Microfluidic devices for high-throughput screening of cell-cell interactions

Abstract

A method and microfluidic device are disclosed for successfully co-encapsulating two or more cells in a high-throughput and high-efficiency manner. The cells are arranged into two or more ordered streams that flow through separate microchannels of the microfluidic device. The cells in the ordered streams are sufficiently spaced such that a single droplet containing exactly one cell from a first cell-ordered stream and at least one cell from a second cell-ordered stream is generated at the junction of the microfluidic device. The single droplet containing two or more cells is useful for performing assays (e.g., high-throughput cell-cell interaction assays). TIFF2025535563000014.tif106162

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 423,233, filed November 7, 2022, the entire disclosure of which is incorporated herein by reference for all purposes. [Background technology]

[0002] background Current efforts in high-throughput cell screening focus on developing microfluidic devices to encapsulate single cells in single droplets. Thus, single cells are processed in single droplets to generate data that can be traced back to the cell. However, while single-cell encapsulation is valuable for extracting data at the single-cell level, such single-cell data cannot capture cell-cell interactions (e.g., data related to the interaction between two or more cells). Summary of the Invention

[0003] overview Disclosed herein are methods for generating single droplets from two or more cell-aligned streams such that the single droplets contain an improved ratio of one cell from a first cell-aligned stream and at least one cell from a second cell-aligned stream. In various embodiments, the plurality of single droplets comprises a single cell from the first aligned stream and a single cell from the second aligned stream. In certain embodiments, the proportion of the plurality of single droplets comprising a single cell from the first aligned stream and a single cell from the second aligned stream exceeds the expected proportion predicted using a Poisson distribution.

[0004] The disclosed method, however, allows for high-throughput production of such droplets while achieving high efficiency and minimizing the number of empty droplets. To generate single droplets containing one cell from a first aligned stream and at least one cell from a second aligned stream in a high-throughput and efficient manner, the cells in the first aligned stream and the cells in the second aligned stream are aligned and sufficiently spaced to achieve successful co-encapsulation. In various embodiments, the cells in the aligned streams are aligned in a single file through a process known as inertial focusing, which is caused by Dean forces. Inertial focusing pushes the cells tangentially to the direction of flow until they reach an equilibrium position. When the cells in the aligned streams arrive at the droplet-generation zone of the microfluidic device, also referred to herein as the junction, one cell from the first cell-aligned stream and at least one cell from the second cell-aligned stream are controllably encapsulated within the single droplet.

[0005] Notably, the generation of a single droplet encapsulating one cell from a first cell-aligned stream and at least one cell from a second cell-aligned stream cannot be achieved by simply merging two streams of aligned cells together. Specifically, the rate of inertial focusing of cells increases with increasing flow rate. However, increasing flow rate hinders droplet generation by creating a "jetting" or "co-flow" regime when the aqueous cell stream merges with the oil-phase sheath fluid. Thus, the addition of a second cell-aligned stream, achieved by flowing the second stream fast enough to inertially focus the cells, adds an aqueous flow rate that is too high for droplet generation to occur. Conversely, when the flow rate is reduced to allow droplet generation, the cells in the aligned stream are not sufficiently inertially focused, and therefore cannot be controllably encapsulated into a single droplet. Thus, achieving successful co-encapsulation of at least paired cells requires simultaneous fine-tuning of various competing parameters to achieve sufficient cell alignment and droplet co-encapsulation.

[0006] Disclosed herein are methods for encapsulating two cells in a single droplet, the method comprising: flowing a first aqueous phase comprising a first cell-aligned stream in a first microchannel toward a junction; flowing a second aqueous phase comprising a second cell-aligned stream in a second microchannel toward the junction; flowing an oil phase in a third microchannel toward the junction; and generating a single droplet at the junction formed from the first aqueous phase, the second aqueous phase, and the oil phase, the single droplet comprising cells from the first cell-aligned stream and cells from the second cell-aligned stream. In various embodiments, the method disclosed herein further comprises generating single droplets at the junction to generate a population of single droplets, the population characterized by a proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream, the proportion exceeding the expected proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream, as predicted using a Poisson distribution. In various embodiments, the proportion exceeds the expected proportion by a factor of 2-3. In various embodiments, the method produces single droplets at a rate of at least 5,000 drops per second. In various embodiments, the method produces single droplets at a rate of at least 8,000 drops per second.

[0007] In various embodiments, cells of the first cell-aligning stream are aligned along the central axis or edge of the first microchannel. In various embodiments, cells of the first cell-aligning stream are aligned through inertial focusing while flowing through the first microchannel. In various embodiments, inertial focusing is generated by flowing the first aqueous phase through a curved region of the first microchannel. In various embodiments, the curved region is 150-300 mm in length. In various embodiments, the curved region is 50-150 mm in length. In various embodiments, the curved region is approximately 100 mm in length. In various embodiments, the curved region includes at least one undulating portion comprising at least a 45-degree change in flow vector over the length of the undulating portion. In various embodiments, the curved region includes at least one undulating portion comprising at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in flow vector over the length of the undulating portion. In various embodiments, the curved region includes 60-120 undulating portions.

[0008] In various embodiments, the intercellular spacing for at least 80% of the cells in the first aligned stream is between 1 and 3.5 times the average cell diameter. In various embodiments, the intercellular spacing for at least 60% of the cells in the first aligned stream is between 1.5 and 3 times the average cell diameter. In various embodiments, the standard deviation of the intercellular spacing between consecutive cells in a pair is less than 10 μm when measured over 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first aligned stream. In various embodiments, the intercellular spacing between paired cells in the first aligned stream is adjusted by passing the paired cells between a set of pillars. In various embodiments, the set of pillars is positioned at the entrance of the first microchannel. In various embodiments, the set of pillars at the entrance of the first microchannel includes a gap of 5 to 40 μm between the pillars. In various embodiments, the cells of the second cell alignment stream are aligned along the central axis or edge of the second microchannel. In various embodiments, the cells of the second cell alignment stream are aligned through inertial focusing while flowing through the second microchannel. In various embodiments, the inertial focusing is produced by flowing the second aqueous phase through a curved region of the second microchannel. In various embodiments, the curved region of the second microchannel is 150-300 mm in length. In various embodiments, the curved region is 50-150 mm in length. In various embodiments, the curved region is approximately 100 mm in length. In various embodiments, the curved region of the second microchannel includes at least one undulating portion comprising at least a 45-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region of the second microchannel comprises at least one undulating portion comprising at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in the flow vector over the length of the undulating portion.In various embodiments, the curved region of the second microchannel comprises 60 to 120 undulating portions. In various embodiments, the intercellular spacing for at least 80% of the cells in the second aligned stream is between 1 and 3.5 times the average cell diameter. In various embodiments, the intercellular spacing for at least 60% of the cells in the second aligned stream is between 1.5 and 3 times the average cell diameter. In various embodiments, the standard deviation of intercellular spacing between pairs of consecutive cells is less than 10 μm when measured over 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first aligned stream.

[0009] In various embodiments, the intercellular spacing between paired cells in the second cell-aligned stream is adjusted by passing the paired cells between a second set of pillars. In various embodiments, the second set of pillars is positioned at the entrance of the second microchannel. In various embodiments, the second set of pillars at the entrance of the second microchannel includes a gap between the pillars of 5-40 μm.

[0010] In various embodiments, the ratio between the width of the first microchannel and the average diameter of the cells in the first cell alignment stream is between 1 and 20. In various embodiments, the ratio is between 1.5 and 10. In various embodiments, the ratio is between 1.5 and 7.5. In various embodiments, the ratio is between 2.5 and 5.0. In various embodiments, the cells in the first cell alignment stream are between 5 and 25 μm in diameter. In various embodiments, the first microchannel comprises a channel width between 10 and 100 μm. In various embodiments, the ratio between the average diameter of the cells in the second cell alignment stream and the width of the second microchannel is between 1 and 20. In various embodiments, the ratio is between 1.5 and 10. In various embodiments, the ratio is between 1.5 and 7.5. In various embodiments, the ratio is between 2.5 and 5.0. In various embodiments, the cells in the second cell alignment stream are between 5 and 25 μm in diameter. In various embodiments, the second microchannel comprises a channel width between 10 and 100 μm.

[0011] In various embodiments, the maximum concentration C1 of cells in the first cell alignment stream is: It is defined according to TIFF2025535563000002.tif11128, where D1 represents the diameter of the cells in the first aligned stream, S1 represents the spacing between the cells of a pair in the first aligned stream, W1 represents the width of the first microchannel, and H1 represents the height of the first microchannel.

[0012] In various embodiments, the maximum concentration C2 of cells in the second cell alignment stream is: Defined according to TIFF2025535563000003.tif11128, where D2 represents the diameter of the cells in the second aligned stream, S2 represents the spacing between the cells of a pair in the second aligned stream, W2 represents the width of the second microchannel, and H2 represents the height of the second microchannel.

[0013] In various embodiments, generating a single droplet comprises contacting the flowing first and second aqueous phases with each other, where the contacting creates a single aqueous phase comprising the first cell-aligned stream and the second cell-aligned stream. In various embodiments, contacting the flowing first and second aqueous phases to create the single aqueous phase occurs at or before the junction. In various embodiments, generating a single droplet further comprises contacting the flowing oil phase with the single aqueous phase to form a conical configuration within the junction, where the single droplet is generated at the tip of the conical configuration.

[0014] In various embodiments, the cells from the first cell-aligned stream and the cells from the second cell-aligned stream are different cell types. In various embodiments, the cells from the first cell-aligned stream are T cells. In various embodiments, the cells from the second cell-aligned stream are antigen-presenting cells (APCs). In various embodiments, the single droplet further comprises at least a second cell from the second cell-aligned stream. In various embodiments, the first aqueous phase is flowed at a first rate of 10 μL / min to 60 μL / min. In various embodiments, the first aqueous phase is flowed at a first rate of about 45 μL / min. In various embodiments, the second aqueous phase is flowed at a second rate of 10 μL / min to 60 μL / min. In various embodiments, the second aqueous phase is flowed at a second rate of about 45 μL / min. In various embodiments, the oil phase is flowed at a third rate of 10 μL / min to 60 μL / min. In various embodiments, the oil phase is flowed at a third rate of about 45 μL / min.

[0015] In various embodiments, the second aqueous phase is flowed at a second velocity that is faster than the first velocity of the first aqueous phase, such that a single droplet contains only a single cell from the first cell-aligned stream and two or more cells from the second cell-aligned stream.

[0016] In various embodiments, the methods disclosed herein further include detecting an interaction between cells from the first cell-aligned stream and cells from the second cell-aligned stream. In various embodiments, the first aqueous phase or the second aqueous phase further includes a reagent for detecting the interaction. In various embodiments, the reagent includes a fluorescent marker, a bead, or a nucleic acid barcode. In various embodiments, detecting the interaction includes detecting a biomarker analyte indicative of the interaction. In various embodiments, detecting the interaction includes detecting the interaction in a single droplet.

[0017] Also disclosed herein is a method for encapsulating two or more cells in a plurality of droplets, the method comprising: flowing a first aqueous phase comprising a first cell-aligned stream in a first microchannel; flowing a second aqueous phase comprising a second cell-aligned stream in a second microchannel; flowing an oil phase in a third microchannel; and flowing the first aqueous phase, the second aqueous phase, and the oil phase together to generate a plurality of droplets, wherein at least 20% of the droplets in the plurality of droplets comprise a single cell from the first cell-aligned stream and at least one cell from the second cell-aligned stream. In various embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the droplets in the plurality of droplets comprise a single cell from the first cell-aligned stream and at least one cell from the second cell-aligned stream.

[0018] In various embodiments, the multiple droplets are characterized by a proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream, where the proportion exceeds the expected proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream predicted using a Poisson distribution. In various embodiments, the proportion exceeds the expected proportion by a factor of 2-3. In various embodiments, the method generates multiple droplets at a rate of at least 5,000 droplets per second. In various embodiments, the method generates multiple droplets at a rate of at least 8,000 droplets per second.

[0019] In various embodiments, cells of the first cell alignment stream are aligned along a central axis of the first microchannel. In various embodiments, cells of the first cell alignment stream are aligned through inertial focusing while flowing through the first microchannel. In various embodiments, inertial focusing is generated by flowing the first aqueous phase through a curved region of the first microchannel. In various embodiments, the curved region is 150-300 mm in length. In various embodiments, the curved region is 50-150 mm in length. In various embodiments, the curved region is approximately 100 mm in length. In various embodiments, the curved region includes at least one undulating portion comprising at least a 45-degree change in flow vector over the length of the undulating portion. In various embodiments, the curved region includes at least one undulating portion comprising at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in flow vector over the length of the undulating portion. In various embodiments, the curved region includes 60-120 undulating portions.

[0020] In various embodiments, the intercellular spacing for at least 80% of the cells in the first aligned stream is between 1 and 3.5 times the average cell diameter. In various embodiments, the intercellular spacing for at least 60% of the cells in the first aligned stream is between 1.5 and 3 times the average cell diameter. In various embodiments, the standard deviation of the intercellular spacing between consecutive cells in a pair is less than 10 μm when measured over 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first aligned stream. In various embodiments, the intercellular spacing between paired cells in the first aligned stream is adjusted by passing the paired cells between a set of pillars. In various embodiments, the set of pillars is positioned at the entrance of the first microchannel. In various embodiments, the set of pillars at the entrance of the first microchannel includes a gap of 5 to 40 μm between the pillars.

[0021] In various embodiments, the cells of the second cell alignment stream are aligned along the central axis of the second microchannel. In various embodiments, the cells of the second cell alignment stream are aligned through inertial focusing while flowing through the second microchannel. In various embodiments, the inertial focusing is generated by flowing the second aqueous phase through a curved region of the second microchannel. In various embodiments, the curved region of the second microchannel is 150-300 mm in length. In various embodiments, the curved region is 50-150 mm in length. In various embodiments, the curved region is approximately 100 mm in length. In various embodiments, the curved region includes at least one undulating portion comprising at least a 45-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region includes at least one undulating portion comprising at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region includes 60-120 undulating portions. In various embodiments, the intercellular spacing for at least 80% of the cells in the second aligned stream is between 1 and 3.5 times the average cell diameter. In various embodiments, the intercellular spacing for at least 60% of the cells in the second aligned stream is between 1.5 and 3 times the average cell diameter. In various embodiments, the standard deviation of the intercellular spacing between consecutive cells in a pair is less than 10 μm when measured over 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first aligned stream. In various embodiments, the intercellular spacing between paired cells in the second aligned stream is adjusted by passing the paired cells between a set of pillars. In various embodiments, the set of pillars is positioned at the entrance of the second microchannel. In various embodiments, the set of pillars at the entrance of the first microchannel includes a gap of 5 to 40 μm between the pillars.

[0022] In various embodiments, the ratio between the width of the first microchannel and the average diameter of the cells in the first cell alignment stream is between 1 and 20. In various embodiments, the ratio is between 1.5 and 10. In various embodiments, the ratio is between 1.5 and 7.5. In various embodiments, the ratio is between 2.5 and 5.0. In various embodiments, the cells in the first cell alignment stream are between 5 and 25 μm in diameter. In various embodiments, the first microchannel comprises a channel width between 10 and 100 μm. In various embodiments, the ratio between the average diameter of the cells in the second cell alignment stream and the width of the second microchannel is between 1 and 20. In various embodiments, the ratio is between 1.5 and 10. In various embodiments, the ratio is between 1.5 and 7.5. In various embodiments, the ratio is between 2.5 and 5.0. In various embodiments, the cells in the second cell alignment stream are between 5 and 25 μm in diameter. In various embodiments, the second microchannel comprises a channel width between 10 and 100 μm.

[0023] In various embodiments, the maximum concentration C1 of cells in the first cell alignment stream is: TIFF2025535563000004.tif11128, where D1 represents the average diameter of cells in the first aligned stream, S1 represents the spacing between a pair of cells in the first aligned stream, W1 represents the width of the first microchannel, and H1 represents the height of the first microchannel.

[0024] In various embodiments, the maximum concentration C2 of cells in the second cell alignment stream is: Defined according to TIFF2025535563000005.tif11128, where D2 represents the average diameter of cells in the first aligned stream, S2 represents the spacing between pairs of cells in the first aligned stream, W2 represents the width of the first microchannel, and H2 represents the height of the first microchannel.

[0025] In various embodiments, generating a single droplet comprises contacting a flowing first aqueous phase and a flowing second aqueous phase, wherein the contacting creates a single aqueous phase comprising the first cell-aligned stream and the second cell-aligned stream. In various embodiments, contacting the flowing first aqueous phase and the flowing second aqueous phase to create a single aqueous phase occurs at or before the junction. In various embodiments, generating a single droplet comprises contacting a flowing oil phase with the single aqueous phase to form a conical configuration within the junction, wherein the single droplet is generated at the tip of the conical configuration.

[0026] In various embodiments, the cells from the first cell-aligned stream and the cells from the second cell-aligned stream are different cell types. In various embodiments, the cells from the first cell-aligned stream are T cells. In various embodiments, the cells from the second cell-aligned stream are antigen-presenting cells (APCs). In various embodiments, the single droplet further comprises at least a second cell from the second cell-aligned stream. In various embodiments, the first aqueous phase is flowed at a first rate of 10 μL / min to 60 μL / min. In various embodiments, the first aqueous phase is flowed at a first rate of about 45 μL / min. In various embodiments, the second aqueous phase is flowed at a second rate of 10 μL / min to 60 μL / min. In various embodiments, the second aqueous phase is flowed at a second rate of about 45 μL / min. In various embodiments, the oil phase is flowed at a third rate of 10 μL / min to 60 μL / min. In various embodiments, the oil phase is flowed at a third rate of about 45 μL / min. In various embodiments, the second aqueous phase is flowed at a second velocity that is faster than the first velocity of the first aqueous phase, such that a single droplet contains only a single cell from the first cell-aligned stream and two or more cells from the second cell-aligned stream.

[0027] In various embodiments, the methods disclosed herein further include detecting an interaction in a single droplet between cells from the first cell-aligned stream and cells from the second cell-aligned stream. In various embodiments, the first aqueous phase or the second aqueous phase further includes a reagent for detecting the interaction. In various embodiments, the reagent includes a fluorescent marker, a bead, or a nucleic acid barcode. In various embodiments, detecting the interaction in the single droplet includes detecting a biomarker analyte indicative of the interaction.

[0028] Also disclosed herein is a microfluidic device for encapsulating pairs of cells in droplets, the device comprising a first microchannel, a second microchannel, and a third microchannel fluidly connected to one another through a junction, the first microchannel comprising a curved region comprising a channel width of 10-100 μm, and the second microchannel comprising a curved region comprising a channel width of 10-100 μm. In various embodiments, the curved region of the first microchannel and the curved region of the second microchannel are 150-300 mm in length. In various embodiments, the first microchannel comprises a non-curved region located closer to the junction than the curved region of the first microchannel. In various embodiments, the second microchannel comprises a non-curved region located closer to the junction than the curved region of the second microchannel. In various embodiments, the channel widths of the curved regions of the first microchannel and the second microchannel are 15-75 μm. In various embodiments, the radius of curvature of the curved region of the first microchannel increases continuously along the entire length of the curved region. In various embodiments, the radius of curvature of the curved region of the second microchannel increases continuously along the entire length of the curved region. In various embodiments, the radius of curvature of the curved region of the first microchannel remains constant along the entire length of the curved region. In various embodiments, the curved region of the first microchannel includes a first undulating portion with a first radius of curvature and a second undulating portion with a second radius of curvature. In various embodiments, the first radius of curvature is different from the second radius of curvature. In various embodiments, the curved region includes at least one undulating portion including at least a 45-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region includes at least one undulating portion including at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region includes between 60 and 120 undulating portions. In various embodiments, the radius of curvature of the curved region of the second microchannel remains constant along the entire length of the curved region.In various embodiments, the curved region of the first microchannel includes a first undulating portion with a first radius of curvature and a second undulating portion with a second radius of curvature.

[0029] In various embodiments, the first radius of curvature is different from the second radius of curvature. In various embodiments, the curved region comprises 60-120 undulations. In various embodiments, the first microchannel further comprises a set of pillars positioned at an inlet of the first microchannel. In various embodiments, the set of pillars comprises a gap of 5-40 μm between the pillars. In various embodiments, the second microchannel further comprises a set of pillars positioned at an inlet of the second microchannel. In various embodiments, the set of pillars comprises a gap of 5-40 μm between the pillars. [Brief explanation of the drawings]

[0030] These and other features, aspects, and advantages of the present invention will be better understood in conjunction with the following description and the accompanying drawings. As a note, where practical, like or similar reference numerals may be used within the drawings and may indicate like or similar functionality. For example, a letter following a reference numeral, such as "aqueous well 105A," indicates that the text specifically refers to the element having that particular reference numeral. A reference numeral in text without a following letter, such as "aqueous well 105," refers to any or all elements in the drawings bearing that reference numeral (e.g., "aqueous well 105" in the text refers to the reference numerals "aqueous well 105A" and / or "aqueous well 105B" in the drawings). [Figure 1A] FIG. 1 shows an exemplary schematic of a microfluidic device for encapsulating two or more cells in a single droplet, according to one embodiment. [Figure 1B] 1 shows a first cell alignment stream, a second cell alignment stream, and encapsulation of two cells in a single droplet, according to one embodiment. [Figure 2]1 depicts a flow diagram for the alignment and encapsulation of two cells into a single droplet, according to one embodiment. [Figure 3A] 1 depicts an exemplary curved region of a serpentine microfluidic channel for aligning cells, according to one embodiment. [Figure 3B] See legend to Figure 3A. [Figure 3C] 1 depicts an exemplary curved region of a spiral microfluidic channel for aligning cells, according to one embodiment. [Figure 4] 1 shows an exemplary channel using pillars to separate cells into ordered streams, according to one embodiment. [Figure 5] 1 depicts an exemplary junction of a microfluidic device, according to one embodiment. [Figure 6A] 1 depicts an exemplary microfluidic device with a serpentine microchannel. [Figure 6B] An exemplary curved region of a serpentine microchannel of the exemplary microfluidic device shown in FIG. 6A is depicted at a higher magnification. [Figure 7A] 1 depicts an exemplary microfluidic device with a spiral microchannel. [Figure 7B] An exemplary curved region of the spiral microchannel of the exemplary microfluidic device shown in FIG. 7A is depicted at a higher magnification. [Figure 8] 1 shows the successful loading of two different cells in a single droplet according to one embodiment. [Figure 9A] An exemplary bright field image captured of two cell-aligned streams is shown. [Figure 9B] 1 shows exemplary intercellular distances relative to cell diameters for cells in an aligned stream. [Figure 10] 1 shows Jurkat T cells and K562 cells co-encapsulated in a single droplet to perform a cell-cell interaction assay. [Figure 11] 1 depicts the dimensions of an exemplary microfluidic device. [Figure 12]Figure 12A depicts a still image of droplet formation in an exemplary microfluidic device at 2 ms. Five droplets are circled in solid lines. Figure 12B depicts a still image of droplet formation in an exemplary microfluidic device at 3 ms (e.g., 1 ms after the still image shown in Figure 12A). The five droplets originally shown in Figure 12A are also shown in Figure 12B, circled in solid lines. Eight additional droplets are shown circled in dotted lines. [Figure 13] Depicts a paired cell droplet containing a T cell paired with a K562 cell that functions as an antigen-presenting cell (APC). DETAILED DESCRIPTION OF THE INVENTION

[0031] Detailed Description definition Terms used in the claims and this specification, unless otherwise specified, are defined as set forth below.

[0032] As used herein, "about" will be understood by one of ordinary skill in the art and will vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to a person of ordinary skill in the art given the context in which it is used, "about" will mean plus or minus up to 10% of the particular value.

[0033] The terms "subject" or "patient" are used interchangeably and include organisms, human or non-human, mammal or non-mammal, male or female.

[0034] In some embodiments, the discrete entities described herein are droplets. The terms "emulsion," "drop," "droplet," and "microdroplet" are used interchangeably herein to refer to small, generally spherical structures containing at least a first fluid phase, such as an aqueous phase (e.g., water), bounded by a second fluid phase (e.g., oil) that is immiscible with the first fluid phase. In some embodiments, droplets according to the present disclosure may contain a first fluid phase, such as oil, bounded by a second immiscible fluid phase, such as an aqueous fluid (e.g., water). In some embodiments, the second fluid phase is the carrier fluid of the immiscible phase, and thus droplets according to the present disclosure may be provided as water-in-oil emulsions or oil-in-water emulsions. Droplets may be sized and / or shaped as described herein for discrete entities. For example, droplets according to the present disclosure generally range in diameter from 1 μm to 1000 μm, inclusive. Droplets according to the present disclosure may be used to encapsulate cells, nucleic acids (e.g., DNA), enzymes, reagents, reaction mixtures, and various other components. The term emulsion may be used to refer to emulsions provided in, on, or by a microfluidic device and / or emulsions flowed from or applied by a microfluidic device.

[0035] As used herein, the term "fluid" is given its original meaning: liquid or gas. A fluid cannot maintain a defined shape and flows over an observable time frame, filling a container in which it is placed. Thus, a fluid may have any suitable consistency that allows it to flow. If two or more fluids are present, each fluid may be independently selected by one of skill in the art from essentially any fluid (such as a liquid or gas). Certain embodiments provide multiple droplets. In some embodiments, multiple droplets may be formed from a first fluid and substantially surrounded by a second fluid. As used herein, a droplet is "surrounded" by a fluid if a closed loop can be drawn around the droplet through only the fluid. A droplet is "completely surrounded" if a closed loop can be drawn around the droplet through only the fluid, regardless of direction. A droplet is "substantially surrounded" if a loop can be drawn around the droplet, depending on the direction, that passes only through the fluid (e.g., in some cases, the loop around the droplet will contain almost only that fluid, but may also contain a second fluid or a second droplet, etc.).

[0036] In most, but not all, embodiments, the droplets and the fluid containing them are substantially immiscible, although in some cases they may be miscible. In some cases, a hydrophilic liquid may be suspended in a hydrophobic liquid, a hydrophobic liquid may be suspended in a hydrophilic liquid, air bubbles may be suspended in a liquid, etc. Typically, hydrophobic and hydrophilic liquids are substantially immiscible with each other, with the hydrophilic liquid having a greater affinity for water than the hydrophobic liquid. Examples of hydrophilic liquids include, but are not limited to, water and other aqueous solutions containing water, such as cell or biological media, ethanol, saline solutions, etc.

[0037] The phrase "at a junction" refers to a step occurring at or within a microfluidic junction formed by the confluence of two or more microchannels. In various embodiments, "at a junction" refers to a step occurring immediately downstream of the junction (e.g., within 1 mm, within 2 mm, within 3 mm, within 4 mm, or within 5 mm downstream of the junction). In certain embodiments, the junction is described herein in terms of droplet generation and may also be referred to as a "droplet generation region."

[0038] The phrase "ordered stream of cells" refers to the alignment of cells flowing through a microfluidic channel. In some embodiments, a flowing cell stream is a "cell-ordered stream" if a line drawn parallel to the direction of the cell flow vector successfully intersects each cell in the flowing stream. In some embodiments, a flowing cell stream is a "cell-ordered stream" if a line drawn parallel to the direction of the cell flow vector successfully intersects at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the flowing stream. In some embodiments, a flowing cell stream is a "cell-ordered stream" if a line drawn parallel to the direction of the cell flow vector successfully intersects the center of each cell in the flowing stream.

[0039] The phrase "average cell diameter" refers to the average diameter, e.g., mean diameter, of a particular type of cell in a cell-aligned stream within a microfluidic channel. As further described herein, the average cell diameter can affect the intercellular spacing between paired cells in a cell-aligned stream within a microfluidic channel.

[0040] overview Described herein are methods for aligning and co-encapsulating two or more cells in a single droplet in a high-throughput and efficient manner. The methods further involve examining interactions between the two or more cells co-encapsulated in a single droplet. Also disclosed herein are microfluidic systems for aligning a cell stream and co-encapsulating two or more cells in a single droplet to examine interactions between the two or more cells. When analysis is performed in a high-throughput manner, such microfluidic systems can identify cells of interest while maintaining the integrity of those cells.

[0041] In various embodiments, a method for co-encapsulating two or more cells in a single droplet in a high-throughput and efficient manner generally involves the following steps: 1. Aligning two cell streams through a curved region of a microchannel, where the cells are subjected to inertial focusing forces in the curved region, resulting in alignment of the cells (e.g., at equilibrium with the cells lined up in a single line and substantially equidistant within the microchannel). 2. Providing two cell-aligned streams at a microchannel junction where they meet an oil phase, and by fine-tuning the speed of the oil phase, the oil phase separates each cell stream by a distance of one cell, resulting in co-encapsulation of paired cells, one cell from the first aligned stream and at least one cell from the second aligned stream in a single droplet.

[0042] Disclosed herein is a method for conducting an assay in a single droplet containing two cells, comprising: flowing a first aqueous phase containing a first cell-aligned stream in a first microchannel toward a junction, flowing a second aqueous phase containing a second cell-aligned stream in a second microchannel toward the junction, flowing an oil phase in a third microchannel toward the junction, and generating a single droplet at the junction formed from the first aqueous phase, the second aqueous phase, and the oil phase, the single droplet containing cells from the first cell-aligned stream and cells from the second cell-aligned stream. Also disclosed herein is a method for encapsulating paired cells in multiple droplets, comprising: flowing a first aqueous phase containing a first cell-aligned stream in a first microchannel, flowing a second aqueous phase containing a second cell-aligned stream in a second microchannel, flowing an oil phase in a third microchannel, and flowing the first aqueous phase, the second aqueous phase, and the oil phase together to generate multiple droplets. In various embodiments, the disclosed methods achieve high co-encapsulation efficiencies, where at least 15% of the droplets in a plurality of droplets comprise a single cell from the first cell-aligned stream and a single cell from the second cell-aligned stream, hi various embodiments, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the droplets in a plurality of droplets comprise a single cell from the first cell-aligned stream and a single cell from the second cell-aligned stream.

[0043] As described in further detail herein, a cell-cell interaction assay is performed on a single droplet containing two or more cells. For example, the method disclosed herein involves further encapsulating a reagent for performing a cell-cell interaction assay into the single droplet during or after co-encapsulation. In the droplet where two or more cells interact, the reagent reports on the activation markers of the two or more cells and / or on molecules secreted in the environment. In various embodiments, the method disclosed herein further involves identifying or sorting cells of interest based on the detection of the reagent.

[0044] Exemplary method for encapsulating two cells in a single droplet Generally, a method for encapsulating two cells (or two or more cells) in a single droplet involves at least two steps: 1) aligning two cell streams in two separate microchannels, and 2) generating a single droplet containing cells from a first cell-aligned stream and cells from a second cell-aligned stream at the junction where the two separate microchannels meet. As described herein, parameters that are conducive to aligning cells into an aligned stream (e.g., the concentration of cells in the aqueous fluid, the flow rate of the aqueous phase containing the cells, the flow rate of the oil phase, the microchannel width, and the inter-cell spacing within the cell-aligned stream) may not be conducive to co-encapsulating two or more cells in a single droplet. Therefore, parameters are fine-tuned to achieve a fluid flow regime that achieves both alignment of the cell streams and co-encapsulation of two or more cells in a single droplet.

[0045] Generally, in microfluidic devices, cell alignment occurs within curved regions of the microchannels of the microfluidic device. Generally, cell alignment within curved regions of the microchannel is due to inertial focusing forces arising from the curvature of the curved regions of the microchannel. These secondary forces induce secondary flows, also known as Dean flows. For example, under sufficient Dean flows, the equilibrium positions within the microchannel become unstable as the secondary flows impinge on particles. This can leave a single lateral equilibrium position within the microchannel, such as the curved inner wall, thereby aligning cells within the aligned stream. Further details of cell alignment and Dean flows are described in Martel JM, et al. Inertial focusing in microfluidics. Annu Rev Biomed Eng. 2014 Jul 11;16:371-96, which is incorporated herein by reference in its entirety.

[0046] Co-encapsulation of two or more cells into a single droplet occurs at a junction in a microfluidic device. Generally, a junction represents the confluence of two or more microchannels, where a first microchannel and a second microchannel each carry a cell-aligned stream (e.g., ordered as described above by inertial focusing forces arising from curved regions of the microchannels). Additionally, at least one microchannel carrying an immiscible oil phase (immiscible with the aqueous fluid carrying the cell-aligned stream) flows into the junction. Thus, the confluence of two or more microchannels carrying cell-aligned streams with one or more microchannels carrying an immiscible oil phase results in the generation of a single droplet containing two or more cells (e.g., one cell from the first cell-aligned stream and at least one cell from the second cell-aligned stream). Notably, the flow of the aqueous fluid carrying the cell alignment stream and the flow of the immiscible oil phase are carefully controlled to avoid entering the "jetting" or "co-flow" regime where droplet formation fails.

[0047] Reference is now made to Figure 1A, which shows an exemplary schematic of a microfluidic device for encapsulating two or more cells in a single droplet, according to one embodiment. Figure 1A is shown to introduce a first aqueous well 105A, a second aqueous well 105B, a first microchannel 115A, a curved region 110A of the first microchannel 115A, a second microchannel 115B, a curved region 110B of the second microchannel 115B, a first oil phase well 120A, a third microchannel 125A fluidly connected to the first oil phase well 120A, a second oil phase well 120B, a fourth microchannel 125B fluidly connected to the second oil phase well 120B, a junction 130, a collection well 140, and a sixth microfluidic channel 135 fluidly connecting the junction 130 to the collection well 140. Generally, operation of the microfluidic device shown in FIG. 1A involves the flow of solutions from the left (eg, from wells 105A and 105B) to the right (eg, to collection well 140).

[0048] 1A shows one exemplary embodiment of a microfluidic device for encapsulating two or more cells in a single droplet. In some embodiments, the device may be configured differently. As an example, the microfluidic device need not include two separate oil phase wells 120A and 120B, but instead includes a single oil phase well fluidly connected to provide the oil phase to junction 130. Such exemplary microfluidic devices are shown in FIGS. 6A and 7A.

[0049] 1A shows two sets of aqueous wells 105, microchannels 115, and curved regions 110 of the microchannels 115 leading to junction 130. In various embodiments, the microfluidic device may include additional sets of aqueous wells 105 connected to additional microchannels 115 with curved regions 110. For example, the microfluidic device may include three, four, five, six, seven, eight, nine, or ten sets of aqueous wells 105 connected to additional microchannels 115 with curved regions 110. This allows for the co-encapsulation of different types of cells and / or reagents provided through the different aqueous wells leading to junction 130. For example, in various embodiments, the microfluidic device may allow for the co-encapsulation of two cells, three cells, four cells, five cells, six cells, seven cells, eight cells, nine cells, or ten or more cells in a single droplet.

[0050] Generally, cells of a first cell type are provided in first aqueous well 105A. Cells of a second cell type are provided in second aqueous well 105B. Under microfluidic control, cells of the first cell type are driven from first aqueous well 105A through at least curved region 110A of first microchannel 115A, where curved region 110A of first microchannel 115A exerts an inertial focusing force on the cells of the first cell type to generate a first cell-aligned stream as they enter junction 130. Similarly, under microfluidic control, cells of a second cell type are driven from second aqueous well 105B through at least curved region 110B of second microchannel 115B, where curved region 110B of second microchannel 115B exerts an inertial focusing force on the cells of the second cell type to generate a second cell-aligned stream as they enter junction 130. For purposes of simple diagrammatical illustration, curved regions 110A and 110B are shown as serpentine portions of first microchannel 115A and second microchannel 115B, but as discussed in more detail herein (and shown, for example, in Figures 6A and 9A), curved regions 110A and 110B may be significantly more complex in various embodiments.

[0051] 1A , the first microchannel 115A may include a non-curved region 118A leading to the junction 130. Thus, the non-curved region 118A is closer to the junction 130 than the curved region 110A of the first microchannel 115A. Similarly, the second microchannel 115B may include a non-curved region 118B leading to the junction 130. Thus, the non-curved region 118B is closer to the junction 130 than the curved region 110B of the second microchannel 115B. Here, the non-curved regions 118A and 118B leading to the junction 130 allow the respective cell-aligned streams to approach the junction 130 with reduced, limited, or no inertial focusing forces.

[0052] An immiscible oil phase is provided in oil phase well 120. Here, under microfluidic control, the oil phase flows through third microchannel 125A and / or fourth microchannel 125B to join at junction 130. Reference is now made to FIG. 1B, which shows an enlarged view of junction 130. Specifically, FIG. 1B shows a first cell-aligned stream 150A entering junction 130 through first microchannel 115A, a second cell-aligned stream 150B entering junction 130 through second microchannel 115B, and the immiscible oil phase entering junction 130 through third microchannel 125A and fourth microchannel 125B. The flowing aqueous fluid, including the two cell-aligned streams, is sandwiched by the flowing oil phase, thereby causing droplet formation downstream of junction 130. Specifically, as shown in FIG. 1B, a single droplet 155 contains two or more cells 160, where one cell originates from a first cell-aligned stream 150A and another cell originates from a second cell-aligned stream 150B.

[0053] Exemplary oils for use as the immiscible oil phase of an emulsion may be selected based on chemical properties such as, for example, molecular structure, content, solvent strength, viscosity, boiling point, coefficient of thermal expansion, oil-in-water solubility, water-in-oil solubility, dielectric constant, polarity, water-in-oil surface tension, and / or oil-in-water surface tension. Examples of oils include fluorinated oils, non-fluorinated oils, alkanes (e.g., hexane, decane, and octane), mineral oil, vegetable oil, edible oil, mineral oil, oleic acid, embryo-tested mineral oil, light mineral oil, heavy mineral oil, PCR mineral oil, AS4 silicone oil, AS 100 silicone oil, AR20 silicone oil, AR 200 silicone oil, AR 1000 silicone oil, AP 100 silicone oil, AP 1000 silicone oil, AP 150 silicone oil, AP 200 silicone oil, CR 200 silicone oil, DC 200 silicone oil, DC702 silicone oil, and DC 710 Silicone oil, octanol, decanol, acetophenone, perfluoro oil, perfluorononane, perfluorodecane, perfluorodimethylcyclohexane, perfluoro-1-butanesulfonyl fluoride, perfluoro-1-octanesulfonyl fluoride, perfluoro-1-octanesulfonyl fluoride, nonafluoro-1-butanesulfonyl chloride, nonafluoro-tert-butyl alcohol, perfluorodecanol, perfluorohexane, perfluorooctanol, perfluorodecene, perfluorohexene, perfluorooctene, fuel oil, halocarbon oil 28, halocarbon oil 700, hydrocarbon oil, glycerol, 3M Fluoriner™ fluids (FC-40, FC-43, FC-70, FC-72, FC-77, FC-84, FC-87, FC-3283), oils containing trifluoroacetic acid, oils containing hexafluoroisopropanol, Krytox oils (e.g., oils containing hexafluoropropylene epoxide and / or polymers thereof), oils containing polyhexafluoropropylene oxide and / or polymers thereof, Krytox GPL oils, oils containing perfluoropolyethers, oils containing perfluoroalkyl ethers, oils containing perfluoropolyalkyl ethers, SolvayGalden oil, oils containing hydrofluoroethers (e.g., HFE-7500, HFE-7100, HFE-7200, HFE-7600), oils containing perfluoroalkylamines (e.g., Fluorinert FC-3283, and Fluorinert FC-40), soybean oil, castor oil, coconut oil, cedar oil, clove bud oil, fir oil, linseed oil, safflower oil, sunflower oil, almond seed oil, anise oil, clove oil, cottonseed oil, corn oil, croton oil, olive oil, palm oil, peanut oil, bay oil, borage oil, bergamot oil, cod liver oil, macadamia nut oil, and camada oil. oil), chamomile oil, citronella oil, eucalyptus oil, fennel oil, lavender oil, lemon oil, nutmeg oil, orange oil, petitgrain oil, rose oil, tarragon oil, tung oil, basil oil, birch oil, black pepper oil, birch tar oil, carrot seed oil, cardamom oil, cassia oil, sage oil, cognac oil, copaiba balsam oil, cypress oil, eucalyptus oil, dill oil, grapefruit oil Root oil, ginger oil, juniper oil, lavender oil, lovage oil, marjoram oil, mandarin oil, myrrh oil, neroli oil, olibanum oil, onion oil, paraffin oil, origanum oil, parsley oil, peppermint oil, pimenta leaf oil, sage oil, rosemary oil, rose oil, sandalwood oil, sassafras oil, spearmint oil, thyme oil, trance oil, verbena oil, and rapeseed oil.

[0054] Returning to FIG. 1A , the droplets containing two or more cells continue to flow through microchannel 135 toward collection well 140. In various embodiments, while flowing through microchannel 135, the droplets may be subjected to an assay, such as a cell-cell interaction assay, to determine whether two or more cells have interacted with one another. As described in further detail herein, a cell-cell interaction assay may involve capturing a signal (e.g., a fluorescent signal) emitted in the single droplet when a cell-cell interaction occurs. Thus, in such embodiments, a detection module, i.e., a detector such as, for example, an optical imager or a fluorescent imager, may be configured to capture the signal emitted in the single droplet. For example, the detection module may be positioned to capture the signal emitted in the single droplet while the droplet is flowing through microchannel 135 toward collection well 140.

[0055] Reference is now made to Figure 2, which depicts a flow diagram for the alignment and encapsulation of two cells into a single droplet according to one embodiment. As shown in Figure 2, step 210 involves flowing a first aqueous phase comprising a first cell alignment stream in a first microchannel toward a junction. Step 220 involves flowing a second aqueous phase comprising a second cell alignment stream in a second microchannel toward the junction. Step 230 involves flowing an oil phase in a third microchannel toward the junction. Step 240 involves generating a single droplet comprising cells from the first alignment stream and cells from the second alignment stream from the first aqueous phase, the second aqueous phase, and the oil phase.

[0056] Cell alignment in a stream The embodiments described herein involve generating a cell-ordered stream within a microfluidic channel. In various embodiments, the disclosed methods and apparatus involve generating two or more cell-ordered streams within two or more microfluidic channels. For example, an embodiment may involve generating exactly two cell-ordered streams within two microfluidic channels. In other scenarios, an embodiment may involve generating three cell-ordered streams within three microfluidic channels, four cell-ordered streams within four microfluidic channels, five cell-ordered streams within five microfluidic channels, six cell-ordered streams within six microfluidic channels, seven cell-ordered streams within seven microfluidic channels, eight cell-ordered streams within eight microfluidic channels, nine cell-ordered streams within nine microfluidic channels, or ten cell-ordered streams within ten microfluidic channels. As used herein, "ordered stream of cells" refers to the alignment of cells flowing within a microfluidic channel. For example, a flowing cell stream is a "cell-aligned stream" if a line drawn parallel to the direction of the cell flow vector successfully traverses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the cells in the flowing stream.

[0057] While the following description relates to cell alignment within a single microchannel, the description may equally apply to cell alignment within multiple microchannels. Generally, cells within a microchannel are aligned into a cell-aligned stream through a process known as inertial focusing, which is driven by Dean forces. Inertial focusing pushes cells tangentially to the direction of flow until they reach an equilibrium position. In various embodiments, the equilibrium position may be along the central axis of the microchannel. Thus, in such embodiments, cells in the cell-aligned stream are aligned along the central axis of the microchannel. In various embodiments, the equilibrium position may not be along the central axis of the microchannel, but may instead be along the side or edge of the microchannel due to laminar fluid flow.

[0058] Generally, inertial focusing due to secondary forces (e.g., Dean forces) occurs due to the curvature of a microchannel (e.g., curved region 110A or curved region 110B shown in FIG. 1A ). For example, Dean flow occurs due to velocity differences across the cross section of a microchannel, such as the velocity difference in a parabolic flow where the fluid at the center of the channel moves faster than the fluid near the walls. The additional momentum carried by the faster-moving fluid at the center of the channel carries the fluid toward the outer wall of the channel curvature as it enters the curve. By conservation laws, this creates a recirculation of the fluid along the upper and lower surfaces of the channel toward the center of the channel curvature. Overall, embodiments of curved regions within a microchannel exert inertial focusing forces on flowing cells, thereby aligning the flowing cells and creating a cell-aligned stream within the microchannel.

[0059] In various embodiments, the curved region of the microfluidic channel includes at least an undulating portion, where the directional flow vector of the cells changes over the length of the undulating portion. The change in the directional flow vector of the cells causes an inertial focusing force to be exerted on the flowing cells. In various embodiments, the curved region includes at least one undulating portion that includes at least a 45-degree change in the flow vector over the length of the undulating portion. In various embodiments, the curved region includes at least one undulating portion that includes at least a 60-degree change, at least a 90-degree change, at least a 120-degree change, at least a 150-degree change, or at least a 180-degree change in the flow vector over the length of the undulating portion.

[0060] Reference is now made to FIG. 3A, which depicts an exemplary curved region of a serpentine microfluidic channel for cell alignment according to one embodiment. For example, curved region 115 shown in FIG. 3A could refer to curved region 115A or curved region 115B shown in FIG. 1A. FIG. 3A illustrates a serpentine microfluidic channel with multiple asymmetric curves. Specifically, cell flow enters from the left side, labeled as the "cell inlet." The cells flow through multiple undulating sections (e.g., undulating sections 310A, 310B, 310C, 310D, and 310E). By directing the flow through undulating sections that impart inertial focusing forces, the flowing cells exit on the right side as a cell-aligned stream.

[0061] FIG. 3A also shows the directional flow vectors as cells flow through the various undulating sections 310. Specifically, referring to the directional flow vector corresponding to undulating section 310A, the flow vector begins in an upward 90° direction (assuming a 0° direction is horizontally to the right). As cells continue to flow through undulating section 310A, the flow vector changes. For example, the flow vector may proceed from an upward 90° direction, reduce to 45°, then 0°, then −45°, and then reduce to −90° at the end of undulating section 310A. Referring to the directional flow vector corresponding to undulating section 310B, the flow vector may begin in a downward −90° direction and then rapidly reverse to an upward 90° direction. The rapid directional flow change over the length of undulating section 310B may further aid in the alignment of the cell stream. 3A, cells may further flow through undulating portions 310C and 310D, which may be replicas of undulating portions 310A and 310B, respectively. Finally, cells may further flow through undulating portion 310E, which may be replicas of undulating portion 310A and / or undulating portion 310C.

[0062] While Figure 3A depicts five undulations in an asymmetrically curved microchannel, in various embodiments, additional or fewer undulations may be present in the curved region 115 of the microchannel. In various embodiments, the curved region 115 includes between 30 and 180 undulations. In various embodiments, the curved region 115 includes between 35 and 170 undulations, between 40 and 160 undulations, between 45 and 150 undulations, between 50 and 140 undulations, between 55 and 130 undulations, between 60 and 120 undulations, between 65 and 110 undulations, between 70 and 100 undulations, or between 75 and 90 undulations. In certain embodiments, the curved region 115 includes between 60 and 120 undulations.

[0063] 3A depicts two distinct undulating portions (e.g., undulating portion 310A and undulating portion 310B) replicated across the length of curved region 115. Here, undulating portion 310A and undulating portion 310B differ in that they have different radii of curvature. Specifically, undulating portion 310A may have a first radius of curvature that is greater than the radius of curvature of undulating portion 310B. Thus, as cells flow through curved region 115, they experience different inertial focusing forces due to the first radius of curvature of undulating portion 310A and the second radius of curvature of undulating portion 310B. In various embodiments, undulating portion 310A and undulating portion 310B (and the replicated undulating portions) have the same radius of curvature. Thus, in such embodiments, the radius of curvature of curved region 115 of the microchannel may be constant across the entire length of curved region 115.

[0064] Reference is now made to FIG. 3B, which depicts an exemplary curved region of a serpentine microfluidic channel for cell alignment according to a second embodiment. Here, the curved region 115 shown in FIG. 3B may include unit 340A, which includes undulating portions 310 (e.g., 310A, 310B, 310C, 310D, and 310E) shown in FIG. 3A. Furthermore, the curved region 115 shown in FIG. 3B may further include an additional unit 340B, which includes an additional undulating portion. Here, the additional unit 340B may be a mirror image of the undulating portion of unit 340A. Thus, cells flow from the upper left, labeled "cell inlet," through the undulating portion of unit 340A, and then through the undulating portion of unit 340B to the outlet, labeled "cell alignment stream." Generally, the curved region 115 shown in Figure 3B includes additional undulations compared to the curved region 115 shown in Figure 3A, which means that cells flowing through the curved region 115 in Figure 3B are subjected to inertial focusing forces along a longer length of the curved portion 115 in Figure 3B. The longer length of the curved region aids in consistent alignment of cells within the microchannel.

[0065] Reference is now made to FIG. 3C , which depicts an exemplary curved region of a spiral microfluidic channel for aligning cells, according to one embodiment. Generally, in a spiral microfluidic channel, inertial focusing forces increase as the radius of curvature in the microchannel decreases (e.g., a tighter curve in the microchannel creates a greater imbalance between outward and inward forces, which preferentially pushes cells toward the inner wall of the curve). As shown in FIG. 3C , a cell inlet may feed cells into curved region 115. As the radius of curvature of curved region 115 increases with each loop, inertial focusing becomes less efficient and flow resistance increases, which reduces the overall flow rate and further reduces inertial focusing. The equilibrium position for inertial focusing may be along the inner wall of the microchannel.

[0066] FIG. 3C depicts a spiral microfluidic channel with two loops (e.g., one internal loop and one external loop leading to an outlet labeled "cell alignment stream"). In various embodiments, the spiral microfluidic channel may include additional loops. For example, the spiral microfluidic channel may include three, four, five, six, seven, eight, nine, or ten loops before leading to the outlet. FIG. 3C further illustrates the directional flow vector of a cell over half the length of the loop. For example, the directional flow vector begins at the bottom of the loop and heads left (e.g., 180°). As the cell migrates along the length of the channel, the flow vector continuously decreases from 180° to 135°, then to 90° at the midpoint, then to 45°, and then to 0° before exiting through the outlet. Thus, over half the length of the loop, the flow vector changes a full 180°. Furthermore, over the entire length of the loop, the flow vector changes a full 360°.

[0067] In various embodiments, the cell alignment streams are 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 110 or more or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or or more, 1000 or more, 1200 or more, 1500 or more, 1800 or more, 2000 or more, 2500 or more, 3000 or more, 4000 or more, 5000 or more, 10,000 or more, 15,000 or more, 20,000 or more, 25,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 100,000 or more, 200,000 or more, 300,000 or more, 400,000 or more, 500,000 or more, or 1 million or more cells. In certain embodiments, the cell alignment stream comprises 10-1000 cells, 20-900 cells, 30-800 cells, 40-700 cells, 50-600 cells, 60-500 cells, 70-400 cells, 80-300 cells, 90-200 cells, or 100-150 cells.

[0068] As described herein, intercellular spacing refers to the distance between any pair of cells in an ordered stream (e.g., between the center of a first cell and the center of a second cell). In certain embodiments, intercellular spacing refers to the distance between two consecutive cells in an ordered stream (e.g., two consecutive cells with no other cells between them). Intercellular spacing may be described in terms of cell diameter, such as the average cell diameter of the cells in the ordered stream. In various embodiments, the intercellular spacing between any pair of consecutive cells is between 0.5 times the average cell diameter and 5.5 times the average cell diameter. In certain embodiments, the intercellular spacing between any pair of consecutive cells is between 1 times the average cell diameter and 5 times the average cell diameter, between 1.2 times the average cell diameter and 4 times the average cell diameter, between 1.5 times the average cell diameter and 3.5 times the average cell diameter, or between 2 times the average cell diameter and 3 times the average cell diameter. In certain embodiments, the intercellular spacing between any pair of consecutive cells is about one cell diameter, about two cell diameters, about three cell diameters, about four cell diameters, or about five cell diameters. In various embodiments, the cell diameter (e.g., average cell diameter) of the cells is about 5 μm to about 25 μm. Further examples of cell diameters are disclosed herein.

[0069] In various embodiments, for a cell-aligned stream, the intercellular spacing for at least 80% of the cells in the aligned stream is between 1 and 3.5 times the average cell diameter. In various embodiments, for a cell-aligned stream, the intercellular spacing for at least 60% of the cells in the aligned stream is between 1.5 and 3 times the average cell diameter. In various embodiments, for a cell-aligned stream, the standard deviation of intercellular spacing between pairs of consecutive cells is less than 10 μm when measured over 10 pairs of adjacent cells in the first cell-aligned stream. In various embodiments, for a cell-aligned stream, the standard deviation of intercellular spacing between pairs of consecutive cells is less than 10 μm when measured over 20, 30, 40, 50, 60, 70, 80, 90, or 100 pairs of adjacent cells in the first cell-aligned stream. In various embodiments, for a cell alignment stream, the standard deviation of intercellular spacing between pairs of consecutive cells is less than 9 μm, less than 8 μm, or less than 7 μm when measured across 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first cell alignment stream.

[0070] Parameters for cell alignment As described herein, tunable parameters are set to achieve cell alignment and successful co-encapsulation of two or more cells in a single droplet. Examples of tunable parameters include the concentration of cells in the aqueous fluid, the flow rate of the cell-containing aqueous phase, the flow rate of the oil phase, the microchannel width, and the inter-cell spacing in the cell alignment stream. Exemplary sets of parameters useful for aligning cell streams and / or co-encapsulating two or more cells in a single droplet are disclosed herein.

[0071] Microfluidic channel dimensions and cell diameter In general, the successful ordering of cells into a stream may depend on the microfluidic channel width and also on the cell concentration. For example, as the microchannel width is increased, the maximum concentration of cells that can be processed may decrease. Conversely, as the microchannel width is reduced, the flow resistance increases, thereby limiting the volume of cell solution that can be processed per unit time. Therefore, the microchannel width should be optimized to enable the maximum volumetric throughput and the maximum cell concentration.

[0072] While the following description refers to a single microchannel, those skilled in the art will understand that the description may also refer to either or both of the microchannels (e.g., first microchannel 115A and / or second microchannel 115B) as described in FIG. 1A . In various embodiments, the appropriate width of the curved region of the microchannel (e.g., first microchannel and / or second microchannel) for aligning a cell stream depends on the diameter of the cells. The cell diameter may refer to the average cell diameter of the cells in a population of cells. In various embodiments, the width of the microchannel (e.g., the curved region of the microchannel) for aligning a cell stream is from about 1 to about 20 times the cell diameter. In other words, the ratio between the width of the microchannel and the average diameter of the cells in the cell-aligning stream is from 1 to 20. In various embodiments, the width of the microchannel (e.g., the curved region of the microchannel) for aligning a cell stream is from about 2 to about 8 times the cell diameter, from about 3 to about 7 times the cell diameter, or from about 4 to about 6 times the cell diameter. In various embodiments, the width of the microchannel (e.g., the curved region of the microchannel) for aligning the cell stream is about 1x, about 1.5x, about 2x, about 2.5x, about 3x, about 3.5x, about 4x, about 4.5x, about 5x, about 5.5x, about 6x, about 6.5x, about 7x, about 7.5x, about 8x, about 8.5x, about 9x, about 9.5x, about 10x, about 11.5x, about 12x, about 12.5x, about 13x, about 13.5x, about 14x, about 14.5x, about 15x, about 15.5x, about 16x, about 16.5x, about 17x, about 17.5x, about 18x, about 18.5x, about 19x, about 19.5x, or about 20x the cell diameter. In certain embodiments, the ratio between the width of the microchannel and the average diameter of the cells in the cell alignment stream is between 1.5 and 7.5. In certain embodiments, the ratio between the width of the microchannel and the average diameter of the cells in the cell alignment stream is between 2.5 and 5.0.In certain embodiments, the ratio between the width of the microchannel and the average diameter of the cells in the cell alignment stream is between 10.0 and 20.0, such as between 11.0 and 19.0, between 12.0 and 18.0, between 13.0 and 17.0, or between 14.0 and 16.0.

[0073] In various embodiments, the cell diameter of the cells (e.g., the average cell diameter of a population of cells to be arrayed) is about 5 μm to about 25 μm. In various embodiments, the cell diameter of the cells (e.g., the average cell diameter of a population of cells to be arrayed) is about 6 μm to about 24 μm, about 7 μm to about 23 μm, about 8 μm to about 22 μm, about 9 μm to about 21 μm, about 10 μm to about 20 μm, about 11 μm to about 19 μm, about 12 μm to about 18 μm, about 13 μm to about 17 μm, or about 14 μm to about 16 μm. In various embodiments, the cell diameter of the cells (e.g., the average cell diameter of the population of cells to be arrayed) is about 5 μm, about 6 μm, about 7 μm, about 8 μm, about 9 μm, about 10 μm, about 11 μm, about 12 μm, about 13 μm, about 14 μm, about 15 μm, about 16 μm, about 17 μm, about 18 μm, about 19 μm, about 20 μm, about 21 μm, about 22 μm, about 23 μm, about 24 μm, or about 25 μm.

[0074] In various embodiments, the width of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 5 μm to about 200 μm. In various embodiments, the width of the microchannel is about 10 μm to about 100 μm, about 15 μm to about 95 μm, about 20 μm to about 90 μm, about 25 μm to about 85 μm, about 30 μm to about 80 μm, about 35 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of the microchannel is about 50 μm to about 100 μm (e.g., about 55 μm to about 95 μm, about 60 μm to about 90 μm, about 65 μm to about 85 μm, or about 70 μm to about 80 μm). In various embodiments, the width of the microchannel is about 50 μm to about 75 μm. In certain embodiments, the width of the microchannel is about 50 μm. In certain embodiments, the width of the microchannel is about 75 μm. In various embodiments, a single microchannel may vary in width, which may further facilitate aligning cells into a stream. For example, a first portion of a single microchannel may have a width of about 50 μm, and a second portion of the single microchannel may have a width of about 75 μm.

[0075] Another tunable parameter includes the length of the microchannel. Specifically, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) may be tuned to allow more time for the Dean forces to inertially focus the cells. However, increasing the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) also increases the flow resistance and reduces the flow velocity.

[0076] In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 10 mm to about 500 mm. In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 125 mm to about 400 mm, about 150 mm to about 300 mm, or about 175 mm to about 200 mm. In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 100 mm to about 300 mm, about 125 mm to about 275 mm, about 150 mm to about 250 mm, about 175 mm to about 225 mm, about 180 mm to about 220 mm, about 185 mm to about 215 mm, about 190 mm to about 210 mm, or about 195 mm to about 205 mm. In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 30 mm to about 400 mm, about 40 mm to about 350 mm, or about 50 mm to about 300 mm. In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 40 mm to about 200 mm, about 50 mm to about 150 mm, about 60 mm to about 140 mm, about 70 mm to about 130 mm, about 80 mm to about 120 mm, about 85 mm to about 115 mm, about 90 mm to about 110 mm, or about 95 mm to about 105 mm. In various embodiments, the length of the curved region of the microchannel (e.g., the first microchannel and / or the second microchannel) is about 100 mm.

[0077] cell concentration Generally, cell concentration is a tunable parameter that affects the alignment of cell streams. For example, providing too low a cell concentration can result in too low a throughput or too slow an analysis. Conversely, using too high a cell concentration can cause cell aggregation and disrupt the flow, resulting in more than one cell being trapped in a droplet from a single cell stream.

[0078] In various embodiments, the cell concentration may depend on the values ​​of other tunable parameters. For example, the appropriate cell concentration may be determined based on any of 1) cell diameter, 2) cell-to-cell spacing, 3) microchannel width, and / or 4) microchannel height. In certain embodiments, the appropriate cell concentration depends on 1) cell diameter, 2) cell-to-cell spacing, 3) microchannel width, and / or 4) microchannel height.

[0079] In various embodiments, the concentration C1 of cells in the first cell alignment stream is calculated according to formula (1): TIFF2025535563000006.tif11128, where D1 represents the average diameter of cells in the first aligned stream, S1 represents the spacing between a pair of cells in the first aligned stream, W1 represents the width of the first microchannel, and H1 represents the height of the first microchannel. Similarly, the concentration C2 of cells in the second aligned stream is calculated using Equation (2): It can be defined according to TIFF2025535563000007.tif11128, where D2 represents the average diameter of cells in the second aligned stream, S2 represents the spacing between pairs of cells in the second aligned stream, W2 represents the width of the second microchannel, and H2 represents the height of the second microchannel.

[0080] In various embodiments, the above-described formulas (1) and (2) refer to a maximum cell concentration C1 and a maximum cell concentration C2, respectively, within each microchannel. In various embodiments, the above-described formulas (1) and (2) refer to a maximum cell concentration C1 and a maximum cell concentration C2, respectively, added to each well (e.g., aqueous well). Thus, staying below the maximum cell concentration C1 and the maximum cell concentration C2 may ensure alignment of both the first cell stream and the second cell stream.

[0081] In various embodiments, the concentration of cells defined in Equation (1) and Equation (2) may be adjusted according to the desired droplet diameter and volume, for example, the denominator of either Equation (1) or Equation (2) may be equal to half the volume of the desired droplet volume.

[0082] Intercellular spacing Generally, the intercellular spacing of cells in an ordered stream affects the number of cells encapsulated in a single cell. As used herein, intercellular spacing refers to the distance between a pair of consecutive cells in an ordered stream (e.g., when there are no other cells between the pair of cells). In various embodiments, the desired intercellular spacing of cells in an ordered stream depends on the volume of the droplets formed at the junction in the microfluidic center. In various embodiments, the desired intercellular distance between two cells is a distance that, when multiplied by the cross-sectional area of ​​the microchannel, is about ¼ * droplet volume to about 1 * droplet volume. In various embodiments, the desired intercellular distance between two cells is a distance that, when multiplied by the cross-sectional area of ​​the microchannel, is about ½ * droplet volume to about 1 * droplet volume. For square or rectangular microchannels, the cross-sectional area of ​​the microchannel is the product of the height and width of the microchannel. For circular microchannels, the cross-sectional area of ​​the microchannel is the product of π and the square of the radius of the microchannel. By controlling the intercellular spacing in this manner, individual cells within the cell-aligned stream are successfully encapsulated into separate, single droplets. If the intercellular spacing is smaller than desired, more than one cell from the aligned stream will be encapsulated within the droplet. Conversely, if the intercellular spacing is larger than desired, multiple droplets will be formed that do not contain a single cell.

[0083] In various embodiments, the average intercellular spacing between cells of the first aligned stream (e.g., in the first microchannel) and the average intercellular spacing between cells of the second aligned stream (e.g., in the second microchannel) are substantially equal (e.g., within 10% of each other), ensuring that cells from the first aligned stream arrive at the junction at substantially the same time as corresponding cells from the second aligned stream, resulting in co-encapsulation of cells from the first aligned stream and corresponding cells from the second aligned stream.

[0084] In various embodiments, cells within an ordered stream may have a distribution of intercellular spacing. In other words, a first pair of cells within an ordered stream may have an intercellular spacing that is different from the intercellular spacing of a second pair of cells. In various embodiments, the average intercellular spacing of paired cells within an ordered stream is between about 0.5 times the average cell diameter and about 6 times the average cell diameter. In various embodiments, consecutive paired cells within an ordered stream may independently have an intercellular spacing of between about 0.6 times the average cell diameter and about 5.5 times the average cell diameter, between about 0.7 times the average cell diameter and about 5 times the average cell diameter, between about 0.8 times the average cell diameter and about 4.5 times the average cell diameter, between about 1 time the average cell diameter and about 4 times the average cell diameter, between about 1.5 times the average cell diameter and about 3.5 times the average cell diameter, between about 2 times the average cell diameter and about 3 times the average cell diameter, or between about 2.25 times the average cell diameter and about 2.75 times the average cell diameter.

[0085] In various embodiments, the average intercellular distance between paired cells within the ordered stream is between about 5 μm and about 100 μm. In various embodiments, consecutive paired cells within the ordered stream may independently have an intercellular distance of between about 6 μm and about 90 μm, between about 7 μm and about 80 μm, between about 8 μm and about 70 μm, between about 9 μm and about 60 μm, between about 10 μm and about 50 μm, between about 11 μm and about 40 μm, between about 12 μm and about 30 μm, or between about 15 μm and about 20 μm.

[0086] flow rate The flow rates of the aqueous and / or oil phases can be fine-tuned to ensure successful cell stream alignment and co-encapsulation of two or more cells into a single droplet. As used herein, a first aqueous phase refers to an aqueous fluid flowing through a first microchannel carrying cells of a first cell type. A second aqueous phase refers to an aqueous fluid flowing through a second microchannel carrying cells of a second cell type. An oil phase refers to an immiscible fluid flowing through two or more microchannels that merges with the first and second aqueous phases at a junction (e.g., to form a single droplet).

[0087] In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 5 μL / min to 100 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 10 μL / min to 75 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, or about 25 μL / min to 35 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 10 μL / min to 40 μL / min, about 15 μL / min to 35 μL / min, about 20 μL / min to 30 μL / min, or about 22.5 μL / min to 27.5 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 8 μL / min to 80 μL / min, about 10 μL / min to 60 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, about 25 μL / min to 35 μL / min, or about 27.5 μL / min to 32.5 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 60 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 45 μL / min. In various embodiments, the first aqueous phase is flowed through the first microchannel at a rate of about 30 μL / min.

[0088] In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 5 μL / min to 100 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 10 μL / min to 75 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, or about 25 μL / min to 35 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 10 μL / min to 40 μL / min, about 15 μL / min to 35 μL / min, about 20 μL / min to 30 μL / min, or about 22.5 μL / min to 27.5 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 8 μL / min to 80 μL / min, about 10 μL / min to 60 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, about 25 μL / min to 35 μL / min, or about 27.5 μL / min to 32.5 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 60 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 45 μL / min. In various embodiments, the second aqueous phase is flowed through the second microchannel at a rate of about 30 μL / min.

[0089] In various embodiments, the first and second aqueous phases are flowed through their respective microchannels at the same flow rate. In various embodiments, the first and second aqueous phases are flowed through their respective microchannels at different flow rates. For example, the second aqueous phase may be flowed at a second velocity that is faster than the first velocity of the first aqueous phase. By flowing the second aqueous phase faster, more cells of the second cell type carried by the second aqueous phase may be encapsulated in a single droplet. For example, a single droplet may contain only a single cell from the first cell-aligned stream (flowed at the first flow rate) and two or more cells from the second cell-aligned stream (flowed at the second, faster flow rate).

[0090] In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 5 μL / min to 100 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 10 μL / min to 75 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, or about 25 μL / min to 35 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 10 μL / min to 40 μL / min, about 15 μL / min to 35 μL / min, about 20 μL / min to 30 μL / min, or about 22.5 μL / min to 27.5 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 8 μL / min to 80 μL / min, about 10 μL / min to 60 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, about 25 μL / min to 35 μL / min, or about 27.5 μL / min to 32.5 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 60 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 45 μL / min. In various embodiments, the oil phase is flowed through the oil microchannel at a rate of about 30 μL / min.

[0091] Cell alignment using pillars In various embodiments, the microfluidic device includes additional features, such as one or more pillars, that assist in aligning cells into a stream. Generally, one or more pillars of a microfluidic device are useful for manipulating the movement of single cells within the microfluidic device. For example, the pillars may manipulate the movement of single cells to prevent cells from clumping together and clogging the microchannel. As another example, the pillars may manipulate the movement of single cells to help achieve specific intercellular spacing, which may further assist in aligning the cell stream.

[0092] In various embodiments, one or more pillars are located at the inlets of the microchannels of the microfluidic device. For example, referring to FIG. 1A , one or more pillars may be located in aqueous well 105A at the inlet of first microchannel 115A. As another example, one or more pillars may be located in aqueous well 105B at the inlet of second microchannel 115B. In certain embodiments, aqueous well 105A and aqueous well 105B include one or more pillars located at the inlets to first microchannel 115A and second microchannel 115B, respectively. Thus, as cells in aqueous well 105 enter their respective microchannels 115, the one or more pillars disrupt the flow of cells to prevent cells from clumping and forming clumps within the microchannels 115. Specifically, the one or more pillars disrupt the flow of cells to achieve a specific intercellular spacing within the microchannels 115.

[0093] In various embodiments, the one or more pillars at the entrance of the microchannel are arranged in a row. The rows of pillars may be arranged substantially perpendicular or perpendicular to the flow of cells. In various embodiments, the set of pillars comprises at least two rows of pillars. In various embodiments, the set of pillars comprises at least three rows, at least four rows, at least five rows, at least six rows, at least seven rows, at least eight rows, at least nine rows, at least ten rows, at least fifteen rows, at least twenty rows, at least twenty-five rows, at least thirty rows, at least thirty-five rows, at least forty rows, at least forty-five rows, or at least fifty rows of pillars.

[0094] In various embodiments, a set of pillars at the entrance to a microchannel includes a gap between each pair of pillars. For example, pairs of pillars within the same row may have a specific gap. Generally, the gap between pillars within a row is small enough to allow cells to pass through to the center of the gap, and laminar flow causes cells to flow into the center of the gap in the next row of pillars, and so on. In various embodiments, the gaps between pairs of pillars within a row are uniform in distance. In various embodiments, a set of pillars includes a gap between pillars of 2-80 μm, 3-60 μm, 4-50 μm, 5-40 μm, 6-30 μm, or 8-20 μm. In certain embodiments, a set of pillars includes a gap of 5-40 μm. In various embodiments, the gap between pairs of pillars in successive rows becomes successively smaller. For example, in successive rows, the gap between pairs of pillars is about 5%-50% smaller than the gap between pairs of pillars in the immediately preceding row. In some scenarios, in successive rows, the gap between pairs of pillars is about 10% to about 40% smaller, about 15% to about 35% smaller, about 20% to about 30% smaller, or about 22.5% to about 27.5% smaller than the gap between pairs of pillars in the immediately preceding row.

[0095] In various embodiments, the columns of pillars are shifted (e.g., offset) relative to adjacent columns of pillars. Thus, through successive columns of pillars, where one column is shifted relative to the previous column of pillars, cells flowing through the pillars are directed toward a common point in the direction of the shift. In various embodiments, the columns of pillars are shifted by a ratio of the gap distance between a pair of pillars in the same column. For example, assuming the gap distance between a pair of pillars in the same column (e.g., the first column) is X, the second column of pillars adjacent to the first column may be shifted by a value of Z*X, where Z represents a ratio and is less than 1. In various embodiments, Z is 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95.

[0096] Reference is now made to Figure 4, which shows an exemplary flow path using pillars to separate cells into ordered streams, according to one embodiment. Specifically, Figure 4 shows a vertically oriented column of pillars, with columns of pillars shifted relative to the previous column. Here, the cell flow path shows cells flowing by laminar flow through gaps in the columns of pillars to the central axis before entering the microchannel on the right.

[0097] Encapsulation of cells from an ordered stream into a single droplet Disclosed herein is a method for co-encapsulating two or more cells from two or more ordered streams into a single droplet at a junction in a microfluidic device. A first cell-ordered stream flows in an aqueous phase in a first microchannel to the junction, a second cell-ordered stream flows in an aqueous phase in a second microchannel to the junction, and an oil phase flows through a third microchannel to the junction. The joining of the aqueous and oil phases at the junction causes the formation of a single droplet.

[0098] In various embodiments, a method for co-encapsulating two or more cells involves substantially simultaneously converging two or more aqueous and oil phases at a junction (e.g., the various phases meet each other simultaneously, or within 100 milliseconds (ms), 10 ms, or 1 ms). In various embodiments, a method for co-encapsulating two or more cells involves first contacting two or more aqueous phases with each other. For example, the method may involve contacting a flowing first aqueous phase with a second aqueous phase, where the contacting creates a single aqueous phase comprising a first cell-aligned stream from the first aqueous phase and a second cell-aligned stream from the second aqueous phase. In various embodiments, contacting the flowing first and second aqueous phases to create a single aqueous phase occurs at or before the junction. For example, see FIG. 1B, which depicts an exemplary junction 130. Here, first cell alignment stream 150A flows in a first aqueous phase in first microchannel 115A, and second cell alignment stream 150B flows in a second aqueous phase in second microchannel 115B. The aqueous phases of first microchannel 115A and second microchannel 115B join at a location prior to junction 130 to form a single aqueous phase.

[0099] As shown in Figure 1B, the aqueous phases merge before meeting the oil phases flowing in microchannels 125A and 125B. In such embodiments, the method for co-encapsulating two or more cells further comprises contacting the flowing oil phase with a single aqueous phase to form a conical configuration within the junction, where the conical configuration is formed by the interaction of the immiscible oil phase with the aqueous phase. Thus, a single droplet is generated at the tip of the conical configuration.

[0100] In various embodiments, the oil phase is flowed through the junction at a rate of about 5 μL / min to 100 μL / min. In various embodiments, the oil phase is flowed at a rate of about 10 μL / min to 75 μL / min, about 15 μL / min to 50 μL / min, about 20 μL / min to 40 μL / min, or about 25 μL / min to 35 μL / min. In various embodiments, the oil phase is flowed at a rate of about 10 μL / min to 40 μL / min, about 15 μL / min to 35 μL / min, about 20 μL / min to 30 μL / min, or about 22.5 μL / min to 27.5 μL / min.

[0101] In various embodiments, the methods disclosed herein are useful for generating populations of single droplets. In various embodiments, the methods involve generating at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least 1 million, at least 10 million, at least 50 million, or at least 100 million single droplets. In various embodiments, the methods involve generating between 10 and 10,000 single droplets. In various embodiments, the methods involve generating 50-5,000, 100-1,000, 200-900, 300-800, 400-700, or 500-600 single droplets.

[0102] Generally, the population of single droplets is characterized by the proportion of single droplets that contain cells from the first aligned stream (e.g., of a first cell type) and at least one cell from the second aligned stream (e.g., of a second cell type). The proportion of single droplets that contain cells from the first aligned stream and at least one cell from the second aligned stream exceeds the expected proportion of single droplets that contain cells from the first aligned stream and cells from the second aligned stream, as predicted using a Poisson distribution. For example, based on Poisson's law, approximately 10% of single droplets would contain cells from the first aligned stream and at least one cell from the second aligned stream. The Poisson prediction is expressed by the Poisson distribution equation: Calculated using TIFF2025535563000008.tif11128, where k is the number of occurrences (in this case, the number of cells in each droplet) and λ is the expected number of cells in a droplet. The expected number of cells in a droplet is the cell concentration divided by the droplet volume. As an example, considering droplets with a diameter of 60 μm and 4 million T cells / mL, the Poisson equation calculates that approximately 28% of droplets contain one Jurkat cell. For the same droplet diameter and 9 million K562 cells / mL, the Poisson equation calculates that approximately 36% of droplets contain one K562 cell. Because these two events are independent, the number of droplets containing Jurkat cells and K562 cells is 28% * 36% = approximately 10%.

[0103] In various embodiments, the methods and apparatus disclosed herein generate populations of single droplets characterized by a proportion of single droplets that contain cells from the first aligned stream and at least one cell from the second aligned stream, the proportion exceeding the expected proportion predicted using a Poisson distribution by at least 1.5 times. In various embodiments, the rate exceeds the expected rate predicted using a Poisson distribution by at least 1.6x, at least 1.7x, at least 1.8x, at least 1.9x, at least 2.0x, at least 2.1x, at least 2.2x, at least 2.3x, at least 2.4x, at least 2.5x, at least 2.6x, at least 2.7x, at least 2.8x, at least 2.9x, at least 3.0x, at least 3.1x, at least 3.2x, at least 3.3x, at least 3.4x, at least 3.5x, at least 3.6x, at least 3.7x, at least 3.8x, at least 3.9x, at least 4.0x, at least 4.1x, at least 4.2x, at least 4.3x, at least 4.4x, at least 4.5x, at least 4.6x, at least 4.7x, at least 4.8x, at least 4.9x, or at least 5.0x.

[0104] In various embodiments, the rate exceeds the expected rate predicted using a Poisson distribution by a factor ranging from 1.3 to 10.0. In various embodiments, the rate exceeds the expected rate predicted using a Poisson distribution by a factor ranging from 1.4 to 9.0, by a factor ranging from 1.5 to 8.0, by a factor ranging from 1.6 to 7.0, by a factor ranging from 1.7 to 6.0, by a factor ranging from 1.8 to 5.0, by a factor ranging from 1.9 to 4.0, or by a factor ranging from 2.0 to 3.0.

[0105] Exemplary Microfluidic Devices Microfluidic devices, such as those disclosed herein, are designed and used according to a range of parameters (e.g., concentration of cells in the aqueous fluid, flow rate of the cell-containing aqueous phase, flow rate of the oil phase, microchannel width, and cell-to-cell spacing within the cell-aligned stream) to successfully achieve co-encapsulation of two or more cells into a single droplet. Specifically, the microfluidic device is used to find a range of flow rates that are fast enough for inertial focusing (e.g., to achieve a cell-aligned stream) but slow enough to achieve droplet generation. Furthermore, the microfluidic device is also designed to process cells in a high-throughput manner, thereby ensuring cell viability during processing.

[0106] A tunable parameter of a microfluidic device includes the width of the microfluidic channel. Specifically, reducing the width of the microchannel increases the rate of inertial focusing by reducing the distance cells must travel to reach an equilibrium position within the microchannel. However, reducing the microchannel width comes with a trade-off: increasing flow resistance reduces the flow rate, thereby reducing cell processing throughput. Another tunable parameter includes the length of the microchannel. Specifically, the channel length may be adjusted to allow more time for Dean forces to inertially focus cells. However, increasing the length of the microchannel also increases the flow resistance, thus reducing the flow rate at a given operating pressure.

[0107] Disclosed herein is a microfluidic device for encapsulating pairs of cells in droplets, the microfluidic device including a first microchannel, a second microchannel, and a third microchannel fluidly connected to one another through a junction.

[0108] Exemplary junctions of microfluidic devices with serpentine or spiral microchannels Reference is made to FIG. 5, which depicts an exemplary junction of a microfluidic device, such as an exemplary microfluidic device including a serpentine channel (as described below with respect to FIGS. 6A and 6B) or an exemplary microfluidic device including a spiral channel (as described below with respect to FIGS. 7A and 7B). Here, FIG. 5 depicts a first microchannel 580A (e.g., for carrying a first cell alignment stream), a second microchannel 580B (e.g., for carrying a second cell alignment stream), an oil channel 590 (for carrying an immiscible phase), a junction 585, a nozzle region 588 after junction 585, and a post-nozzle region 592 for collecting droplets. Generally, operation of the microfluidic device shown in FIG. 5 involves flowing a solution from the right (e.g., through first microchannel 580A and second microchannel 580B) to the left (e.g., to post-nozzle region 592). Additionally, FIG. 5 includes dimensional designations. For example, "AA" refers to the width of oil channel 590. "BB" refers to the width of junction 585. "CC" refers to the width of the nozzle region 588. "DD" refers to the length of the nozzle region 588. "EE" refers to the width of the post-nozzle region 592.

[0109] 5, first microchannel 580A and second microchannel 580B may meet at junction 585 or in a region immediately prior to junction 585. This region then connects to junction 585 to form droplets. A nozzle region 588 leads away from junction 585 to a post-nozzle region 592 that collects the formed droplets.

[0110] Referring first to first microchannel 580A, in various embodiments, the width of first microchannel 580A is about 5 μm to about 200 μm. In various embodiments, the width of first microchannel 580A is about 10 μm to about 100 μm, about 15 μm to about 95 μm, about 20 μm to about 90 μm, about 25 μm to about 85 μm, about 30 μm to about 80 μm, about 35 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of first microchannel 580A is about 50 μm to about 100 μm (e.g., about 55 μm to about 95 μm, about 60 μm to about 90 μm, about 65 μm to about 85 μm, or about 70 μm to about 80 μm). In various embodiments, the width of first microchannel 580A is about 50 μm to about 75 μm. In certain embodiments, the width of first microchannel 580A is about 50 μm. In certain embodiments, the width of first microchannel 580A is about 75 μm.

[0111] In various embodiments, the width of first microchannel 580A tapers toward junction 585. For example, first microchannel 580A may have a first width distal to junction 585 relative to a second width of first microchannel 580A closer to junction 585. The first width of first microchannel 580A may be greater than the second width of first microchannel 580A. In various embodiments, the first width of first microchannel 580A (e.g., distal to junction 585) is between about 40 μm and about 100 μm, between about 45 μm and about 80 μm, or between about 50 μm and about 60 μm. In certain embodiments, the first width of first microchannel 580A (e.g., distal to junction 585) is about 50 μm. In various embodiments, the second width of first microchannel 580A (e.g., near junction 585) is about 10 μm to about 40 μm, about 20 μm to about 35 μm, or about 25 μm to about 32 μm. In certain embodiments, the second width of first microchannel 580A (e.g., near junction 585) is about 30 μm. In certain embodiments, the second width of first microchannel 580A (e.g., near junction 585) is about 25 μm. In certain embodiments, the width of first microchannel 580A tapers from a first width of 50 μm to a second width of 30 μm at junction 585.

[0112] In various embodiments, the width of second microchannel 580B is about 5 μm to about 200 μm. In various embodiments, the width of second microchannel 580B is about 10 μm to about 100 μm, about 15 μm to about 95 μm, about 20 μm to about 90 μm, about 25 μm to about 85 μm, about 30 μm to about 80 μm, about 35 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of second microchannel 580B is about 50 μm to about 100 μm (e.g., about 55 μm to about 95 μm, about 60 μm to about 90 μm, about 65 μm to about 85 μm, or about 70 μm to about 80 μm). In various embodiments, the width of second microchannel 580B is about 50 μm to about 75 μm. In certain embodiments, the width of second microchannel 580B is about 50 μm. In certain embodiments, the width of second microchannel 580B is about 75 μm.

[0113] In various embodiments, the width of second microchannel 580B tapers toward junction 585. For example, second microchannel 580B may have a first width distal to junction 585 relative to a second width of second microchannel 580B closer to junction 585. The first width of second microchannel 580B may be greater than the second width of second microchannel 580B. In various embodiments, the first width of second microchannel 580B (e.g., distal to junction 585) is between about 40 μm and about 100 μm, between about 45 μm and about 80 μm, or between about 50 μm and about 60 μm. In certain embodiments, the first width of second microchannel 580B (e.g., distal to junction 585) is about 50 μm. In various embodiments, the second width of second microchannel 580B (e.g., located near junction 585) is about 10 μm to about 40 μm, about 20 μm to about 35 μm, or about 25 μm to about 32 μm. In certain embodiments, the second width of second microchannel 580B (e.g., located near junction 585) is about 30 μm. In certain embodiments, the second width of second microchannel 580B (e.g., located near junction 585) is about 25 μm. In certain embodiments, the width of second microchannel 580B tapers from a first width of 50 μm to a second width of 30 μm at junction 585.

[0114] In various embodiments, the width of the oil microchannel 590 (denoted as "AA" in FIG. 5) is about 5 μm to about 200 μm. In various embodiments, the width of the oil microchannel 590 is about 10 μm to about 150 μm, about 20 μm to about 140 μm, about 30 μm to about 130 μm, about 40 μm to about 125 μm, about 50 μm to about 120 μm, about 60 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of the oil microchannel 590 is about 50 μm to about 100 μm (e.g., about 55 μm to about 95 μm, about 60 μm to about 90 μm, about 65 μm to about 85 μm, or about 70 μm to about 80 μm). In various embodiments, the width of the oil microchannel 590 is about 50 μm to about 75 μm. In certain embodiments, the width of the oil microchannel 590 is about 50 μm. In certain embodiments, the width of the oil microchannel 590 is about 60 μm. In certain embodiments, the width of the oil microchannel 590 is about 75 μm.

[0115] Turning now to junction 585, in various embodiments, the width of junction 585 (denoted as "BB" in FIG. 5) is about 5 μm to about 200 μm. In various embodiments, the width of junction 585 is about 10 μm to about 150 μm, about 20 μm to about 140 μm, about 30 μm to about 130 μm, about 40 μm to about 125 μm, about 50 μm to about 120 μm, about 60 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of junction 585 is about 50 μm to about 100 μm (e.g., about 55 μm to about 95 μm, about 60 μm to about 90 μm, about 65 μm to about 85 μm, or about 70 μm to about 80 μm). In various embodiments, the width of junction 585 is about 50 μm to about 75 μm. In certain embodiments, the width of junction 585 is about 50 μm. In certain embodiments, the width of junction 585 is about 60 μm. In certain embodiments, the width of junction 585 is about 75 μm.

[0116] In various embodiments, the ratio between the width of oil channel 590 (represented in FIG. 5 as "AA") and the width of junction 585 (represented in FIG. 5 as "BB") is about 1 to about 5. In various embodiments, the ratio between the width of oil channel 590 (represented in FIG. 5 as "AA") and the width of junction 585 (represented in FIG. 5 as "BB") is about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 4, or about 2 to about 3. In certain embodiments, the ratio between the width of oil channel 590 (represented in FIG. 5 as "AA") and the width of junction 585 (represented in FIG. 5 as "BB") is about 100 μm / 60 μm (e.g., about 1.6).

[0117] Referring next to the width of the nozzle 588, the width (denoted as "CC" in FIG. 5) can be designed to control the size of the resulting droplets. In various embodiments, the width of the nozzle 588 controls the size of the resulting droplets by about ±20 μm centered around the nozzle width. For example, given a nozzle width of 50 μm, the resulting droplets will be 30-70 μm in size (e.g., depending on the pressure applied to the aqueous channels and their corresponding flow rates). In various embodiments, the width of the nozzle 588 is from about 5 μm to about 200 μm. In various embodiments, the width of nozzle 588 is about 10 μm to about 150 μm, about 20 μm to about 140 μm, about 30 μm to about 130 μm, about 40 μm to about 125 μm, about 50 μm to about 120 μm, about 60 μm to about 75 μm, about 40 μm to about 70 μm, about 45 μm to about 65 μm, or about 50 μm to about 60 μm. In various embodiments, the width of nozzle 588 is about 50 μm to about 75 μm. In certain embodiments, the width of nozzle 588 is about 50 μm. In certain embodiments, the width of nozzle 588 is about 75 μm. In certain embodiments, the width of nozzle 588 is about 100 μm.

[0118] In certain embodiments, the width of nozzle 588 (denoted as "CC" in FIG. 5) is smaller than the width of junction 585 (denoted as "BB" in FIG. 5). In various embodiments, the width of nozzle 588 is at least 1% smaller than the width of junction 585. In various embodiments, the width of nozzle 588 is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20% smaller than the width of junction 585. In some embodiments, the width of nozzle 588 is between about 40 μm and about 55 μm, and the width of junction 585 is between about 55 μm and about 75 μm. In some embodiments, the width of nozzle 558 is about 45 μm to about 52 μm, and the width of junction 585 is about 58 μm to about 65 μm. In some embodiments, the width of nozzle 558 is about 50 μm, and the width of junction 585 is about 60 μm.

[0119] Referring next to the length of nozzle 588, the length of nozzle 588 (denoted as "DD" in FIG. 5) can be designed to ensure droplet formation at higher flow rates of the aqueous and / or oil phases. For example, higher flow rates or higher flow rates can result in an aqueous / oil phase interface in the co-flow or jetting regime, which prevents the formation of single droplets. Therefore, increasing the length of nozzle 588 can increase the flow rate to higher values ​​while still allowing droplet formation to occur. In various embodiments, the length of nozzle 588 is from about 20 μm to about 500 μm. In various embodiments, the length of nozzle 588 is from about 30 μm to about 450 μm, from about 40 μm to about 400 μm, from about 50 μm to about 350 μm, from about 75 μm to about 300 μm, from about 100 μm to about 250 μm, or from about 150 μm to about 225 μm. In certain embodiments, the length of nozzle 588 is about 50 μm. In certain embodiments, the length of the nozzle 588 is about 200 μm.

[0120] Turning now to the post-nozzle region 592, the width of the post-nozzle region 592 (denoted as "EE" in FIG. 5) can be designed to ensure droplets are spaced far enough apart to form without merging with one another. In various embodiments, the width of the post-nozzle region 592 is between about 50 μm and about 1000 μm. In various embodiments, the width of the post-nozzle region 592 is between about 100 μm and about 900 μm, between about 150 μm and about 850 μm, between about 200 μm and about 800 μm, between about 250 μm and about 750 μm, between about 300 μm and about 700 μm, between about 350 μm and about 650 μm, between about 400 μm and about 600 μm, between about 450 μm and about 550 μm, or between about 475 μm and about 525 μm. In certain embodiments, the width of the post-nozzle region 592 is about 500 μm.

[0121] In various embodiments, the width of junction 585 (denoted as "BB" in FIG. 5) is greater than the width of nozzle 588 (denoted as "CC" in FIG. 5). In various embodiments, the ratio between the width of junction 585 (denoted as "BB" in FIG. 5) and the width of nozzle 588 (denoted as "CC" in FIG. 5) is about 1 to about 5. In various embodiments, the ratio between the width of junction 585 and the width of nozzle 588 is about 1 to about 4, about 1 to about 3, about 1 to about 2, about 2 to about 4, or about 2 to about 3. In certain embodiments, the ratio between the width of junction 585 and the width of nozzle 588 is about 60 μm / 50 μm (e.g., about 1.2).

[0122] In various embodiments, the width of the post-nozzle region 592 (designated as "EE" in FIG. 5 ) is greater than the width of the nozzle 588 (designated as "CC" in FIG. 5 ). In various embodiments, the ratio of the width of the post-nozzle region 592 (designated as "EE" in FIG. 5 ) to the width of the nozzle 588 (designated as "CC" in FIG. 5 ) is from about 1 to about 15. In various embodiments, the ratio of the width of the post-nozzle region 592 (designated as "EE" in FIG. 5 ) to the width of the nozzle 588 (designated as "CC" in FIG. 5 ) is from about 2 to about 14.5, from about 3 to about 14, from about 4 to about 13.5, from about 5 to about 13, from about 6 to about 12.5, from about 7 to about 12, from about 8 to about 11.5, or from about 9 to about 11. In certain embodiments, the ratio of the width of the post-nozzle region 592 to the width of the nozzle 588 is about 10.

[0123] Serpentine microchannel microfluidic device 6A depicts an exemplary microfluidic device with a serpentine microchannel. For purposes of introduction, the microfluidic device includes two aqueous wells 610A and 610B, where cells of a first cell type may be introduced into aqueous well 610A in an aqueous fluid, and cells of a second cell type may be introduced into aqueous well 610B in an aqueous fluid.

[0124] As shown in FIG. 6A , aqueous well 610A is fluidly connected to curved region 615A of the first serpentine microchannel. Similarly, aqueous well 610B is fluidly connected to curved region 615B of the second serpentine microchannel. Thus, cells provided through aqueous well 610A flow back and forth through curved region 615A of the serpentine microchannel to junction 640. Curved region 615A provides an inertial focusing force to align the cell stream before the cells reach junction 640, as shown in FIG. 1B . Similarly, cells provided through aqueous well 610B flow back and forth through curved region 615B of the serpentine microchannel to junction 640. Curved region 615B provides an inertial focusing force to align the cell stream before the cells reach junction 640, as shown in FIG. 1B . Reference is now made to FIG. 6B , which depicts, at a higher magnification, an exemplary curved region of the serpentine microchannel of the exemplary microfluidic device shown in FIG. 6A . Specifically, Figure 6B shows a higher magnification of inset 650 shown in Figure 6A. Here, inset 650 illustrates the directional flow of cells along a serpentine microchannel with an asymmetric curve, beginning at a cell inlet. Inset 650 shows cells entering through the cell inlet and flowing through the asymmetric curve. Specifically, the curved region includes multiple undulations that exert an inertial focusing force on the cells. In this particular example, the undulations may have a channel width of 0.075 mm followed by a narrowing to a channel width of 0.050 mm. Here, the radius of curvature of the channel at the 0.075 mm channel width is greater than the radius of curvature of the channel at the 0.05 mm channel width. Cells continue to flow through additional undulations to the "cell outlet," as indicated by the "cell flow" directional arrows. Generally, the directional flow of cells through the upper undulations is from left to right, and the directional flow of cells through the lower undulations is from right to left. Thus, the continuous flow of cells back and forth through the undulating portion results in the alignment of the cell stream.6A, there may be additional undulations through which cells further flow, e.g., from left to right and / or right to left, such that the inertial focusing forces imparted on the cells through these additional undulations further align the cells within the microchannel. In the particular exemplary microfluidic device shown in FIG. 6A, cells within aqueous well 610A flow from right to left through a first set of undulations, then from left to right through a second set of undulations, then from right to left through a third set of undulations, then from left to right through a fourth set of undulations, and then from right to left through a fifth (and final) set of undulations to junction 640. Similarly, cells in aqueous well 610B flow from right to left through the first set of undulating portions, then from left to right through the second set of undulating portions, then from right to left through the third set of undulating portions, then from left to right through the fourth set of undulating portions, and then from right to left through the fifth (and final) set of undulating portions to junction 640.

[0125] Further shown in Figure 6A is oil well 625, into which the oil phase is introduced into the microfluidic device. The oil phase flows from oil well 625 through microchannel 630 to junction 640, where the first cell-aligned stream from well 610A, the second cell stream from well 610B, and the oil phase from oil well 625 combine to form a single droplet that encapsulates two or more cells. An exemplary image of junction 640 during co-encapsulation is shown in Figure 1B.

[0126] Spiral microchannel microfluidic device Figure 7A depicts an exemplary microfluidic device with a spiral microchannel. For purposes of introduction, the microfluidic device includes two aqueous wells 710A and 710B, where cells of a first cell type may be introduced into aqueous well 710A in an aqueous fluid, and cells of a second cell type may be introduced into aqueous well 710B in an aqueous fluid. Generally, operation of the microfluidic device shown in Figure 7A involves flowing solutions from the left (e.g., from wells 710A, 710B, and 725) to the right (e.g., to junction 740 and toward collection well 745).

[0127] As shown in Figure 7A, aqueous well 710A is fluidly connected to curved region 715A of a first spiral microchannel. Similarly, aqueous well 710B is fluidly connected to curved region 715B of a second spiral microchannel. Thus, cells provided through aqueous well 710A flow through curved region 715A of the spiral microchannel to junction 740. Curved region 715A provides an inertial focusing force to align the cell stream before the cells reach junction 740. Similarly, cells provided through aqueous well 710B flow through curved region 715B of the spiral microchannel to junction 740. Curved region 715B provides an inertial focusing force to align the cell stream before the cells reach junction 740.

[0128] Reference is now made to FIG. 7B, which depicts an exemplary curved region of the spiral microchannel of the exemplary microfluidic device shown in FIG. 7A at a higher magnification. Specifically, FIG. 7B shows inset 750 shown in FIG. 7A at a higher magnification. Here, inset 750 illustrates a spiral microchannel that loops with a continuously increasing radius of curvature. In this particular example, the radius of curvature of the microchannel closer to aqueous well 710A is smaller than the radius of curvature of the microchannel closer to the cell outlet shown in FIG. 7B. Thus, as cells flow through the spiral microchannel, inertial focusing forces exerted on the cells organize the cells into a cell-aligned stream.

[0129] Further shown in Figure 7A is oil well 725, into which the oil phase is introduced into the microfluidic device. The oil phase flows from oil well 725 through microchannel 730 to junction 740. At junction 740, the first cell-aligned stream from well 710A, the second cell stream from well 710B, and the oil phase from oil well 725 combined to form a single droplet encapsulating two or more cells.

[0130] Additional Features of Microfluidic Devices In various embodiments, the microfluidic devices disclosed herein, such as those shown in Figures 6A and / or 7A, may include additional features to improve alignment of cell streams and / or co-encapsulation of two or more cells in a single droplet.

[0131] In various embodiments, additional features include one or more pillars. The pillars can be useful for directing the flow of cells from aqueous well 115A and / or aqueous well 115B into their respective microchannels. For example, a set of pillars can help establish intercellular distances in the two cell-aligned streams. Thus, given the appropriate intercellular distances in the two cell-aligned streams, two or more cells can be successfully co-encapsulated into a single droplet at the junction.

[0132] In various embodiments, the set of pillars is located at the entrance to the microchannel. For example, referring again to FIG. 1A, the set of pillars may be located at the entrance to microchannel 115A or microchannel 115B. In certain embodiments, the set of pillars may be located at the end of aqueous channel 105A or 105B, just before the entrance to microchannel 115A and microchannel 115B. In various embodiments, the set of pillars includes a 5-40 μm gap between pillars. An exemplary flow of cells around a set of pillars is shown in FIG. 4.

[0133] High-throughput cell-cell interaction assays As described herein, the method involves co-encapsulating two or more cells in a single droplet to perform high-throughput cell-cell interaction assays. Specifically, a method for performing an assay in a single droplet containing two cells can include: flowing a first aqueous phase containing a first cell-aligned stream in a first microchannel toward a junction, flowing a second aqueous phase containing a second cell-aligned stream in a second microchannel toward the junction, and flowing an oil phase in a third microchannel toward the junction; and generating a single droplet at the junction formed from the first aqueous phase, the second aqueous phase, and the oil phase, the single droplet containing cells from the first cell-aligned stream and cells from the second cell-aligned stream.

[0134] In various embodiments, one or more reagents for conducting a cell-cell interaction assay may be introduced into a single droplet to conduct a cell-cell interaction assay. For example, the reagent may be introduced via the first aqueous phase and / or the second aqueous phase. Thus, by co-encapsulating two or more cells in a single droplet, the reagent is also co-encapsulated in the single droplet. Generally, the reagent is useful for indicating the presence of an interaction between two or more cells. Thus, the method involves detecting an interaction between two or more cells in a single droplet using the reagent. For example, the reagent may be used to detect a biomarker analyte indicative of the interaction. Such a biomarker analyte may be an antibody, a cytokine, a cytolytic protein, a hormone, and / or a small molecule (e.g., less than 2 kDa or 1 kDa).

[0135] In various embodiments, the reagent indicating an interaction between two or more cells may be a fluorescent marker, a bead, a nucleic acid barcode, or a combination thereof. In various embodiments, the bead (or particle) may comprise, for example, polystyrene, polyethylene, or any other suitable polymer. In such cases, the particle may be a polymer-coated particle, such as a polystyrene-coated gold particle or a polyethylene-coated silica particle. In some embodiments, the particle may be a gel particle or a hydrogel particle. The particle may be spherical or non-spherical and may be any suitable size, such as, for example, less than about 10 micrometers, less than about 3 micrometers, less than about 1 micrometer, less than about 300 nm, or less than about 100 nm. In some cases, the particle may be modified to facilitate attachment of an antibody or other agent to the surface of the particle. For example, in one set of embodiments, the particle may be coated with streptavidin, and the antibody may be modified with a biotin-labeled moiety capable of binding to streptavidin, thereby immobilizing the antibody to the surface of the particle.

[0136] In some embodiments, particles capture and detect specific biomarker analytes. For example, particles may be coated with antibodies that exhibit affinity for target biomarker analytes. In various embodiments, such particles may be streptavidin-coated polystyrene beads coated with biotin-labeled capture antibodies. In addition, the detection antibodies are tagged with a signaling entity (e.g., a fluorescent label). If the biomarker analyte is present in a single droplet due to an interaction between two or more cells, it attaches to the capture beads and detection antibodies upon contact. Thus, the biomarker analyte is captured on the beads, and the labeled detection antibodies are further concentrated around the beads. This provides a sufficient signal (e.g., with a sufficient signal-to-noise ratio) to allow detection of the signaling entity, thereby indicating that a cell-cell interaction has occurred.

[0137] In some cases, the particle may be capable of simultaneously capturing and detecting several different biomarker analytes (e.g., molecules secreted or otherwise released by one or more of the co-encapsulated cells). For example, a capture particle may be coated with a first antibody specific for a first antigen and a second antibody specific for a second antigen. As a specific, non-limiting example, a droplet may contain a capture particle with both the first and second antibodies, two or more cells, and a labeled antibody specific for the first antigen labeled with a first signaling entity (e.g., a red fluorescent dye) and a labeled antibody specific for the second antigen labeled with a second signaling entity (e.g., a green fluorescent dye). Thus, if an interaction between two or more cells occurs, resulting in the release of the first and second antigens, the particle can capture both the first and second antigens. The labeled antibody labeled with the first signaling entity and the labeled antibody labeled with the second signaling entity then concentrate around the capture particle (due to the bound first and second antigens), thereby enabling detection of signals originating from the first and second signaling entities. Further details and methods for performing cell-cell interaction assays are described in WO2017165791, which is incorporated by reference in its entirety.

[0138] In various embodiments, bead-based in-droplet barcoding can be performed by including barcoding polystyrene beads or spherical hydrogels during droplet formation. Further details of barcoded beads are described in US20150298091, which is incorporated by reference in its entirety. This allows for next-generation sequencing of many cells while maintaining the relationship between cells injected into the same droplet.

[0139] Co-encapsulated cells The embodiments described herein involve performing an assay in a single droplet containing two or more cells. In various embodiments, the two or more cells may interact with one another, resulting in a change in one or more markers, such as a marker expressed by one of the two or more cells and / or a marker secreted into the environment surrounding the two or more cells.

[0140] As described herein, the methods involve co-encapsulating two or more cells into a single droplet by providing a first cell-aligned stream of a first cell type and a second cell-aligned stream of a second cell type. By fine-tuning various parameters, a single cell of a first cell type and one or more cells of a second cell type can be successfully encapsulated into a single droplet. In various embodiments, the cells from the first cell-aligned stream and the one or more cells from the second cell-aligned stream are cells of the same cell type. In various embodiments, the cells from the first cell-aligned stream and the one or more cells from the second cell-aligned stream are different cells (e.g., cells of different cell types).

[0141] In various embodiments, cells of a first cell type from the first cell-sorted stream are effector cells. An example of an effector cell is an immune cell, such as a B cell, a T cell, a natural killer cell, a lymphocyte, a macrophage, a neutrophil, a basophil, or a mast cell. In certain embodiments, cells of the first cell type are B cells. In certain embodiments, cells of the first cell type are T cells. For example, the T cells may be cytotoxic CD8+ T cells. As another example, the T cells may be CD4+ T cells. In various embodiments, effector cells may be genetically modified or engineered. For example, effector cells may be T cells with genetically modified T cell receptors. For example, in some cases, T cells may be collected from a subject (e.g., healthy or diseased) and modified to contain a chimeric antigen receptor. To determine the target cells to which the modified T cells respond, the T cells may be expanded and then assayed as discussed herein.

[0142] In various embodiments, cells of the second cell type from the second cell-ordered stream are cells that may interact with cells of the first cell type (e.g., effector cells). In certain embodiments, cells of the second cell type are antigen-presenting cells (APCs). Exemplary APCs include macrophages, microglia, dendritic cells, B cells (e.g., memory B cells), tumor cells, or any native or modified cell. Thus, by co-encapsulating effector cells (e.g., T cells) with APCs in a single droplet, high-throughput screening can be performed to identify interactions between specific T cells and specific APCs.

[0143] In various embodiments, a first type of cell (e.g., an effector cell) may interact with a second type of cell by producing or secreting an antibody, cytokine, or cytolytic protein, or other compound (e.g., perforin, granzyme, etc.). Other compounds that may be secreted include other proteins encoded by genes, or other non-protein compounds, such as penicillin, hormones, small molecules (e.g., less than 2 kDa or 1 kDa). In various embodiments, the interaction between a first cell type (e.g., an effector cell) and a second cell type (e.g., an APC) may be direct (e.g., the effector cell directly binds to the APC) and / or indirect. As a non-limiting example, T cells and APCs from a cancer patient may be examined to determine receptors on T cells that can recognize tumor antigens presented by the APC. Further details of exemplary cells (e.g., effector cells) for performing cell-cell interaction assays are described in WO2017165791, the entire contents of which are incorporated by reference.

[0144] Exemplary System Aspects Systems and devices for performing assays in droplets containing two cells are also described herein. Exemplary systems include microfluidic devices, such as the exemplary microfluidic devices described herein. In various embodiments, the system may also include one or more of the following: (a) one or more pumps or control modules for controlling the flow of aqueous fluid and / or oil phases in the microfluidic device; (b) a temperature control module for controlling the temperature of one or more portions of the subject device and / or droplets therein, and operatively connected to the microfluidic device; and / or (c) a detection module, i.e., a detector, such as, for example, an optical or fluorescent imager, operatively connected to the microfluidic device. The one or more detection modules, i.e., a detector, such as, for example, an optical or fluorescent imager, are configured to detect the presence of a signal in one or more droplets (e.g., a signal from a reagent indicative of a cell-cell interaction). In some embodiments, the detector module is configured to recognize one or more components of one or more droplets in one or more flow channels. [Example]

[0145] Example 1: Serpentine channels in microfluidic devices successfully align and encapsulate paired cells in droplets To align and encapsulate paired cells in a single droplet, a microfluidic device with a serpentine microchannel was developed and fabricated. Specifically, a microfluidic device based on FIG. 6A was fabricated. Generally, operation of the microfluidic device shown in FIG. 6A involves flowing solutions from the right (e.g., from wells 610A, 610B, and 625) to the left (e.g., to junction 640 and toward collection well 645).

[0146] The channel width of the serpentine microchannel for aligning the cell streams was 50 μm to 75 μm. Specifically, as shown in Figure 6B, the channel width of the smaller wavy portion was 50 μm. As shown in Figure 6B, the channel width of the larger wavy portion was 75 μm at the apex and then narrowed to 50 μm, where it met the smaller wavy portion.

[0147] Cells of a first cell type (e.g., T cells) were loaded into aqueous well 610A at a concentration of 4 million cells / milliliter. The average cell diameter of the cells of the first cell type was 8 μm. Cells of a second cell type (e.g., antigen-presenting cells, specifically K562 cells engineered to be antigen-presenting cells) were loaded into aqueous well 610B at a concentration of 9 million cells / milliliter. The average cell diameter of the cells of the second cell type was 15 μm.

[0148] The water pressure applied to aqueous wells 610A and 610B was 1450 mbar. T cells provided through aqueous well 610A flowed back and forth through curved region 615A of the serpentine microchannel to junction 640. Curved region 615A provided an inertial focusing force to align the T cell stream before the T cells reached junction 640. Similarly, K-562 cells engineered to be APCs, provided through aqueous well 610B, flowed back and forth through curved region 615B of the serpentine microchannel to junction 640. Curved region 615B provided an inertial focusing force to align the K-562 cell stream before the K-562 cells reached junction 640. The pressure applied to the oil well was 1200 mbar. T cells and K-562 cells underwent co-encapsulation at junction 640.

[0149] Figure 8 shows the successful loading of two different cells (e.g., T cells and K-562 cells engineered to be antigen-presenting cells) in a single droplet. A single droplet could successfully encapsulate T cells and K-562 cells at either a 1:1 ratio or a 1:2 ratio (as labeled in Figure 8). Generally, in the microfluidic device shown in Figure 6A, approximately 26% of all droplets have a 1:1 ratio of T cells and K-562 cells.

[0150] At these cell concentrations, Poisson's law predicts that only 10% of the droplets will contain cells at a 1:1 ratio. Thus, the efficiency results shown in Figure 8 represent at least a 2.5-fold improvement over Poisson's law.

[0151] Example 2: Characterization of cell alignment streams Analyses were performed to determine the characteristics of cell-aligned streams using the microfluidic device described in Example 1. Specifically, analyses were performed to determine the intercellular distances that lead to successful co-encapsulation of cells from two separate aligned streams (e.g., the distance between consecutive cells within an aligned stream).

[0152] Cell-aligned streams were generated by flowing cells through the curved region of a serpentine channel. Bright-field images of the cell-aligned streams were captured before they entered the junction, and the intercellular distances of consecutive cells within the aligned streams were measured. Figure 9A shows an exemplary bright-field image captured of two cell-aligned streams. Exemplary intercellular distances are labeled.

[0153] Figure 9B shows an exemplary distribution of intercellular distances relative to cell diameter for cells in the alignment stream (e.g., average cell diameter 8 μm). Specifically, most cells exhibited intercellular distances of 1-4 times the cell diameter, with some intercellular distances ranging up to approximately 5.5 times the average cell diameter. Table 1 below further shows the standard deviation of intercellular spacing for successfully paired cells. Generally, the standard deviation of intercellular spacing for cells is less than 10 μm.

[0154] Table 1. Calculated intercellular spacing across various numbers of cells. TIFF2025535563000009.tif105160

[0155] Example 3: Exemplary cell-cell assay in a single droplet An experiment was performed to co-encapsulate single Jurkat cells (T cells) with single K562 cells engineered to act as APCs. Jurkat cells were first stained with a bispecific antibody that binds to the cell surface of Jurkat cells and also binds to IL-2 cytokine. IL-2 represents an activation cytokine released by activated Jurkat cells. Excess bispecific antibody was washed away, and then a second fluorescent detection antibody (e.g., red fluorescent) was added to the cell mixture. This fluorescently labeled detection antibody binds only to IL-2 cytokine.

[0156] Using the microfluidic device described in Example 1, a mixture of fluorescent detection antibody and Jurkat cells was provided to a first aqueous well and injected through a first microchannel of the microfluidic device. K562 cells engineered to be APCs were provided to a second aqueous well and injected through a second microchannel of the microfluidic device. Individual Jurkat cells and K562 cells were co-encapsulated in a single droplet. The fluorescent detection antibody was also encapsulated in a single droplet with the cells.

[0157] Figure 10 shows Jurkat T cells and K562 cells co-encapsulated in a single droplet to perform a cell-cell interaction assay. As shown in Figure 10, a single Jurkat cell is fluorescently labeled as blue, and a single K562 cell is fluorescently labeled as green. Activated Jurkat T cells secreted IL-2, which attached to the bispecific antibody on the surface of the Jurkat cells. A fluorescent detection antibody further attached to the opposite side of the IL-2, thus carrying a red fluorophore to the surface of the cells. As shown in Figure 10, fluorescence indicative of cell-cell interaction was detectable (in this case, red fluorescence). Therefore, activated Jurkat T cells were detected via the presence of this fluorescence indicative of cell-cell interaction.

[0158] Example 4: Further exemplary microfluidic devices successfully align and encapsulate paired cells in droplets To align and encapsulate paired cells in a single droplet, additional microfluidic devices with serpentine microchannels were developed and fabricated. Specifically, a microfluidic device based on the serpentine channel shown in Figure 6A was fabricated. Figure 11 further depicts the dimensions at the junction of the exemplary microfluidic device.

[0159] Specifically, the dimensions at the junctions of the exemplary microfluidic device are as follows: The width of the first microchannel 1180A tapers from 50 μm to 30 μm at the junction 1185. The width of the second microchannel 1180B tapers from 50 μm to 30 μm at the junction 1185 The width of the junction 1185 is 60 μm. The width of the oil microchannel 1190 is 100 μm The length of the nozzle 1188 is 200 μm The width of the nozzle 1188 is 50 μm The width of the post-nozzle region 1192 is 500 μm The ratio of the width of the nozzle 1188 to the width of the post-nozzle region 1192 is 50 μm to 500 μm (e.g., 1 to 10) The ratio of the width of the oil channel 1190 to the width of the junction 1185 is 100 μm to 60 μm (e.g., a ratio of 5 to 3). Includes.

[0160] The microfluidic device was tested for ultra-high-throughput generation of paired cell droplets. Specifically, the microfluidic device achieved paired cell droplet generation at a rate of at least 8,000 droplets per second, reaching 10,000 droplets per second depending on droplet size. For example, for droplets of approximately 60 μm, the device achieved droplet generation at 6,600 droplets per second. For droplets of approximately 55 μm, the device achieved droplet generation at 8,000 droplets per second.

[0161] Specifically, Figure 12A depicts a still image of droplet formation in an exemplary microfluidic device at 2 ms. Five droplets are circled in solid lines. Figure 12B depicts a still image of droplet formation in an exemplary microfluidic device at 3 ms (e.g., 1 ms after the still image shown in Figure 12A). The five droplets originally shown in Figure 12A are also shown in Figure 12B, circled in solid lines. Eight additional droplets are shown circled in dotted lines. Thus, the droplet generation rate is 8 droplets per millisecond, or 8,000 droplets per second.

[0162] Figure 13 depicts paired cell droplets containing T cells paired with K562 cells that function as antigen-presenting cells (APCs). Overall, approximately 30% of all droplets have one T cell paired with at least one K562 cell, which is approximately three times higher than the expected percentage predicted using a Poisson distribution.

Claims

1. 1. A method for encapsulating two cells in a single droplet, comprising: flowing a first aqueous phase comprising a first cell-aligning stream in a first microchannel toward a junction; flowing a second aqueous phase comprising a second cell-aligning stream in a second microchannel toward the junction; flowing an oil phase in a third microchannel towards the junction; generating the single droplet at the junction formed from the first aqueous phase, the second aqueous phase, and the oil phase, the single droplet containing cells from the first cell alignment stream and cells from the second cell alignment stream.

2. 2. The method of claim 1, further comprising generating additional single droplets at the junction to generate a population of single droplets, the population characterized by a proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream, the proportion exceeding an expected proportion of single droplets comprising cells from the first aligned stream and cells from the second aligned stream as predicted using a Poisson distribution.

3. 3. The method of claim 2, wherein said percentage exceeds said expected percentage by a factor of 2 to 3.

4. 3. The method of claim 2, wherein the method generates single droplets at a rate of at least 5,000 droplets per second.

5. 3. The method of claim 2, wherein the method generates single droplets at a rate of at least 8,000 droplets per second.

6. The method of any one of claims 1 to 5, wherein the cells of the first cell-aligned stream are aligned along a central axis or an edge of the first microchannel.

7. 7. The method of any one of claims 1 to 6, wherein cells of said first cell alignment stream are aligned through inertial focusing while flowing through said first microchannel.

8. 8. The method of claim 7, wherein the inertial focusing is produced by flowing the first aqueous phase through a curved region of the first microchannel.

9. 9. The method of claim 8, wherein the curved region is 150 to 300 mm in length.

10. 9. The method of claim 8, wherein the curved region is 50 to 150 mm in length.

11. 9. The method of claim 8, wherein the curved region is about 100 mm in length.

12. 12. The method of any one of claims 8 to 11, wherein the curved region comprises at least one undulating portion that comprises at least a 45 degree change in flow vector over the length of the undulating portion.

13. 12. The method of any one of claims 8-11, wherein the curved region comprises at least one undulating portion comprising at least a 60 degree change, at least a 90 degree change, at least a 120 degree change, at least a 150 degree change, or at least a 180 degree change in flow vector over the length of the undulating portion.

14. The method of any one of claims 8 to 13, wherein the curved region comprises 60 to 120 undulating portions.

15. 15. The method of any one of claims 1 to 14, wherein the intercellular spacing for at least 80% of the cells in said first aligned stream is between 1 times the average cell diameter and 3.5 times the average cell diameter.

16. 15. The method of any one of claims 1 to 14, wherein the intercellular spacing for at least 60% of the cells in said first aligned stream is between 1.5 times the average cell diameter and 3 times the average cell diameter.

17. 15. The method of any one of claims 1 to 14, wherein the standard deviation of intercellular spacing between consecutive cells in a pair is less than 10 μm when measured across 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in said first cell-aligned stream.

18. 18. The method of any one of claims 15 to 17, wherein the inter-cell spacing between paired cells in the first cell-aligned stream is adjusted by passing the paired cells between a set of pillars.

19. 20. The method of claim 18, wherein the set of pillars is positioned at the entrance of the first microchannel.

20. 20. The method of claim 18 or 19, wherein the set of pillars at the entrance to the first microchannel comprises a gap between pillars of 5 to 40 μm.

21. 21. The method of any one of claims 1 to 20, wherein the cells of the second cell alignment stream are aligned along a central axis or an edge of the second microchannel.

22. 22. The method of any one of claims 1 to 21, wherein cells of said second cell alignment stream are aligned through inertial focusing while flowing through said second microchannel.

23. 23. The method of claim 22, wherein the inertial focusing is produced by flowing the second aqueous phase through a curved region of the second microchannel.

24. 24. The method of claim 23, wherein the curved region of the second microchannel is 150 to 300 mm in length.

25. 24. The method of claim 23, wherein the curved region is 50 to 150 mm in length.

26. 24. The method of claim 23, wherein the curved region is about 100 mm in length.

27. 27. The method of any one of claims 23 to 26, wherein the curved region of the second microchannel comprises at least one undulating portion comprising at least a 45 degree change in flow vector over the length of the undulating portion.

28. 27. The method of any one of claims 23-26, wherein the curved region of the second microchannel comprises at least one undulating portion comprising at least a 60 degree change, at least a 90 degree change, at least a 120 degree change, at least a 150 degree change, or at least a 180 degree change in flow vector over the length of the undulating portion.

29. The method of any one of claims 23 to 28, wherein the curved region of the second microchannel comprises 60 to 120 undulating portions.

30. 30. The method of any one of claims 1 to 29, wherein the intercellular spacing for at least 80% of the cells in said second aligned stream is between 1 times the average cell diameter and 3.5 times the average cell diameter.

31. 30. The method of any one of claims 1 to 29, wherein the intercellular spacing for at least 60% of the cells in said second aligned stream is between 1.5 times the average cell diameter and 3 times the average cell diameter.

32. 32. The method of claim 30 or 31, wherein the standard deviation of intercellular spacing between consecutive cells in a pair is less than 10 μm when measured across 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in the first cell-aligned stream.

33. 33. The method of any one of claims 30-32, wherein the intercellular spacing between the paired cells in the second cell-aligned stream is adjusted by passing the paired cells between a second set of pillars.

34. 34. The method of claim 33, wherein the second set of pillars is positioned at the entrance of the second microchannel.

35. 35. The method of claim 33 or 34, wherein the second set of pillars at the entrance to the second microchannel comprises gaps between pillars of 5 to 40 μm.

36. 36. The method of any one of claims 1 to 35, wherein the ratio between the width of the first microchannel and the average diameter of cells in the first cell-aligning stream is 1 to 20.

37. 37. The method of claim 36, wherein the ratio is between 1 and 10.

38. 37. The method of claim 36, wherein the ratio is between 1.5 and 7.

5.

39. 37. The method of claim 36, wherein the ratio is between 2.5 and 5.

0.

40. 40. The method of any one of claims 36 to 39, wherein the cells of said first cell-aligned stream are 5 to 25 μm in diameter.

41. The method of any one of claims 36 to 40, wherein the first microchannel comprises a channel width of 10 to 100 μm.

42. 42. The method of any one of claims 1 to 41, wherein the ratio between the average diameter of cells in the second cell-aligned stream and the width of the second microchannel is 1 to 20.

43. 43. The method of claim 42, wherein the ratio is between 1 and 10.

44. 43. The method of claim 42, wherein the ratio is between 1.5 and 7.

5.

45. 43. The method of claim 42, wherein the ratio is between 2.5 and 5.

0.

46. 46. ​​The method of any one of claims 42 to 45, wherein the cells of said second cell-aligned stream are 5 to 25 μm in diameter.

47. 46. ​​The method of any one of claims 42 to 45, wherein the second microchannel comprises a channel width of 10 to 100 μm.

48. the maximum concentration of cells in the first cell alignment stream, C 1 but, where D 1 represents the average diameter of cells in the first aligned stream, and S 1 represents the spacing between the cells of the pair of first aligned streams, and W 1 represents the width of the first microchannel, and H 1 48. The method of any one of claims 1-47, wherein represents the height of the first microchannel.

49. the maximum concentration of cells in the second cell alignment stream, C 2 but, where D 2 represents the average diameter of cells in the second aligned stream, and S 2 represents the spacing between the cells of the pair of second aligned streams, and W 2 represents the width of the second microchannel, and H 2 The method of any one of claims 1 to 47, wherein represents the height of the second microchannel.

50. generating the single droplets contacting the flowing first and second aqueous phases with each other to create a single aqueous phase comprising the first cell-aligned stream and the second cell-aligned stream.

50. The method of any one of claims 1 to 49, comprising:

51. 51. The method of claim 50, wherein said contacting of said flowing first and second aqueous phases to create said single aqueous phase occurs at a location at or before said junction.

52. generating the single droplets contacting the flowing oil phase with the single aqueous phase to form a conical configuration within the junction, wherein the single droplet is generated at the tip of the conical configuration; 52. The method of claim 50 or 51, further comprising:

53. 53. The method of any one of claims 1 to 52, wherein the cells from the first cell sorting stream and the cells from the second cell sorting stream are different cells.

54. 54. The method of claim 53, wherein said cells from said first cell-aligned stream are T cells.

55. 54. The method of claim 53, wherein said cells from said second cell sorting stream are antigen-presenting cells (APCs).

56. 56. The method of any one of claims 1 to 55, wherein said single droplet further comprises at least a second cell from said second cell-aligned stream.

57. 57. The method of any one of claims 1 to 56, wherein the first aqueous phase is flowed at a first rate of between 10 μL / min and 60 μL / min.

58. 58. The method of claim 57, wherein the first aqueous phase is flowed at a first rate of about 45 μL / min.

59. 57. The method of any one of claims 1 to 56, wherein the second aqueous phase is flowed at a second rate of between 10 μL / min and 60 μL / min.

60. 60. The method of claim 59, wherein the second aqueous phase is flowed at a second rate of about 45 μL / min.

61. 57. The method of any one of claims 1 to 56, wherein the oil phase is flowed at a third rate of 10 μL / min to 60 μL / min.

62. 62. The method of claim 61, wherein the oil phase is flowed at a third rate of about 45 μL / min.

63. 57. The method of any one of claims 1 to 56, wherein the second aqueous phase is flowed at a second velocity that is faster than the first velocity of the first aqueous phase, such that the single droplet contains only a single cell from the first cell-aligned stream and two or more cells from the second cell-aligned stream.

64. detecting an interaction between the cells from the first cell alignment stream and the cells from the second cell alignment stream.

57. The method of any one of claims 1 to 56, further comprising:

65. 65. The method of claim 64, wherein the first aqueous phase or the second aqueous phase further comprises a reagent for detecting the interaction.

66. 66. The method of claim 64 or 65, wherein the reagent comprises any of a fluorescent marker, a bead, or a nucleic acid barcode.

67. 67. The method of any one of claims 64 to 66, wherein detecting said interaction comprises detecting a biomarker analyte indicative of said interaction.

68. 68. The method of any one of claims 64 to 67, wherein detecting the interaction comprises detecting the interaction in the single droplet.

69. 1. A method for encapsulating two or more cells in a plurality of droplets, comprising: flowing a first aqueous phase comprising a first cell alignment stream in a first microchannel; flowing a second aqueous phase comprising a second cell alignment stream in a second microchannel; flowing the oil phase in a third microchannel; Flowing the first aqueous phase, the second aqueous phase, and the oil phase together to generate the plurality of droplets, wherein at least 20% of the droplets in the plurality of droplets comprise a single cell from the first cell alignment stream and at least one cell from the second cell alignment stream.

70. 70. The method of claim 69, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the droplets in said plurality of droplets comprise a single cell from said first cell-aligned stream and at least one cell from said second cell-aligned stream.

71. 71. The method of claim 69 or 70, wherein the plurality of droplets is characterized by a proportion of single droplets containing cells from the first aligned stream and cells from the second aligned stream, and wherein the proportion exceeds an expected proportion of single droplets containing cells from the first aligned stream and cells from the second aligned stream predicted using a Poisson distribution.

72. 72. The method of claim 71, wherein said percentage exceeds said expected percentage by a factor of 2 to 3.

73. 72. The method of claim 71, wherein droplets of the plurality of droplets are generated at a rate of at least 5,000 droplets per second.

74. 72. The method of claim 71, wherein droplets of the plurality of droplets are generated at a rate of at least 8,000 droplets per second.

75. 75. The method of any one of claims 69 to 74, wherein the cells of the first cell alignment stream are aligned along a central axis of the first microchannel.

76. 75. The method of any one of claims 69-74, wherein cells of said first cell alignment stream are aligned through inertial focusing while flowing through said first microchannel.

77. 77. The method of claim 76, wherein the inertial focusing is produced by flowing the first aqueous phase through a curved region of the first microchannel.

78. 78. The method of claim 77, wherein the curved region is 150 to 300 mm in length.

79. 78. The method of claim 77, wherein the curved region is 50 to 150 mm in length.

80. 78. The method of claim 77, wherein the curved region is about 100 mm in length.

81. 81. The method of any one of claims 77-80, wherein the curved region comprises at least one undulating portion comprising at least a 45 degree change in flow vector over the length of the undulating portion.

82. 81. The method of any one of claims 77-80, wherein the curved region comprises at least one undulating portion comprising at least a 60 degree change, at least a 90 degree change, at least a 120 degree change, at least a 150 degree change, or at least a 180 degree change in flow vector over the length of the undulating portion.

83. 83. The method of any one of claims 77-82, wherein the curved region comprises 60 to 120 undulating portions.

84. 84. The method of any one of claims 69-83, wherein the intercellular spacing for at least 80% of the cells in said first aligned stream is between 1 times the average cell diameter and 3.5 times the average cell diameter.

85. 84. The method of any one of claims 69-83, wherein the intercellular spacing for at least 60% of the cells in said first aligned stream is between 1.5 times the average cell diameter and 3 times the average cell diameter.

86. 86. The method of claim 84 or 85, wherein the standard deviation of intercellular spacing between consecutive cells in a pair is less than 10 μm when measured across 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in said first cell-aligned stream.

87. 87. The method of any one of claims 84-86, wherein the intercellular spacing between paired cells in the first cell-aligned stream is adjusted by passing the paired cells between a set of pillars.

88. 88. The method of claim 87, wherein the set of pillars is positioned at an entrance to the first microchannel.

89. 89. The method of claim 87 or 88, wherein the set of pillars at the entrance to the first microchannel comprises a gap between pillars of 5 to 40 μm.

90. 90. The method of any one of claims 69 to 89, wherein the cells of the second cell alignment stream are aligned along a central axis of the second microchannel.

91. 91. The method of any one of claims 69 to 90, wherein cells of said second cell alignment stream are aligned through inertial focusing while flowing through said second microchannel.

92. 92. The method of claim 91, wherein the inertial focusing is produced by flowing the second aqueous phase through a curved region of the second microchannel.

93. 93. The method of claim 92, wherein the curved region of the second microchannel is 150 to 300 mm in length.

94. 94. The method of claim 92 or 93, wherein the curved region comprises at least one undulating portion comprising at least a 45 degree change in flow vector over the length of the undulating portion.

95. 94. The method of claim 92 or 93, wherein the curved region comprises at least one undulating portion comprising at least a 60 degree change, at least a 90 degree change, at least a 120 degree change, at least a 150 degree change, or at least a 180 degree change in flow vector over the length of the undulating portion.

96. 96. The method of any one of claims 92-95, wherein the curved region comprises 60 to 120 undulating portions.

97. 97. The method of any one of claims 69-96, wherein the intercellular spacing for at least 80% of the cells in said second aligned stream is between 1 times the average cell diameter and 3.5 times the average cell diameter.

98. 97. The method of any one of claims 69-96, wherein the intercellular spacing for at least 60% of the cells in said second aligned stream is between 1.5 times the average cell diameter and 3 times the average cell diameter.

99. 99. The method of claim 97 or 98, wherein the standard deviation of intercellular spacing between consecutive cells in a pair is less than 10 μm when measured across 10 pairs, 20 pairs, 30 pairs, 40 pairs, 50 pairs, 60 pairs, 70 pairs, 80 pairs, 90 pairs, or 100 pairs of adjacent cells in said first cell-aligned stream.

100. 100. The method of any one of claims 97-99, wherein the intercellular spacing between paired cells in the second cell-aligned stream is adjusted by passing the paired cells between a set of pillars.

101. 100. The method of claim 99, wherein the set of pillars is positioned at the entrance of the second microchannel.

102. 102. The method of claim 99 or 101, wherein the set of pillars at the entrance to the first microchannel comprises a gap between pillars of 5 to 40 μm.

103. 103. The method of any one of claims 69 to 102, wherein the ratio between the width of the first microchannel and the average diameter of cells in the first cell-aligned stream is 1 to 20.

104. 104. The method of claim 103, wherein the ratio is between 1 and 10.

105. 104. The method of claim 103, wherein the ratio is between 1.5 and 7.

5.

106. 104. The method of claim 103, wherein the ratio is between 2.5 and 5.

0.

107. 107. The method of any one of claims 103 to 106, wherein the cells of said first cell-aligned stream are 5 to 25 μm in diameter.

108. The method of any one of claims 103 to 107, wherein the first microchannel comprises a channel width of 10 to 100 μm.

109. 109. The method of any one of claims 69 to 108, wherein the ratio between the average diameter of cells in the second cell-aligned stream and the width of the second microchannel is 1 to 20.

110. 110. The method of claim 109, wherein the ratio is between 1 and 10.

111. 110. The method of claim 109, wherein the ratio is between 1.5 and 7.

5.

112. 110. The method of claim 109, wherein the ratio is between 2.5 and 5.

0.

113. 113. The method of any one of claims 109-112, wherein the cells of said second cell-aligned stream are 5-25 μm in diameter.

114. The method of any one of claims 109-112, wherein the second microchannel comprises a channel width of 10 to 100 μm.

115. the maximum concentration of cells in the first cell alignment stream, C 1 but, where D 1 represents the diameter of the cells of the first aligned stream, and S 1 represents the spacing between the cells of the pair of first aligned streams, and W 1 represents the width of the first microchannel, and H 1 The method of any one of claims 69-114, wherein represents the height of the first microchannel.

116. the maximum concentration of cells in the second cell alignment stream, C 2 but, where D 2 represents the diameter of the cells of the first aligned stream, and S 2 represents the spacing between the cells of the pair of first aligned streams, and W 2 represents the width of the first microchannel, and H 2 The method of any one of claims 69-115, wherein represents the height of the first microchannel.

117. generating the single droplet, contacting the flowing first and second aqueous phases to produce a single aqueous phase comprising the first cell-aligned stream and the second cell-aligned stream.

117. The method of any one of claims 69 to 116, comprising:

118. 118. The method of claim 117, wherein said contacting of said flowing first and second aqueous phases to create said single aqueous phase occurs at a location at or before said junction.

119. generating the single droplet, contacting the flowing oil phase with the single aqueous phase to form a conical configuration within the junction, wherein the single droplet is generated at the tip of the conical configuration; 119. The method of claim 117 or 118, further comprising:

120. 120. The method of any one of claims 69 to 119, wherein said cells from said first cell alignment stream and said cells from said second cell alignment stream are different cells.

121. 121. The method of claim 120, wherein said cells from said first cell-sorted stream are T cells.

122. 121. The method of claim 120, wherein said cells from said second cell sorting stream are antigen-presenting cells (APCs).

123. 123. The method of any one of claims 69-122, wherein said single droplet further comprises at least a second cell from said second cell-aligned stream.

124. The method of any one of claims 69 to 123, wherein the first aqueous phase is flowed at a first rate of 10 μL / min to 40 μL / min.

125. 125. The method of claim 124, wherein the first aqueous phase is flowed at a first rate of about 45 μL / min.

126. The method of any one of claims 69 to 123, wherein the second aqueous phase is flowed at a second rate of between 10 μL / min and 40 μL / min.

127. 127. The method of claim 126, wherein the second aqueous phase is flowed at a second rate of about 45 μL / min.

128. The method of any one of claims 69 to 123, wherein the oil phase is flowed at a third rate of 10 μL / min to 50 μL / min.

129. 129. The method of claim 128, wherein the oil phase is flowed at a third rate of about 45 μL / min.

130. 124. The method of any one of claims 69-123, wherein the second aqueous phase is flowed at a second velocity that is faster than the first velocity of the first aqueous phase, such that the single droplet contains only a single cell from the first cell-aligned stream and two or more cells from the second cell-aligned stream.

131. detecting an interaction in the single droplet between the cells from the first cell alignment stream and the cells from the second cell alignment stream.

124. The method of any one of claims 69-123, further comprising:

132. 132. The method of claim 131, wherein said first aqueous phase or said second aqueous phase further comprises a reagent for detecting said interaction.

133. 133. The method of claim 131 or 132, wherein the reagent comprises any of a fluorescent marker, a bead, or a nucleic acid barcode.

134. 134. The method of any one of claims 131 to 133, wherein detecting said interaction in said single droplet comprises detecting a biomarker analyte indicative of said interaction.

135. a first microchannel, a second microchannel, and a third microchannel fluidly connected to one another through a junction; the first microchannel comprises a curved region comprising a channel width of 10 to 100 μm; the second microchannel comprises a curved region comprising a channel width of 10 to 100 μm; A microfluidic device for encapsulating cell pairs in droplets.

136. 136. The microfluidic device of claim 135, wherein the curved region of the first microchannel and the curved region of the second microchannel are 150 to 300 mm in length.

137. 136. The microfluidic device of claim 135, wherein the curved region is 50 to 150 mm in length.

138. 136. The microfluidic device of claim 135, wherein the curved region is about 100 mm in length.

139. The microfluidic device of any one of claims 135 to 138, wherein the first microchannel comprises a non-curved region located closer to the junction than the curved region of the first microchannel.

140. 139. The microfluidic device of any one of claims 135 to 138, wherein the second microchannel comprises a non-curved region located closer to the junction than the curved region of the second microchannel.

141. The microfluidic device of any one of claims 135 to 140, wherein the channel width of the curved region of the first microchannel and the channel width of the curved region of the second microchannel are between 15 and 75 μm.

142. 142. The microfluidic device of any one of claims 135 to 141, wherein the radius of curvature of the curved region of the first microchannel increases continuously along the entire length of the curved region.

143. 142. The microfluidic device of any one of claims 135 to 141, wherein the radius of curvature of the curved region of the second microchannel increases continuously along the entire length of the curved region.

144. 142. The microfluidic device of any one of claims 135 to 141, wherein the radius of curvature of the curved region of the first microchannel remains constant along the entire length of the curved region.

145. 142. The microfluidic device of any one of claims 135 to 141, wherein the curved region of the first microchannel comprises a first wavy portion having a first radius of curvature and a second wavy portion having a second radius of curvature.

146. 146. The microfluidic device of claim 145, wherein the first radius of curvature is different from the second radius of curvature.

147. 147. The microfluidic device of any one of claims 144 to 146, wherein said curved region comprises at least one undulating portion comprising at least a 45 degree change in flow vector over the length of said undulating portion.

148. 147. The microfluidic device of any one of claims 144-146, wherein the curved region comprises at least one undulating portion comprising at least a 60 degree change, at least a 90 degree change, at least a 120 degree change, at least a 150 degree change, or at least a 180 degree change in flow vector over the length of the undulating portion.

149. The microfluidic device of any one of claims 144-148, wherein the curved region comprises between 60 and 120 undulating portions.

150. 149. The microfluidic device of any one of claims 144 to 148, wherein the radius of curvature of the curved region of the second microchannel remains constant along the entire length of the curved region.

151. The microfluidic device of any one of claims 144 to 148, wherein the curved region of the first microchannel comprises a first wavy portion having a first radius of curvature and a second wavy portion having a second radius of curvature.

152. 152. The microfluidic device of claim 151, wherein the first radius of curvature is different from the second radius of curvature.

153. The microfluidic device of any one of claims 150-152, wherein the curved region comprises between 60 and 120 undulating portions.

154. The microfluidic device of any one of claims 135 to 153, wherein the first microchannel further comprises a set of pillars positioned at the inlet of the first microchannel.

155. 155. The microfluidic device of claim 154, wherein the set of pillars comprises a gap between pillars of 5 to 40 μm.

156. The microfluidic device of any one of claims 135 to 153, wherein the second microchannel further comprises a set of pillars positioned at the inlet of the second microchannel.

157. 157. The microfluidic device of claim 156, wherein the set of pillars comprises a gap between pillars of 5 to 40 μm.

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