or closed), and pressure can be applied to the filled control channels 152A, 152B to actuate the valves. In these implementations, all of the valve portions controlled by each control channel can be actuated simultaneously or nearly simultaneously. In other implementations, the incompressible fluid is partially or wholly removed from the control channels 152A, 152B as the valve portions return to their open states.

[0041] 2A and 2B, in some implementations, the cell flow layer 110 can include a plurality of back channels 142 disposed adjacent to the cell growth trenches 120, such that each set of cell growth trenches 120 is disposed between one of the growth channels 112 and one of the back channels 142. Each of the cell growth trenches 120 can open to one of the back channels 142. Generally, the opening of each cell growth trench 120 is narrow enough to allow growth medium and other fluids to flow through the opening into the back channel 142, but not allow cells populating the cell growth trench 120 to pass through the opening. As discussed further herein, the back channels 142 can be used to populate the cell growth trenches 120.

[0042] The various channels of the cell flow layer 110 and the control layer 150 include openings through which fluid can flow in and out. In some implementations, the inlet portion 114A of the proliferation channel 112 includes a first inlet opening 115A and a second inlet opening 115B, as shown in the top two proliferation channels 112 of FIG. 2A. In other implementations, the inlet portion 114A of the proliferation channel 112 includes only a first inlet opening 115A, as shown in the lower proliferation channel 112. The inlet portion 124A of each harvest channel 122 includes an inlet opening 125. In some implementations, the inlet portion 114A can have three or more inlet openings. In some implementations, the inlet portion 124A can have two or more inlet openings.

[0043] The outlet portions similarly include outlet openings through which fluid can flow in and out. The outlet portions 114B of the growth channels 112 each include an outlet opening 117. The outlet portions 124B of the harvest channels 122 each include an outlet opening 127. The first control channel 152A and the second control channel 152B each include an opening through which the control channels 152A, 152B can be filled with fluid and pressurized. The first control channel 152A includes an opening 153A and the second control channel 152B includes an opening 153B. The back channels 142 adjacent to each set of cell growth trenches 120 each include a back channel outlet opening 143.

[0044] The various inlet openings, outlet openings, and control channel openings are generally all three-dimensional and rise vertically to the top wall of the control layer 150 as shown in FIGS. 1A and 1B. Various devices, mechanisms, etc. can be connected to the openings in the top wall of the control layer 150 to inject cells, fluids, etc. into the openings. In some examples, pumps (e.g., peristaltic pumps, syringe pumps, pressure tubes, etc.) can be connected to the openings to inject cells, growth medium, washing fluids, etc., and to pressurize and depressurize (via flowed incompressible fluid) the control channels 152A and 152B. In the illustrated implementation, the backchannel outlet opening 143 is open to the atmosphere outside the device 100. However, in other implementations, the backchannel outlet opening 143 may be located entirely within the cell flow layer 110 and may fluidly connect the backchannel 142 to the main portion 118 of the growth channel 112.

[0045] 4A-4C, the inlet portion 114A of the growth channel 112 can be washed using two inlet openings 115A and 115B. As shown in FIG. 4A, one or both of the inlet openings 115A, 115B can be used to inject cells and growth medium into the main portion 118 of the growth channel 112. Because the first control channel 152A is not pressurized, the inlet valve portion 116A of the growth channel 112 is in its open state. Cells, growth medium, or other substances injected into the first inlet opening 115A can flow into the main portion 118 of the growth channel 112. Thereafter, as shown in FIG. 4B, a certain amount of material may remain in the inlet portion 114A (e.g., biofilm, cells lodged tightly in the cracks and / or crevices of the PDMS and cannot be removed by vigorous flushing of growth medium or replaced by cell growth). During use of device 100, inlet valve portion 116A typically remains open, so this excess material must be removed from inlet portion 114A to prevent contamination of growth channel 112 with excess cells and debris during use of device 100.

[0046] As shown in FIG. 4C, the first control channel 152A can be pressurized to close the inlet valve portion 116A of the proliferation channel 112. When the inlet valve portion 116A is closed, any fluid injected into the inlet portion 114A cannot flow through the inlet valve portion 116A to the main portion 118. A wash fluid can then be injected into the first inlet opening 115A. Because the inlet valve portion 116A is closed and the inlet portion 114A includes a second inlet opening 115B, the wash fluid can flow through the inlet portion 114A and exit from the second inlet opening 115B. The wash fluid can remove excess cells and other materials from the inlet portion 114A, so that the inlet valve portion 116A can be open during use of the device 100 without contaminating the proliferation channel 112.

[0047] A variety of different types of fluids can be used to wash the inlet portion 114A. In some implementations, the washing fluid is undiluted bleach. In some implementations, the washing fluid is diluted bleach (e.g., 10% (volume / volume) bleach). In some implementations, a multi-step washing process can be used that requires multiple different washing fluids. For example, bleach (diluted or undiluted) or another strong washing fluid can be used first to wash the inlet portion 114A to remove any remaining material in the inlet portion 114A. Next, ethanol (which can be dilute (10% (volume / volume)) or undiluted) is injected into the inlet portion 114A to remove excess bleach from the inlet portion 114A. Finally, growth medium is injected into the inlet portion 114A to remove excess ethanol and restore nutrient balance. In some implementations, water is used instead of ethanol.

[0048] This multi-step cleaning process can be beneficial because in many applications of device 100, growth medium is flowed (injected, pumped, etc.) into growth channel 112 during use. Thus, the multi-step cleaning process can (1) utilize a strong cleaning fluid, such as bleach, to ensure that all excess cells and other material is removed, and (2) ensure that no excess or residual bleach remains in inlet portion 114A after cleaning, such that bleach does not inadvertently flow into growth channel 112 during use.

[0049] 5A and 5B show various dimensions of the cell growth trenches and backchannel. The width 502 of each of the cell growth trenches is between about 0.1 μm and about 50.0 μm. The height of each of the cell growth trenches is between about 0.1 μm and about 50.0 μm. Different cell growth trenches extending from the same growth channel may have different widths. For example, FIG. 5A shows multiple cell growth trenches including cell growth trenches 120A, 120B, 120C, and 120D. Trench 120A has the smallest width of the four trenches, and increases in size through 120B, 120C, and 120D. In general, the cell growth trenches may have any desired width depending on the application. Adjacent pairs of cell growth trenches are spaced apart by a distance 504. This distance may be between about 0.1 μm and about 20.0 μm.

[0050] FIG. 5B shows a series of cell growth trenches (including cell growth trenches 120A, 120B, 120C, and 120D) that open at their rear ends to backchannel 142. Cell growth trench 120A includes opening 121A. Cell growth trench 120B includes opening 121B. Cell growth trench 120C includes opening 121C. Cell growth trench 120D includes opening 121D. Each of these openings 121A-121D can have a width 506 between about 1.0 μm and about 300.0 μm, and a height between about 0.1 μm and about 20.0 μm. Backchannel 142 can have a width 508 between about 1.0 μm and about 300.0 μm, and a height between about 0.1 μm and about 50.0 μm.

[0051] FIG. 6 shows a time series of one of the cell growth trenches 120 showing the growth of a single starting cell of an isogenic line. At time zero, the cell growth trench 120 contains a single cell. After 9 hours, the single cell has grown to two cells. At 18 hours, the cell growth trench 120 contains three cells. At 27 hours, the cell growth trench 120 contains four cells. Finally, at 36 hours, the cell growth trench 120 contains six cells. As shown in FIG. 6, the narrow cell growth trench 120 allows the cell population to group together in a linear, one-dimensional grouping, e.g., grow in a line within the cell growth trench 120. This one-dimensional grouping allows individual cells to be imaged, analyzed, monitored, etc., and is also useful for tracking the lineage of the cells.

[0052] As the cell population proliferates, the cells begin to approach the end of the cell proliferation trench 120 closest to the proliferation channel 112, as shown in Figure 6. When the cell proliferation trench 120 becomes full, the next division will push the last cells out into the proliferation channel 112. The cell population grows two n where n is the number of generations of cells. Generally, the cell growth trench 120 is long enough to accommodate between about 5 cells and about 10 cells, and isogenicity can be achieved between about 2 and about 10 generations of growth. Because isogenicity is achieved very quickly, robustly, and passively through cell growth, it is not necessary to individually load the cell growth trench 120 with a single cell or multiple cells from the same lineage (e.g., a pooled mixed lineage population can be randomly seeded within the cell trench, as discussed further herein).

[0053] FIG. 7 shows a flow chart of a method 700 of using the device 100. FIGS. 8A-8C show the stages of the device 100 at different steps of the method 700. In step 702, cells and growth medium are injected into the growth channel 112 via the inlet portion 114A. The cells and growth medium may be injected via one or both of the inlet openings 115A and 115B. The cells and growth medium are injected while the bridge valve portion 134 is closed. As shown in FIG. 8A, the first control channel 152A is not pressurized, so the inlet valve portions 116A and 126A are open. Thus, the cells and growth medium can flow into the main portion 118 of the growth channel 112 through the inlet valve portion 116A. Because the bridge valve portion 134 is closed, the cells and growth medium do not flow through the bridge channel 132 to the harvest channel 122.

[0054] In step 704, the inlet valve portions 116A, 126A and the outlet valve portions 116B, 126B are closed by pressurizing the first control channel 152A. As shown in FIG. 8B, when the first control channel 152A is pressurized, the inlet valve portions 116A, 126A and the outlet valve portions 116B, 126B move to a closed state. When these valve portions are in their closed state, cells and growth medium remain within the main portion 118 of the growth channel 112 and are prevented from flowing into the inlet portion 114A or the outlet portion 114B. Because the bridge valve portion 134 remains closed, cells and growth medium cannot flow through the bridge channel 132.

[0055] In step 706, the inlet portion 114A is cleaned by injecting a cleaning fluid into either the first inlet opening 115A or the second inlet opening 115B. As shown in FIG. 8B, the first control channel 152A is pressurized and the inlet valve portion 116A is closed, so that the cleaning fluid flows between the two inlet openings 115A and 115B through the inlet portion 114A. Thus, the cleaning fluid removes contaminants such as residual cells, growth medium, and bacteria. In some embodiments, cleaning the inlet portion 114A includes using a three-step method, as described herein. In these implementations, bleach is first flowed through the inlet portion 114A to remove contaminants. Then, ethanol is flowed through the inlet portion 114A to remove excess bleach, and finally, growth medium is flowed through the inlet portion 114A to remove excess ethanol and restore nutrient balance. In some implementations, water is used instead of ethanol.

[0056] When cells and growth medium are injected into device 100, there are several techniques that can be used to cause cells to populate cell growth trench 120. In some implementations, cells populate cell growth trench 120 via diffusion. In these implementations, inlet valve portions 116A and 126A, outlet valve portions 116B and 126B, and bridge valve portion 134 all remain closed. Cells in main portion 118 of growth channel 112 can then diffuse into cell growth trench 120. Closing bridge valve portion 134 ensures that cells do not inadvertently diffuse through bridge channel 132 into harvest channel 122. Closing inlet valve portions 116A and 126A, and outlet valve portions 116B and 126B ensures that cells do not inadvertently diffuse out of main portion 118 of growth channel 112. Closing inlet valve portions 116A and 126A and outlet valve portions 116B and 126B also prevents all fluid from entering or exiting main portion 118 of growth channel 112, which can slow or prevent some or all of the cells from diffusing into cell growth trenches 120. This diffusion process can occur before, during, or after inlet portion 114A is washed, so long as inlet valve portions 116A and 126A and outlet valve portions 116B and 126B are closed.

[0057] In another implementation, centrifugation can be used to load the cell growth trench 120. This technique can be useful for dilute cultures that cannot be concentrated and therefore have difficulty diffusing into the cell growth trench 120. In these implementations, once the cells and growth medium are injected into the growth channel 112, the device 100 can be spun using a centrifuge or other rotating mechanism to cause the cells to flow into the cell growth trench. Because the device 100 is spinning, it is generally not possible to pressurize the first control channel 152A and the second control channel 152B, and all valve portions remain open during the centrifuge-based loading process. Thus, generally, the inlet portion 114A must be cleaned after using a centrifuge. It is also necessary to clean the harvest channel 122 after using a centrifuge.

[0058] In implementations in which the cell flow layer 110 includes a back channel 142, cells can be drawn into the cell growth trench 120 using branched convection of fluid through the cell growth trench 120. In these implementations, the bridge valve portion 134 remains closed. Loading via branched convection can occur before the inlet portion 114A is flushed, so long as the inlet valve portions 116A and 126A and the outlet valve portions 116B and 126B are closed during the inlet flush process.

[0059] Referring back to FIG. 7, once inlet portion 114A has been cleaned, in step 708, inlet valve portion 116A and outlet valve portion 116B may be opened to allow additional growth medium to flow into main portion 118 of growth channel 112. In step 710, the cells in cell growth trench 120 may be imaged, monitored, analyzed, etc. At this stage of method 700, device 100 is in the configuration shown in FIG. 8A. As part of assembling cells into cell growth trench 120, additional growth medium may be flowed into growth channel 112. However, growth medium is typically flowed continuously into main portion 118 of growth channel 112 during analysis of cells in cell growth trench 120. Thus, inlet valve portion 116A and outlet valve portion 116B of growth channel 112 are typically open during analysis of cells in cell growth trench 120. However, in other implementations, the inlet valve portion 116A and the outlet valve portion 116B of the proliferation channel 112 are closed after additional growth medium flows into the main portion 118 of the proliferation channel 112. In these implementations, imaging and / or analysis is performed while the inlet valve portion 116A and the outlet valve portion 116B of the proliferation channel 112 are closed.

[0060] Once the cells have assembled in the cell proliferation trench 120, a variety of different types of analyses can be performed. The cells can be imaged over time using any number of different microscopy techniques, including but not limited to fluorescence, phase contrast, bright field, light sheet, or any type of super-resolution imaging modality. To assay for extracellular secretions, antibody-conjugated beads or other analyte detection systems can be flowed into the main portion 118 of the proliferation channel 112. Additionally, oil or another fluid immiscible with water can be flowed into the main portion 118 of the proliferation channel 112 to confine the cells and aqueous medium within the cell proliferation trench 120, effectively creating an enclosed reaction compartment. After such extracellular factors have been assayed by imaging, the oil can be purged from the main portion 118 of the proliferation channel 112 and replaced with growth medium to resume growth of all the cells within the cell proliferation trench 120.

[0061] In step 712, the harvest channel 122 can be washed by flowing a washing fluid into the harvest channel 122 through the inlet opening 125. Because the target cells will eventually be transferred to the harvest channel 122, potential contaminants generally need to be removed from the harvest channel 122 before the target cells are introduced. Washing the harvest channel 122 generally involves the same three-step washing process applied to the inlet portion 114A of the growth channel 112. First, a washing fluid (e.g., diluted or undiluted bleach) can be flowed through the harvest channel 122. Next, ethanol is flowed through the harvest channel 122 to remove residual bleach. Finally, growth medium is flowed through the harvest channel 122 to remove residual ethanol. In some implementations, water is used instead of ethanol.

[0062] 8A, second control channel 152B is pressurized and bridge valve portion 134 is closed so that fluid used during the washing process flows throughout harvest channel 122 without entering proliferation channel 112. The washing fluid removes cells and other contaminants that may be present in harvest channel 122, as well as in any upstream and downstream connectors, tubing, etc., that may have adhered due to assembly of the chip in a non-sterile environment.

[0063] Cleaning of collection channel 122 may generally occur at any time after cells have been loaded into cell growth trenches 120. Thus, cleaning of collection channel 122 may occur during or after imaging and / or analysis of the cells in step 710. Prior to cleaning collection channel 122, inlet valve portion 126A and outlet valve portion 126B of collection channel 122 must be closed.

[0064] Once one or more target cells are identified during the monitoring and analysis phase, the one or more target cells can be easily extracted from the device 100 using the bridge channel 132 and the collection channel 122. In step 714, as shown in FIG. 8C, the inlet valve portions 116A and 126A and the outlet valve portions 116B and 126B are first closed and the bridge valve portion 134 is opened. After the inlet valve portions 116A and 126A and the outlet valve portions 116B and 126B are closed, the bridge valve portion 134 is opened to ensure that the cells are not affected by residual flow through the inlet valve portions 116A and 126A and the outlet valve portions 116B and 126B. In step 716, the one or more target cells are moved from the cell growth trench 120 to the collection channel 122. The one or more target cells can flow from the cell growth trench 120, via the bridge channel 132, to the main portion 128 of the collection channel 122. Because the inlet valve portions 116A, 126A and the outlet valve portions 116B, 126B are closed, one or more target cells cannot inadvertently flow into the inlet portions 114A, 124A or the outlet portions 114B, 124B.

[0065] One or more target cells may be moved using any suitable technique. In some implementations, electrowetting or dielectrophoresis (which may include optoelectronic tweezers) may be used. In other implementations, optical tweezers may be used. Optical tweezers are optical instruments that use a focused laser beam to exert a force on small particles. In some implementations, optical tweezers may be implemented using a laser (e.g., a neodymium-doped yttrium aluminum garnet laser), a beam expander, a variety of different lenses and mirrors that direct the laser beam to a desired location, and a microscope objective and condenser that form a desired beam shape and intensity profile. During use with device 100, the components of the optical tweezers may generally be positioned in any orientation relative to device 100. The optical tweezers' laser beam propagates into cell flow layer 110 and captures or grabs the cells to be moved. Device 100 may be moved relative to a stationary optical tweezers, and / or the optical tweezers' laser beam itself may be steered to manipulate the captured cells. The optical tweezers can then move the target cell from one of the cell growth trenches 120 , through one of the bridge channels 132 , and into the main portion 128 of the collection channel 122 .

[0066] Closure of the inlet valve portions 116A, 126A and the outlet valve portions 116B, 126B prevents residual convection and minimizes drag forces on the cells, thus allowing reliable cell transport via optical tweezers at low laser power. Furthermore, arrays of optical tweezers can be fabricated through the implementation of beam splitting, mode shaping, and adaptive wavefront correction (i.e., holographic optical trapping). Holographic optical trapping offers the advantage of being able to transport multiple cells simultaneously.

[0067] In general, any number of cells of interest can be transferred to the collection channel 122. In some implementations, a single cell of interest can be extracted from a single cell growth trench 120. In other implementations, a single cell of interest can be extracted from each of a plurality of cell growth trenches 120. In additional implementations, multiple cells of interest can be extracted from a single cell growth trench 120. In yet further implementations, multiple cells of interest can be extracted from each of a plurality of cell growth trenches 120. In still other implementations, a single cell can be extracted from each of a first set of one or more cell growth trenches 120 and multiple cells can be extracted from each of a second set of one or more cell growth trenches 120.

[0068] In step 718, bridge valve portion 134 is closed and outlet valve portion 126B of collection channel 122 is opened. In step 720, one or more target cells are extracted from outlet portion 124B of collection channel 122. Any suitable technique can be used to move one or more target cells from main portion 128 of collection channel 122 to outlet portion 124B of collection channel 122, for example, by flowing growth medium through collection channel 122 or by using optical tweezers.

[0069] In some implementations, one or more cells of interest may be extracted from the proliferation channel 122 and then transferred to the cell proliferation trench 130 of the harvest channel 122. The cells of interest may be maintained and monitored in the cell proliferation trench 130 and then transferred to the outlet portion 124B for extraction. Depending on the type of analysis being performed, it may be beneficial to extract the cells of interest from the proliferation channel 112 but not immediately extract the cells of interest from the harvest channel 122. In these implementations, the cells of interest extracted from the harvest channel 122 may include progeny of the cells of interest that were transferred to the cell proliferation trench 130 of the harvest channel 122.

[0070] In some implementations, once the cells of interest have been extracted from the device 100, the collection channel 122 can be flushed to ensure that all remaining cells are removed from the collection channel 122. This flushing can be accomplished, for example, by flowing growth medium through the collection channel 122.

[0071] In some implementations, not all of the cells of interest are transferred from cell growth trench 120 to collection channel 122 and then extracted. In these implementations, a portion of the cells in cell growth trench 120 are extracted. Collection channel 122 is then flushed, inlet valve portion 126A and outlet valve portion 126B of collection channel 122 are closed, and bridge valve portion 134 is reopened, allowing additional cells of interest to be transferred from cell growth trench 120 to collection channel 122.

[0072] In some implementations of method 700, when any one of inlet valve portion 116A, outlet valve portion 116B, inlet valve portion 126A, or outlet valve portion 126B is opened or closed, the remainder of the inlet and outlet valve portions are opened or closed. Thus, when any of the steps of method 700 refer to opening or closing any one of the inlet and outlet valve portions, the step also includes opening or closing another inlet and outlet valve portion. However, bridge valve portion 134 is generally controlled separately from inlet valve portion 116A, 126A, and outlet valve portion 116B, 126B. In other implementations, any one of inlet valve portion 116A, outlet valve portion 116B, inlet valve portion 126A, or outlet valve portion 126B may be controlled independently from the remainder of inlet valve portion 116A, outlet valve portion 116B, inlet valve portion 126A, or outlet valve portion 126B. In still other implementations, bridge valve portion 134 may be controlled together with any one of inlet valve portion 116A, outlet valve portion 116B, inlet valve portion 126A, or outlet valve portion 126B.

[0073] Thus, as shown in Figures 8A-8C, the various valve portions of the cell flow layer 110 allow cells to be analyzed over extended periods of time and easily extracted from the device with minimal effort. The inlet valve portion 116A allows for washing of the inlet portion 114A of the proliferation channel 112 after the cells have flowed into the main portion 118 of the proliferation channel 112. Additionally, the inlet valve portion 116A and the outlet valve portion 116B allow for growth medium to flow into the proliferation channel 112 during analysis of the cells in the cell growth trench 120. Finally, the bridge valve portion 134 allows the collection channel 122 to remain isolated from the proliferation channel 112 until analysis is complete. Thus, the migration path of one or more cells of interest (the proliferation channel 112 and the collection channel 122) is kept substantially or completely free of debris and contaminants (e.g., residual cells, bacteria, etc.). Additionally, bridge valve portion 134 allows collection channel 122 and any off-chip tubing attached to collection channel 122 to be washed, as well as inlet portion 114A of growth channel 112. Thus, one or more cells of interest can be easily transferred from cell growth trench 120 to outlet portion 124B of collection channel 122 and extracted from device 100 without contamination.

[0074] The arrangement of the linear cell growth trench 120 perpendicular to the growth channel 112 allows for the flushing of cell lineage progeny by the orthogonally flowing medium. The continuous discharge of progeny from cell lineages trapped within the cell growth trench 120 uniquely allows for monitoring of cells over many generations (e.g., 10-100 consecutive generations for mammalian cells and perhaps more than 1000 consecutive generations for bacteria for cells located at the end of the cell trench) since cells do not accumulate locally. Combined with washing the inlet portion 114A and the collection channel 122, the device 100 avoids the massive accumulation of cells that can occur due to the exponential doubling of cells every generation. After 30 generations of growth, more than one billion progeny can accumulate from each initial cell. This accumulation is avoided in the device 100. The combination of the features of the device 100 allows for the acquisition of uncontaminated single cells from cell populations imaged over many generations. This allows for a wide range of applications that were not possible before.

[0075] The device 100 can be used in a variety of different applications. The first application is to detect small but genetically stable differences in a wide range of properties of different cells. Most cellular behaviors are statistically distributed such that a given genotype causes a wide range of different phenotypes. For example, the expression of a protein in a cell can vary significantly even between genetically identical cells growing in the same environment. In genetic screens, rare genetic variants with desirable properties are often outnumbered by cells that only transiently exhibit the desirable phenotype. The device 100 allows tracking of each genetic variant over many generations of growth in multiple parallel cells, thus providing a substantial statistical sample. This statistical sample allows the separation of genetically inherited traits from transient phenotypic diversity, thus enabling the identification of rare variants of interest within a large population of cells that transiently mimic interesting behaviors.

[0076] Any trait that can be observed directly or indirectly by microscopy can be used to record behavior, even when the genetically encoded differences between mutants are small compared to the phenotypic heterogeneity in single cells. This allows for the detection of small but genetically stable differences in a broad range of traits, including but not limited to gene expression, cell growth rate, morphology, cellular localization patterns, enzyme activities, DNA replication and modification, chromosome segregation patterns, metabolic state, and cell envelope, or any combination thereof.

[0077] The properties can be tracked to improve a variety of biomaterials, including ribonucleic acid (RNA) and proteins, including but not limited to fluorescent or luminescent proteins, regulatory elements such as activators and repressors, optogenetically activated control proteins, enzymes and antibodies, mRNA, small RNAs and guide RNAs for clustered regularly interspaced short palindromic repeats (CRISPR).

[0078] Tracking genetic differences may include tracking changes in gene expression and response networks within cells to assess pathways and expression programs; performing whole-cell assays to measure cell physiology, morphology, and proliferation rates as well as differences between daughter cells upon cell division; detecting changes in the number or spatial distribution of organelles or other subcellular structures; and detecting cell-cell interactions and cellular secretions into each trench.

[0079] Furthermore, device 100 allows for the interrogation of genetic variants of interest without cloning the variants, which is typically very time-consuming, resource-demanding, and may even be impossible for complex genetic mutations spanning one or more chromosomes, plasmids, etc. Thus, device 100 allows cells of interest to be immediately available for further amplification, storage, downstream live cell functional assays, and other uses.

[0080] The second application is to identify epigenetic behavior over long time scales (e.g., multiple generations). Many cellular behaviors are epigenetic, e.g., behavior changes over multigenerational time scales. Identifying such behavior requires a range of observations over multiple generations, possibly tens or even hundreds of generations of cell growth. The device 100 allows for the acquisition of cells after observing these long-term epigenetic changes. This enables a variety of methods, including genetic screening for chromatin remodeling, cell fate determination, bistable circuits and multigenerational oscillators, or other epigenetic behaviors that can be observed by long-term imaging.

[0081] A third application is to detect the response of cells to different environments. Many cellular behaviors depend on growth conditions. The device 100 allows multigenerational imaging in many different environments, making it possible to screen and document for the first time how all observable properties of cells and cellular processes change between environments. After monitoring many genetic variants in parallel, cells for the variant of interest can be extracted.

[0082] A fourth application is to perform a variety of assays. By physically extracting cells, the device 100 allows assays beyond DNA sequencing to be performed on the extracted cells, such as genome-wide terminal assays. By extracting one of the two daughter cells at each division, a genome-wide time course across a cell lineage (e.g., tracking genome-wide traits) can be completed while simultaneously observing traits of the daughter cell left behind by the device 100.

[0083] Although the present disclosure has been described with reference to one or more specific implementations, those skilled in the art will recognize that many modifications can be made thereto without departing from the spirit and scope of the present disclosure. Each of these implementations and their obvious variations are considered to be within the spirit and scope of the present disclosure. It is also considered that additional implementations according to aspects of the present disclosure can combine any number of features from any of the implementations described herein.

Claims

1. 1. A microfluidic device for use in analyzing cells and extracting one or more cells of interest, comprising: A substrate; a cell flow layer coupled to the substrate, a proliferation channel having a main portion and an inlet valve portion, the inlet valve portion of the proliferation channel configured to help selectively control flow into the main portion of the proliferation channel; a collection channel having a main portion and an outlet valve portion, the outlet valve portion of the collection channel configured to assist in selectively controlling flow out of the main portion of the collection channel; one or more bridge channels connecting the main portion of the proliferation channel and the main portion of the collection channel, each of the one or more bridge channels including a bridge valve portion configured to assist in selectively controlling flow between the proliferation channel and the collection channel; A cell flow layer comprising: a control layer coupled to the cell flow layer, the control layer configured to assist in actuating (i) the bridge valve portion of each of the one or more bridge channels, (ii) the inlet valve portion of the proliferation channel, and (iii) the outlet valve portion of the harvest channel; Equipped with further comprising one or more cell growth trenches fluidly connected to the main portion of the growth channel; each of the one or more cell growth trenches is disposed adjacent a first side of the main portion of the growth channel; A microfluidic device, characterized in that the main portion of the collection channel is positioned adjacent to a second side of the main portion of the proliferation channel opposite a first side of the main portion of the proliferation channel, and each of the one or more cell proliferation trenches is configured to contain one or more cells therein.

2. when each of the bridge valve portions is in a first position, each of the one or more bridge channels is open such that fluid can flow between the proliferation channel and the harvest channel; 2. The microfluidic device of claim 1 , wherein when the bridge valve portion of each of the one or more bridge channels is in a second position, each of the one or more bridge channels is closed such that fluid is prevented from flowing between the proliferation channel and the harvest channel.

3. when the inlet valve portion of the proliferation channel is in a first position, fluid is prevented from flowing into the main portion of the proliferation channel; The microfluidic device of claim 1 , wherein when the inlet valve portion of the proliferation channel is in a second position, fluid can flow into the main portion of the proliferation channel.

4. when the outlet valve portion of the collection channel is in a first position, fluid is prevented from flowing out of the main portion of the collection channel; 10. The microfluidic device of claim 1, wherein when the outlet valve portion of the collection channel is in a second position, fluid is allowed to flow out of the main portion of the collection channel.

5. the proliferation channel includes an inlet portion having a first inlet opening and a second inlet opening; The microfluidic device of claim 1, characterized in that when the inlet valve portion of the proliferation channel is in a first position, fluid can flow between the first inlet opening and the second inlet opening through the inlet portion of the proliferation channel, but cannot flow between the inlet portion of the proliferation channel and the main portion of the proliferation channel.

6. the cell flow layer includes a top wall, the top wall being spaced from the substrate, and the top walls of each of the growth channels, the one or more cell growth trenches, the collection channel, and the one or more bridge channels are formed by the top wall of the cell flow layer; 2. The microfluidic device of claim 1, wherein the control layer further comprises: (i) a first control channel configured to assist in actuating the bridge valve portion of each of the one or more bridge channels; and (ii) a second control channel configured to assist in actuating the inlet valve portion of the proliferation channel and the outlet valve portion of the harvest channel.

7. 7. The microfluidic device of claim 6, wherein the bridge valve portion of each of the one or more bridge channels is at least partially formed by at least a portion of the top wall of each of the one or more bridge channels.

8. the first control channel of the control layer at least partially overlaps the top wall of each of the one or more bridge channels; 8. The microfluidic device of claim 7, wherein the top wall of each of the one or more bridge channels is configured such that when the first control channel is pressurized, the top wall of each of the one or more bridge channels collapses toward the substrate to prevent fluid from flowing through each of the one or more bridge channels.

9. The microfluidic device of claim 6, wherein the inlet valve portion of the proliferation channel is at least partially formed by at least a portion of the top wall of the proliferation channel adjacent the main portion of the proliferation channel.

10. the second control channel of the control layer at least partially overlaps the portion of the top wall of the proliferation channel that forms the inlet valve portion of the proliferation channel; The microfluidic device of claim 9, characterized in that the portion of the upper wall of the proliferation channel that forms the inlet valve portion of the proliferation channel is configured to collapse toward the substrate when the second control channel is pressurized, preventing fluid from entering the main portion of the proliferation channel.

11. 7. The microfluidic device of claim 6, wherein the outlet valve portion of the collection channel is at least partially formed by at least a portion of the top wall of the collection channel adjacent the main portion of the collection channel.

12. the second control channel of the control layer at least partially overlaps the portion of the top wall of the collection channel that forms the outlet valve portion of the collection channel; 12. The microfluidic device of claim 11, wherein the portion of the top wall of the collection channel forming the outlet valve portion of the collection channel is configured to collapse toward the substrate upon pressurization of the second control channel to prevent fluid from exiting the main portion of the collection channel.

13. 2. The microfluidic device of claim 1, wherein: (i) the cell flow layer and the control layer are formed from polydimethylsiloxane (PDMS); or (ii) the substrate is formed from glass; or (iii) (i) the cell flow layer and the control layer are formed from polydimethylsiloxane (PDMS) and (ii) the substrate is formed from glass.

14. 2. The microfluidic device of claim 1, wherein (i) the main portion of the proliferation channel and the main portion of the collection channel each have a substantially square cross-section, and (ii) the inlet valve portion of the proliferation channel, at least a portion of the one or more bridge channels, and the outlet valve portion of the collection channel each have a substantially dome-shaped cross-section.

15. 2. The microfluidic device of claim 1, wherein the proliferation channel includes an outlet valve portion that selectively enables flow out of the main portion of the proliferation channel without passing through the one or more bridge channels, and the collection channel includes an inlet valve portion that selectively enables flow toward the main portion of the collection channel without passing through the one or more bridge channels.

16. The microfluidic device of claim 1 , wherein each of the one or more cell growth trenches has a length of about 25.0 μm and a width of about 1.5 μm.

17. 10. The microfluidic device of claim 1 , wherein each of the one or more cell growth trenches is spaced apart from an adjacent one of the one or more cell growth trenches by a distance of about 4.0 μm.

18. 10. The microfluidic device of claim 1 , wherein each of the one or more bridge channels has a length of about 200.0 μm and a width of about 50.0 μm and about 100.0 μm.

19. A method for analyzing cells and extracting one or more cells of interest using a microfluidic device, comprising: The microfluidic device comprises: a proliferation channel having a main portion and an inlet valve portion; one or more cell growth trenches fluidly connected to the growth channel; a harvest channel fluidly connected to the proliferation channel, the harvest channel having a main portion and an outlet valve portion; one or more bridge channels connecting the proliferation and collection channels; having each of the one or more cell growth trenches is disposed adjacent a first side of the main portion of the growth channel; the main portion of the collection channel is disposed adjacent a second side of the main portion of the proliferation channel opposite a first side of the main portion of the proliferation channel; each of the one or more cell growth trenches is configured to contain one or more cells therein; injecting one or more cells and growth medium into an inlet portion of the growth channel, causing the one or more cells and the growth medium to flow into the main portion of the growth channel and fill at least one of the one or more cell growth trenches; closing the inlet valve portion of the proliferation channel; analyzing the one or more cells in the at least one of the one or more cell growth trenches to identify one or more cells of interest; opening one or more bridge valve portions to allow fluid to pass through the one or more bridge channels between the main portion of the proliferation channel and the main portion of the harvest channel; transferring the one or more target cells from the at least one of the one or more cell growth trenches to the main portion of the collection channel via the main portion of the growth channel and one or more of the one or more bridge channels; closing the one or more bridge valve portions; opening the outlet valve portion of the collection channel; transferring the one or more target cells from the main portion of the collection channel to an outlet portion of the collection channel; collecting the one or more target cells from the outlet portion of the collection channel; A method comprising:

20. prior to injecting the one or more cells and growth medium into the inlet portion of the growth channel; closing the one or more bridge valve portions of the microfluidic device to prevent fluid from passing between the main portion of the growth channel and the main portion of the harvest channel through the one or more bridge channels; 20. The method of claim 19 further comprising:

21. 20. The method of claim 19, further comprising washing the inlet portion of the proliferation channel to remove contaminants from the inlet portion of the proliferation channel.

22. 20. The method of claim 19, wherein the inlet portion of the proliferation channel has a first inlet opening and a second inlet opening, both of which are fluidly connected to the main portion of the proliferation channel through the inlet portion of the proliferation channel, and washing the inlet portion of the proliferation channel comprises injecting a washing fluid into the first inlet opening of the inlet portion of the proliferation channel, causing the washing fluid to flow through the inlet portion of the proliferation channel inlet and exit the inlet portion of the proliferation channel inlet at the second inlet opening of the inlet portion of the proliferation channel.

23. Injecting the cleaning fluid into the first inlet opening comprises: injecting a bleaching agent into the first inlet opening to remove contaminants from the inlet portion of the growth channel; injecting ethanol or water into the first inlet opening to remove residual bleach from the inlet portion of the growth channel; injecting a growth medium into the first inlet opening to remove residual ethanol or water from the inlet portion of the growth channel; 23. The method of claim 22, comprising:

24. when the inlet valve portion of the proliferation channel is closed, fluid is prevented from flowing between the inlet portion of the proliferation channel and the main portion of the proliferation channel; 20. The method of claim 19, wherein when the outlet valve portion of the collection channel is closed, fluid is prevented from flowing between the main portion of the collection channel and the outlet portion of the collection channel.

25. (i) moving the one or more target cells includes capturing the one or more target cells with optical tweezers and moving the one or more target cells with the optical tweezers; (ii) the one or more cells in the at least one of the one or more cell growth trenches are configured to grow into isogenic cells; (iii) analyzing the one or more cells comprises performing microscopy on the one or more cells; or 20. The method of claim 19, characterized in that (iv) any combination of (i) to (iii).

26. After collecting the one or more target cells from the outlet portion of the collection channel, identifying additional cells of interest in the at least one of the one or more cell growth trenches; flushing the collection channel; harvesting the additional cells of interest from the at least one of the one or more cell growth trenches; 20. The method of claim 19 further comprising:

27. Harvesting the additional cells of interest comprises: closing the outlet valve portion of the collection channel; opening the one or more bridge valve portions; transferring the additional cells of interest from the at least one of the one or more cell growth trenches to a main portion of the collection channel via the main portion of the growth channel and one or more of the one or more bridge channels; closing the one or more bridge valve portions; opening the outlet valve portion of the collection channel; transferring the one or more additional cells of interest from the main portion of the collection channel to the outlet portion of the collection channel; collecting the one or more additional cells of interest from the outlet portion of the collection channel; and 27. The method of claim 26, comprising:

Images