Sorting and selection of cells using biodegradable hydrogels

Abstract

Systems and methods are described for sorting and selecting cells by immobilizing or surrounding the cells within a degradable hydrogel chamber, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel including a surface, (ii) a spatial energy modulation element in optical communication with the surface, and (iii) a detector in optical communication with the surface and operably associated with the spatial energy modulation element; (b) loading the channel with cells and one or more polymer precursors such that the cells are disposed on or proximate to the surface thereof; (c) immobilizing selected cells based on one or more optical signals derived from the cells, the immobilizing being by synthesizing one or more gel structures; and (d) removing unselected cells from the channel.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit of U.S. Provisional Application No. 63 / 350,813, filed on June 9, 2022, which is hereby incorporated by reference in its entirety.

Background Art

[0002] (Background) Due to its high information content, image - based analysis and sorting of cells have attracted great interest in many areas, such as cancer diagnosis, drug development, immunocytochemistry, phenotype - genotype analysis, etc. (e.g., LaBelle et al., Trends in Biotechnology, 39(6):614 - 623(2021); Boutros et al., Cell, 163:1314 - 1325(2015); Caicedo et al., Curr.Opin.Biotechnology, 39:134 - 142(2016); Wang et al., Proc.Natl.Acad.Sci., 116(22):10842 - 10851(2019); Ploem - Zaaijer et al., Cytometry, 15:199 - 206(1994)). As pointed out by LaBelle, current image - based sorting systems generally follow three approaches: (i) imaging in flow through a microfluidic system, (ii) imaging after capture or confinement in a microfluidic system, and (iii) imaging in a microwell array system. These three approaches have important trade - offs among important performance parameters (e.g., throughput, scalability, and capacity). For example, imaging in flow requires sophisticated high - speed optical systems and processing. Microfluidic capture and confinement avoid the problems of imaging in flow systems but reduce throughput and flexibility. Microwell array systems enable excellent image collection but have low throughput.

Prior Art Documents

Non - Patent Documents

[0003] [Non-Patent Document 1] LaBelle et al., Trends in Biotechnology, 39(6):614 - 623(2021) [Non-Patent Document 2] Boutros et al., Cell, 163:1314 - 1325(2015) [Non-Patent Document 3] Caicedo et al., Curr.Opin.Biotechnology, 39:134 - 142(2016) [Non-Patent Document 4] Wang et al., Proc.Natl.Acad.Sci., 116(22):10842 - 10851(2019) [Non-Patent Document 5] Ploem-Zaaijer et al., Cytometry, 15:199 - 206(1994) [Summary of the Invention] [Means for Solving the Problems]

[0004] (Abstract) Considering the above, if an image-based technology that has the advantages of a microarray-based system but is easily scalable and has flexible accommodation and sorting capabilities were available, image-based selection and sorting would thereby progress. In certain aspects, methods of selecting or sorting cells using a degradable gel structure are described herein. In certain aspects, methods of sorting cells are described herein, the methods comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel comprising a surface, (ii) a spatial energy modulation element in optical communication with the surface, and (iii) a detector in optical communication with the surface and operably associated with the spatial energy modulation element, the detector identifying cells and determining the position of the cells on the surface; (b) loading the channel with cells and one or more polymer precursors such that the cells are disposed on or proximate to the surface; (c) fixing the one or more cells selected based on one or more optical signals derived from the one or more cells, the fixing comprising synthesizing one or more gel structures surrounding each of the one or more cells, the synthesizing by projecting light in the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form the gel structures, the position of the gel structures in the channel being determined by the position of the cells surrounded by the gel structures identified by the detector; and (d) removing unselected cells from the channel. In some cases, the method further comprises repeating steps (b) - (c). In some embodiments, steps (b) - (c) are repeated until the fixed cells reach a predetermined density on the surface. In some embodiments, the method further comprises degrading the gel structures of the fixed cells and eluting the selected cells from the channel. In some embodiments, the one or more optical signals indicate cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell migration rate, cell replication rate, or protein secretion.In some particular embodiments, one or more of those optical signals include a series of images from which a cell migration rate can be determined. For example, from a series of images of its surface, the cell positions can be recorded as a function of time, and as a result, the migration speed or velocity can be determined. In other particular embodiments, one or more of those optical signals include a series of images from which cell-cell interactions (e.g., cell death) can be determined or quantified. In some embodiments, those gel structures include photochemically degradable bonds. In some embodiments, those gel structures include enzymatically degradable bonds. In some embodiments, those gel structures include chemically degradable bonds (e.g., disulfide bonds). In some embodiments, the gel structure includes hydrogel chambers each having an interior. In some particular embodiments, cells are trapped or contained within such interiors. In some embodiments, those hydrogel chambers each have an annular-like shape. In some embodiments, the gel structure includes a solid mass that encapsulates the cells.

[0005] In another aspect, a method of performing an assay on cells from a plurality of samples is described herein, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel comprising a surface; (ii) a spatial energy modulation element in optical communication with the surface; and (iii) a detector in optical communication with the surface and operably associated with the spatial energy modulation element, the detector being configured to identify a cell and determine the position of the cell on the surface; (b) loading the channel with cells from the sample and one or more polymer precursors such that the cells are disposed on or proximate to the surface; (c) synthesizing one or more gel structures surrounding each of the one or more cells of the sample, the synthesis comprising projecting light in the channel with the spatial energy modulation element such that the projected light causes crosslinking of the one or more polymer precursors to form the gel structures, wherein the position of the synthesized gel structures in the channel is determined by the position of the cells surrounded by the synthesized gel structures identified by the detector; (d) repeating steps (b) and (c) for each of the plurality of samples; and (e) performing an assay on the cells in the channel to obtain assay results for each of the cells surrounded by the gel structures. In some embodiments, the method further comprises determining the assay results for each of the samples by associating the assay results with the positions of the cells of the samples. In some embodiments, the gel structure is a hydrogel chamber.

[0006] In another aspect, a method for sorting cells is described herein, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel comprising a surface, one or more polymer precursors, and one or more cells; (ii) a spatial energy modulation element in optical communication with the surface; and (iii) a detector in optical communication with the surface and operably associated with the spatial energy modulation element; (b) using the detector to identify the positions of a subset of the one or more cells, the subset of the one or more cells being selected based on one or more optical signals derived from the subset of the one or more cells; (c) controlling the spatial energy modulation element based on the positions of the subset of the one or more cells identified by the detector to project energy into the channel such that the projected energy forms one or more gel structures surrounding the subset of the one or more cells, wherein one or more cells not in the subset of the one or more cells are not surrounded by the one or more gel structures; and (d) removing the one or more cells not in the subset of the one or more cells from the channel. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0017] (Detailed Description) The practice of the systems and methods described herein may use conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, unless otherwise indicated, which are within the skill of those in the art. Such conventional techniques include, but are not limited to, the preparation of synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. Specific illustrations of appropriate techniques can be obtained by reference to the following examples herein. However, other equivalent conventional procedures may also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals (e.g., Genome Analysis: A Laboratory Manual Series (Volumes I - IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all published by Cold Spring Harbor Laboratory Press); Renault and Duchateau, eds., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, eds., Protein Engineering Handbook (Wiley-VCH, 2009), etc.). Guidance for selecting materials and components for performing specific functions can be found in available papers and references regarding scientific apparatus (Moore et al., Building Scientific Apparatus, 3rd Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013) and similar references, but not limited thereto).

[0018] The methods and systems described herein can use degradable gels to select, sort, and isolate cells, particularly based on optically measurable properties. In one aspect, sorting, selection, and separation are achieved by immobilizing individual cells having desirable properties by synthesizing a degradable gel structure that encapsulates or surrounds those target cells. In some embodiments, whenever a desired number of cells having those desirable properties have accumulated (and non-selected cells have been removed), the gel structure that encapsulates or surrounds them can be degraded to release those selected cells. An apparatus for performing such procedures is described more fully below. Briefly, in some embodiments, cells combined with a polymer precursor are loaded into a flow cell channel such that the cells are disposed on the surface, detected (perhaps after an assay step), and then the selected cells (e.g., based on assay results) are immobilized on that surface by surrounding those selected cells in a photosynthesized degradable gel structure. (In some embodiments, the cells and polymer precursor can be sequentially loaded into the channel, e.g., the cells can be loaded and disposed on the channel surface and then the polymer precursor can be loaded). Non-selected cells are removed, and then the gel structure of those selected cells is degraded and those selected cells are recovered. Sorting and selection can be based on a wide variety of properties measured from images and / or assay results (including, but not limited to, size, morphology, organelle characteristics, motility, migration speed, surface protein expression, secreted protein profile, cell-cell interactions, etc.).

[0019] In another aspect, sorting, selection, and separation are achieved by immobilizing individual cells that cannot have desirable properties by synthesizing a gel structure that encapsulates or surrounds such cells but allows cells having desirable properties to elute freely from the flow cell.

[0020] In another aspect, multiple cell samples can be processed simultaneously or subjected to the same assay in the same flow cell channel. In some embodiments of this aspect, multiple cell samples can be sequentially placed on the same surface. After each sample is loaded, the positions of those cells are optically determined and the cells are fixed by synthesizing one or more gel structures surrounding those cells. Thus, for each sample, a map of the positions of the cells and / or gel structures is obtained that enables the assay results for individual cells to be assigned to a particular sample. After all of those samples are loaded and the cells are fixed, one or more assays can be performed simultaneously on all of the cells on that surface. In one aspect, those gel structures may or may not be decomposable, depending in part on whether it is desirable to recover those cells after the assay (assays) are complete. Further, the gel structures synthesized to surround the cells of the sample can have various compositions or shapes such that, for example, if the assay involves instantaneously applying various doses of a compound to the cells, the composition and / or wall thickness of the gel structures are prepared in a manner such that the gel structures having various diffusivities result in the cells of the various samples receiving the various doses of that compound.

[0021] Figures 1A - 1B show embodiments of a cell sorting method using an apparatus described in sufficient detail below. In some embodiments, such an apparatus is a fluid device comprising a channel having a surface on which cells can be placed, imaged, and fixed by synthesizing a gel structure to surround or encapsulate those cells. At the top of Figure 1A, cells (e.g., 102) are loaded (104) into a channel (not shown) such that they are placed on the surface (100), after which the detector of the apparatus records their positions and collects optical signals indicative of cell characteristics (such as size, morphology, area ratio or volume ratio of nucleolus to cytoplasm, surface protein profile, secreted protein profile, etc.). Based on such measurements, cells (e.g., 108a - 108d) having values within a predetermined range for these measurements can be selected. Such selected cells are fixed by photosynthesizing a gel structure (shown as circles around the selected cells 108a - 108d) around each of them (also shown in perspective in Figure 2). The positions of those gel structures are determined by the positions of the cells they surround, and the positions of those cells are in turn determined by optical detection. Such gel structures can have a wide variety of shapes and compositions. In some embodiments, the gel structure can include a solid mass of gel material that contacts and encapsulates the cells. In some embodiments, such a mass can have a well - defined shape (e.g., a solid cylinder, hemisphere, cube, etc.). In some embodiments, the gel structure can include a polymer matrix wall (e.g., a hydrogel chamber described in more detail below) that surrounds and encapsulates the cells without contacting the cells. In some embodiments, the channel can be part of a flow cell, the channel includes a first surface and a second surface, and the hydrogel chamber includes a polymer matrix wall that extends from the first surface to the second surface and surrounds the cells. After the selected cells are fixed by synthesizing the gel structure, unselected cells are removed (110) from the surface (100) such that only the cells fixed by the gel structure remain (111).This process can be repeated such that additional cell populations are loaded (112) into the channels such that they are disposed on the surface (100). The positions of those newly loaded cells are determined and recorded, and optical signals are collected to determine which of those newly loaded cells have the properties for selection. After such determination, the selected cells of the second loading are fixed by synthesizing gel structures (e.g., (114a - 114h)) surrounding each of those cells. The unselected cells are removed (118). These steps are repeated (118) until a predetermined number of selected cells are reached or the surface (100) becomes too crowded for further selection and fixation. For sorting, the gel compositions can be prepared in a manner such that they are capable of being degraded by the various mechanisms described below. As shown in FIG. 1B, the gel structures (120) on the surface (100) can be treated with an agent that degrades or depolymerizes the gel structure and releases the fixed cells (122), after which those cells can be eluted from the flow cell (124).

[0022] As described below, a wide variety of photosynthetic gels and degradable gels are available for implementing the systems and methods described herein. Guidelines for selecting such gels for desirable properties (including, but not limited to, biocompatibility, gelation rate, degradation rate, and similar properties) are provided in the following references, which are incorporated by reference: Kharkar et al., Chem. Soc. Rev., 42:7335-7372 (2013); Kharkar et al., Polymer Chem., 6(31):5565-5574 (2015); Neumann et al., Acta Biomater., 39:1-11 (2016); DeForest et al., Nature Chemistry, 3(12):925-931 (2012); U.S. Patent No. 9,631,092 to Bowman et al.; LeValley et al., ACS Appl. Bio. Mater., 3(10):6944-6958 (2020); Kabb et al., ACS Appl. Mater. Interfaces, 10:16793-16801 (2018); Fairbanks et al., Macromolecules, 44:2444-2450 (2011); Fairbanks et al., Adv. Mater., 21(48):5005-5010 (2009); U.S. Patent Application Publication No. US2016 / 0177030 to Sugiura et al.; Shih et al., Biomacromolecules, 13(7):2003-2012 (2012), etc. In some embodiments, the photosynthesized gel is formed using a photoinitiator for radical polymerization. In some embodiments, examples of photoinitiators include Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), or eosin Y (see, e.g., Choi et al., Biotechniques, 66(1):40-53 (2019)).In some embodiments, hydrogel precursors include hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly(ethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), and poly(vinyl alcohol). In some embodiments, the polymer precursor includes PEG or multi-arm PEG. In some embodiments, the polymer precursor includes an enzymatically degradable crosslinker. In some embodiments, such an enzymatically degradable crosslinker is degradable by esterase or peptidase. In some embodiments, the polymer precursor includes a photocleavable crosslinker. In some embodiments, such a photocleavable crosslinker includes a nitrobenzyl group. In some embodiments, such a photocleavable crosslinker includes a coumarin moiety. In some embodiments, a photocleavable hydrogel is used with the methods described herein. For example, photocleavage of the hydrogel chamber can be selectively performed on demand, such that as a result, a particular hydrogel chamber can be degraded without affecting the non-selected hydrogel chambers. In some embodiments, the hydrogel chambers are non-selectively degraded, such that as a result, all hydrogel chambers in a given channel (or other container) are degraded simultaneously. In some embodiments, such non-selective degradation is performed using a cleavage reagent that specifically cleaves labile bonds in the hydrogel. For example, such a cleavage agent includes a reducing agent. In some embodiments, such non-specific degradation is performed using an enzyme that cleaves bonds or chemical elements in the hydrogel. For example, chemical elements include, but are not limited to, peptides, polysaccharides, and oligonucleotides.

[0023] Figure 2 shows the features of an apparatus for fixing cells by synthesizing a gel structure and an example of a cylindrical hydrogel chamber. (Further description of such an apparatus is provided in FIGS. 8A - 8B). Cells (e.g., 201) combined with one or more polymer precursors are loaded into a channel (200) including a first surface (202) and a second surface (203) such that the cells are disposed on the surface (202). The first surface and the second surface (202 and 203) are parallel and typically include the surfaces of a light - transmissive material (e.g., glass). A detector (204) (along with a system and computer for recording images) detects and / or collects images of those cells (including their positions on the surface (202)). Operationally associated with the detector (204) is a spatial energy modulation element (206) (e.g., a digital micromirror device (DMD)), which projects light energy into the channel (200) to photopolymerize those polymer precursors under programmed control, forming a polymer matrix within the channel (200). Such a polymer matrix can include a solid gel mass encapsulating the cells, or it can include a more complex structure (e.g., a hollow cylinder, or a similar structure) that can surround the cells without directly contacting the cells. Such a hollow structure can include a polymer matrix wall that extends from the first surface (202) to the second surface (203) to form an enclosure (sometimes referred to herein as a "hydrogel chamber" or simply a "chamber") containing the cells. After the cells have been optically analyzed, a gel mass or chamber is synthesized (208) to fix the selected cells. In FIG. 2, those hydrogel chambers are shown for convenience as solid cylinders (225a - 225d) in the channel (200), but they are intended to represent the hollow cylinders (210) shown in the enlarged view. The hollow cylinder, or chamber (210), has a wall (221) with a thickness (216) surrounding an interior (211). After synthesis of the hydrogel chamber surrounding those selected cells, unselected cells are removed (228).

[0024] Figure 3 shows that in some embodiments, cells can be arranged on a surface (300) in a non-random manner by providing a regular array of cell attachment sites (302) on the surface, which can be prepared by various techniques (e.g., Zhu et al., Analytica Chimica Acta, 608(2):186-196 (2008), etc.). In some embodiments, such sites can include antibodies specific for selected cell surface proteins. In some embodiments, such sites can include integrin proteins or integrin peptides, or similar proteins. After loading (304) the cells (e.g., 306), those cells attach to the sites (302) in a regular pattern as shown. Thereafter, a hydrogel chamber (310) can be synthesized to surround and immobilize those cells, and thereafter, further rounds of loading and selection can be performed. In some embodiments, such spatially regular arrays make the imaging and synthesis of gel structures more efficient.

[0025] Figures 4A-4B, 5A-5B, and 6A-6B each show embodiments for sorting cells based on protein secretion profiles, growth rates, and migration rates. In other embodiments, cells can be sorted based on other (or additional) characteristics (e.g., cell-cell interactions, cell death, antigen presentation, etc.).

[0026] Regarding protein secretion, a uniform distribution of protein capture beads (e.g., BioLegend, Inc., San Diego) is adhered to the surface (450) before loading the cells. As shown in the enlarged view (444), the uniform distribution may include beads having antibodies specific for various proteins (e.g., separate types of beads for each of a plurality of cytokines). In one embodiment, sorting based on secretion proceeds as follows: After depositing the cells on the surface (450), the cells are positioned by a detector (445), and a hydrogel chamber is synthesized (446) by a spatial energy modulation element (447). The secreted protein is captured by the protein capture beads and then detected by being labeled with a detection antibody (shown by the dot in the middle (e.g., 457) in the hydrogel chamber (only a single bead type is shown for clarity)). In some embodiments, the porosity of the polymer matrix walls of those chambers is selected to prevent passage of cells but allow free passage of proteins. In other embodiments, such porosity is selected to prevent passage of both cells and proteins. In the latter embodiments, an additional step of depolymerizing those polymer matrix walls is included to provide access means for those detection antibodies. For example, the polymer matrix walls are selected to allow selective photodegradation. The relative amount of secreted protein from each cell is determined (453) by either counting separate types of labeled beads that are close to or proximal to the cells in each chamber or integrating separate fluorescence signals from the beads that are close to or proximal to the cells in each chamber. From the illustration of FIG. 4A, the hydrogel chambers (460, 462, 464, and 466) contain the cells with the highest secretion rate (shown by the amount of filled beads). After the high-secreting cells are identified, they are surrounded again by synthesizing a hydrogel chamber around them (458), and then the non-selected cells are removed (472).As shown, the chambers (482, 484, and 486) contain cells with a high secretion rate. After such removal, the chambers of those high-secretion-rate cells can be disassembled and those cells can be eluted (459). Alternatively, the low-secretion cells can be re-surrounded or fixed by the gel structure, and then the selected high-secretion cells are eluted.

[0027] Figures 5A - 5B show the steps of an embodiment for sorting cells based on growth rate. Cells (e.g., 551) are placed on a surface (550) where they are positioned by a detector system (545) and are enclosed by synthesizing a hydrogel chamber (549) (also shown in the top view in the lower panel) using a spatial energy modulation element (547) (546). The cells are incubated under growth conditions (553) and during that incubation, the hydrogel chambers are periodically imaged by the detector system to determine the number of cells in each chamber. In some embodiments, the cell number is used as a measure of the growth rate and as a result, after a predetermined incubation period, the hydrogel chambers can be identified as containing high-growth-rate cells or low-growth-rate cells based on the cell number as shown in the upper panel of Figure 5B (chambers (562, 564, 566, and 568) are selected as containing cells with a high growth rate). Thereafter, such hydrogel chambers are selectively (i.e., "on demand") disassembled or equivalently depolymerized (558) and then their released high-growth-rate cells are eluted (560). In some embodiments of that growth assay, each hydrogel chamber synthesized has the same shape and area (e.g., in the range of 0.001 mm 2 ~0.01 mm 2 or in the range of 0.001 mm 2 ~1.0 mm 2has an internal area selected from the range of (ring-like). In some embodiments, each hydrogel chamber to be synthesized has the same shape and area for each of the various types of cells to be assayed. For example, cytotoxic T lymphocytes can be confined in a chamber having one area, while helper T lymphocytes can be confined in a chamber having another area. After a desired number of hydrogel chambers have been synthesized, the cells are incubated for a period of time under growth conditions, and then (for example) the cells in each chamber are counted (220) to obtain a measure of the growth ability for each cell. In some embodiments, after synthesizing the chambers to surround the selected cells, unselected cells can be removed by a washing step or by other modification of the reagents in that channel. In some embodiments, the cells can be stained with a membrane or intracellular dye to determine growth by dye dilution such that a measure independent of cell proliferation can be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. The desired number of single cell populations surrounded by the chambers depends on the statistical reliability desired in the measured values. If the target subpopulation exists as only a small part of the total population, a larger number of chambers are required. In some embodiments where mammalian cells are assayed, the number of hydrogel chambers synthesized around a single cell population can exceed 100; or can exceed 1000; or can exceed 10,000; or the number can be in the range of 100 to 100,000; or can be in the range of 1000 to 100,000. After the count values are recorded for each chamber, further assays can be performed on the clonal populations within those chambers to identify the cell types, for example, by evaluation of cell surface proteins, cell protein secretion, transcriptome, and the like. This approach is particularly useful for evaluating populations of immune cells (especially engineered immune cells).

[0028] Figures 6A-6B show the steps of an embodiment for sorting cells based on migration speed. For convenience, Figure 6A does not include an abstracted perspective view of the imaging and synthesis apparatus as in Figure 5A, but those steps use the same equipment. Cells (e.g., 602) are loaded (604) and placed on a surface (600), and then, as indicated by the trajectories in this figure that define the starting position, the completion position, and the path traversed over a predetermined time interval, their detector system determines those positions and tracks their movement. A measure of the cell migration speed can be the length of the path traversed by the cell over that predetermined interval. In some embodiments, cells having the highest migration speed are enclosed in hydrogel chambers (e.g., (612), (612) and (614)), after which the low migration speed cells are removed (616), the chambers of the highly motile cells are disassembled (618), and the released high migration speed cells are eluted (620).

[0029] In some embodiments, as shown in FIGS. 7A-7B, the gel structure can be used to perform assays on single cell populations from multiple samples. The cells of each sample are sequentially loaded, placed on the surface, and enclosed in a gel structure where each sample and its location are associated. In some particular embodiments, the gel structure is a hydrogel chamber. Similar to FIG. 6A, FIG. 7A does not include an abstract perspective view of the imaging and synthesis apparatus as in FIG. 5A, but the steps of this embodiment can use the same equipment. Cells from sample 1 (702) are loaded, placed on the surface (700) (e.g., 701), then their positions are determined, and a hydrogel chamber (e.g., 705) is synthesized (704). Then, cells from sample 2 are loaded (706), placed on the surface (700), then their positions are determined, and a hydrogel chamber (e.g., 707) is synthesized. Finally, cells from sample 3 are loaded (710), placed on the surface (700), then their positions are determined, and a hydrogel chamber (e.g., 711) is synthesized (712). After the last hydrogel chamber is synthesized, the assay can be performed simultaneously on all three samples, and the assay results can be associated with a particular sample and the location of their hydrogel chambers.

[0030] Assays can include, but are not limited to, assays that measure viability, surface protein expression, cell motility, cell migration rate, cell replication rate, protein secretion, cytotoxicity, vector copy number of transduced cells, viral integration sites, transcriptome, etc. In some embodiments, the transcriptome of the enclosed cells is determined. In some embodiments, the protein secretion of the enclosed cells is determined. In some embodiments, the cytokine secretion of the enclosed cells is determined. In some embodiments, the gel structure used to surround or immobilize the cells of a particular sample can be degradable, for example, such that the cells can be eluted or recovered after the assay is performed. In some embodiments, the gel structure that surrounds or immobilizes at least two samples is degradable using orthogonal methods such that the cells of such samples can be eluted or recovered separately. In some embodiments, such orthogonal methods include photocleavable gels and chemically cleavable gels. In some embodiments, the chemically cleavable gel includes disulfide bonds. In some embodiments, the chemically cleavable gel includes pH-sensitive bonds.

[0031] Figure 7C shows an embodiment in which cells can be surrounded by a hydrogel chamber containing two or more orthogonally decomposable hydrogels. Such embodiments can be used to expose selected cells to gel-impermeable reagents (e.g., beads), which can be useful in certain assays (e.g., assays for cytokines or other secreted proteins). Figure 7C shows three cells (722a, 722b, and 722c) disposed in a region of the surface (720) of a flow cell (not shown). Cells (722a and 722b) are selected based on optical properties (e.g., size and shape) and are surrounded (725) by hydrogel chambers (724a and 724b, respectively), the walls of each chamber having a gap (726) of a predetermined position and size. Cell (722c) is removed by washing. In some embodiments (e.g., the embodiments shown), the number and size of those gaps are small enough that the enclosed cells cannot exit their chambers, but are large enough and numerous enough that protein capture beads (e.g., (729)) can diffuse into their chambers in an operable amount (i.e., for statistically reliable measurements) in a reasonable time (e.g., less than 24 hours, or less than 8 hours, or less than 4 hours, or less than 2 hours). As shown in Figure 7C, protein capture beads are loaded (728) into the channels of the flow cell such that they are disposed around and within the hydrogel chambers (e.g., 724a and 724b), after which a polymer precursor is also loaded into the channels and the gaps (732) are closed by photosynthesizing the hydrogel in those gaps. As described more fully below, the shape and size of the initial hydrogel chambers with gaps and subsequent gap filling are controlled by a computer-operated spatial energy modulation element (e.g., DMD). After the gaps are filled, the protein capture beads outside of those chambers can be removed by washing (734).Subsequently, the protein secretion profile can be determined from the protein capture beads inside those hydrogel chambers, for example, using the protocol of the bead manufacturer.

[0032] In the drawings, for convenience, the hydrogel chambers are shown as being isolated without connection to adjacent chambers and having a cylindrical or annular-like shape; however, the spatial energy modulation elements can synthesize chambers of various shapes and sizes as useful for particular applications. In some embodiments of the proliferation assay, each synthesized hydrogel chamber has the same shape and area (e.g., an annular-like having an internal area selected from the range of 0.001 mm 2 ~0.01 mm 2 ).

[0033] As used herein, "channel" means a container capable of holding a fluid (which can be in a stationary or flowing state) and having at least one surface on which beads can be placed and chambers can be synthesized. In some embodiments, the channel can have a first surface and / or a second surface on which chambers can be synthesized and / or on which beads or particles can be placed. As used herein, a reference to a "surface" without reference to either "first" or "second" is intended to include the first surface or the second surface (if both are present in a fluid device, for example, including a flow cell). In some embodiments, the channel can restrict the flow of fluid through the channel from an inlet to an outlet. In other embodiments, the channel can include a non-flowing volume of fluid that can be removed, replaced, or added by an opening or inlet; that is, in some embodiments, the channel can be a well or well-like structure.

[0034] (Systems and Equipment) A system for performing the above method is shown in FIG. 8A. The flow cell (800) is a component of a fluid device that provides one or more channels and liquid handling components under programmable control for delivering beads and reagents to those channels. In this illustration, four channels (802, 804, 806, and 808) are shown, and an enlarged view (812) of a segment (810) of channel 2 (804) is shown below. In the abstracted view of the flow cell (800) of FIG. 8A, the inlets, outlets, and other features of those channels are not shown. On the first surface (814) of channel 2 (804), a plurality of beads (e.g., (818)) are each surrounded by a hydrogel chamber (e.g., (816)). In some embodiments, the porosity of the polymer matrix walls of those hydrogel chambers is selected such that those beads are impermeable, but reagents for forming spatial barcodes are permeable. Thus, the reagents can be introduced into and removed from the interior of those hydrogel chambers by flowing the reagent through the channel (820), but the beads are retained inside. Below the enlarged view (812) of the channel segment (810), an optical system (821) for photosynthesizing hydrogel chambers at the positions of the beads in those channels is shown. Those skilled in the art will recognize that optical systems having configurations different from those of FIGS. 8A and 8B can be used to perform these functions. In some embodiments, one or more DMD-objective lens subsystems for synthesizing hydrogel structures can be used to increase the rate of synthesis by synthesizing multiple structures simultaneously.

[0035] Returning to FIG. 8A, to photosynthesize those hydrogel chambers, a light source (822) generates a light beam (823) of appropriate wavelength light (e.g., UV light), and the light beam passes through an appropriate FET mask or beam shaping or beam steering (Galvo) system to shape the beam to synthesize the desired structure or group of structures in the channel. In some embodiments, a digital micromirror device (DMD) (824) is used, and in other embodiments, a physical FET mask may be used. The position, shape, and thickness of the polymer matrix walls of the chamber are determined, at least in part, from bead position information determined from an image collected by a detector (832). The reflected light from the DMD (824) is shaped using conventional optical elements (e.g., a collimating optical element (828)) and directed through an objective lens system (834) to the channel 2 segment (810). The objective lens (834) and the flow cell (800) are moved relative to each other in the xy direction (836) to photosynthesize the chamber at any position in any of those channels. In some embodiments, the flow cell (800) moves and the optical system (821) is stationary. In some embodiments, the objective lens (834) may also direct a light beam (827) from a light source (829) to a target (e.g., a cell) on the first surface (814) and collect an optical signal (e.g., a fluorescence signal) from an assay performed at the first surface (814). Alternatively, optical signal collection may be performed using a separate objective lens as shown in FIG. 8B. The information collected by the detector (832) or its counterpart in the embodiment of FIG. 8B (particularly, the cell positions in their individual channels) is used by a computer (838) and / or an auxiliary controller to direct the relative positions of the DMD (824), the objective lens (834), and the translation device that controls the flow cell (800) to synthesize hydrogel chambers of appropriate shape and size at appropriate positions.

[0036] Figure 8B shows an alternative optical system, in which the detection portion (850) of the optical system moves (872) independently of the movement (868) of the synthesis portion (852) of the optical system. The detection portion (850) of the optical system includes a detector (856), an objective lens (858), a light source (860), and interconnecting optical elements (e.g., dichroic mirror (862)). Similar to the embodiment of Figure 8A, the detector (856) is operatively associated with a computer (864) and the synthesis portion (852) of the optical system, and provides bead position information to the synthesis portion (852). The computers (864) and (838) are also operatively associated with a stage and / or motor that controls the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the synthesis portion (852) of the optical system is located on the side opposite the detection portion (850) of the first surface (864). Similar to the embodiment of Figure 8A, it includes an objective lens (874), a mirror (876), a collimating optical element (880), a DMD (882), and a light source (878), which are conventional components.

[0037] In some embodiments, the systems described herein include: (i) a channel that includes a surface; (ii) a spatial energy modulation element that optically communicates with the surface; and (iii) a detector that optically communicates with the surface and is operably associated with the spatial energy modulation element, the detector identifying cells and determining their positions on the surface. As used herein, the term “detector” can include, but is not limited to, a microscope element that collects an image of a portion of the channel and magnifies it as needed, and an image analysis element that includes software for identifying cells and associated position information. A computer element uses such information generated by the detector, along with user input, to generate instructions for other elements (e.g., the spatial energy modulation element) to perform various functions, including, but not limited to, synthesizing chambers, “on-demand” disassembling of chambers, selectively photodegrading chambers, etc. The configuration of such embodiments is shown in FIGS. 8A-8B described above. In some embodiments, the channel of the fluid device further includes a second surface (e.g., as shown in FIG. 2), the first and second surfaces being disposed opposite each other across the channel, and the polymeric matrix walls of their chambers extending from the first surface to the second surface to form chambers each having an interior. In some embodiments, the chambers in the channel each enclose a single cell. In some embodiments, both the first and second walls are made of a light-transmissive material (e.g., glass, plastic, etc.), and they are positioned such that the first and second surfaces are substantially parallel to each other. The perpendicular distance between the first and second surfaces can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm.

[0038] In some embodiments, as shown in FIGS. 9A-9B, a plurality of channels can be aligned together in a flow channel. In some embodiments, the number of channels can be in the range of 2 to 12, or in the range of 2 to 8, or in the range of 2 to 6, or in the range of 2 to 4. A flow cell (900) is shown in cross-sectional view and top view. The flow cell (900) includes a bottom or first wall (906) having a first surface (905); an upper or second wall (902) having a second surface (901); and a spacer (904) sealingly sandwiched therebetween, with longitudinally axially holes in the spacer forming channels 1-6, one of those channels being shown in its cross-sectional view by (908) and in its top view by (912). In some embodiments, the spacer (904) can have a thickness in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, and that thickness determines the height inside the channels. The upper wall (902) has an inlet (914) and an outlet (916) for loading and removing reagents and beads into and from channels 1-6 either separately or jointly. In some embodiments, at least one of walls (902) and (906) is made of a light-transmissive material (e.g., glass, plastic, etc.). The flow cell (900) can be operatively associated with a fluid device that delivers reagents and beads to any of channels 1-6 under programmed control. Guidance for particular designs (such as fluid handling and valve operation for such fluid systems) can be found in U.S. Pat. Nos. 8,921,073; 8,173,080; 8,900,828, etc. (which are incorporated herein by reference). FIG. 9B shows the channels of the flow cell (900) together with a random distribution (not to exact scale) of hydrogel chambers having an annular cross-section (e.g., (920)) on their first surfaces.

[0039] As described above, any of the first surface, the second surface of the chamber, or the polymer matrix wall may include capture elements and other functional groups for performing various operations (including, but not limited to, bead capture, cell capture, capture of an analyte (e.g., mRNA, secreted protein, intracellular protein, or genomic sequence), capture of a component of an analytical reagent (e.g., an oligonucleotide target from an antibody), etc.). Derivatizing the surface for such purposes is well known to those skilled in the art, as shown by the following references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above), etc.

[0040] As described above, in some embodiments, the fluid device of the method includes or is operatively associated with a detector, which may share the optical path of the spatial energy modulation element, or may be positioned adjacent to the second wall, or, in embodiments having only the first wall and the first surface, adjacent to the first wall on the side opposite the spatial energy modulation element (e.g., the well). The detector is positioned such that it can detect an optical signal derived from a cell in the channel (e.g., distributed across the entire first surface in the chamber) or an optical signal proximate to the cell. In some embodiments, the first wall and the second wall each include a light-transmissive material, such that, as a result, the spatial energy modulation element can project light energy into the interior of the channel, and as a result, the detector can detect an optical signal (e.g., fluorescence emission or reflected light) derived from a biological component. In some embodiments, the energy projected from the spatial energy modulation element is light energy from a light beam. In some embodiments, the light beam projected by the spatial energy modulation element may have a complex cross-section that enables simultaneous synthesis of multiple chambers (in various embodiments). Light-transmissive materials include, but are not limited to, glass, quartz, plastic, and similar materials.

[0041] The spatial energy modulation element can use light energy for polymerization and can include a physical photomask or a virtual photomask (e.g., a digital micromirror device (DMD)). The following references, which are hereby incorporated by reference, provide guidance in selecting and operating a DMD for photopolymerizing a gel: U.S. Patent No. 10464307 to Chung et al.; U.S. Patent No. 10351819 to Hribar et al.; U.S. Patent No. 9561622 to Das et al.; Huang et al., Biomicrofluidics, 5:034109 (2011), etc.

[0042] (Hydrogel chamber) (Function). A wide variety of photosynthetic gels can be used in connection with the systems and methods described herein. In some embodiments, hydrogels are particularly used because of their compatibility with living cells and their versatility in preparing gels having desirable properties (such as porosity, which mainly determines what is contained by the gel (or polymer matrix) walls and what passes through the gel (or polymer matrix) walls, degradability, mechanical strength, ease of synthesis, and speed of synthesis, etc., but are not limited thereto) according to a method. In some embodiments, the gel or hydrogel is both photosynthetic and photodegradable. In some embodiments, the gel degradation mechanism is compatible with living cells. In some embodiments, the synthesis of the gel chamber or gel wall can be used directly (i.e., independently) or in combination with imaging to size-select cells. A hydrogel chamber or other hydrogel structure (e.g., a barrier or wall) can be synthesized such that in channels having gaps in a polymer matrix wall of a predetermined size, smaller cells can pass through those gaps while larger cells are blocked and retained on one side of those barriers or walls.

[0043] (Porosity). In some embodiments, the porosity of the hydrogel is selected to allow passage of a selected reagent while preventing passage of other reagents or objects (e.g., cells). In some embodiments, the porosity of the hydrogel is selected to prevent passage of biological cells while allowing passage of reagents (including proteins such as polymerases). In some embodiments, such reagents that are permeable through the polymer matrix wall include lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, deoxynucleotide triphosphates, buffers, cell culture media, or divalent cations. In some embodiments, at least one of the polymer matrices includes pores, and the pores are sized to allow diffusion of reagents through at least one of the polymer matrices, but are too small for DNA or RNA for analysis to pass through the pores (having a size greater than 100 nucleotides or 100 base pairs, or greater than 300 nucleotides or 300 base pairs). In some embodiments, cross-linking the polymer chains of the hydrogel structure forms a hydrogel matrix having pores (i.e., a porous hydrogel matrix). In some versions, the size of the pores in these hydrogel structures can be adjusted or regulated and encapsulate sufficiently large genetic material (e.g., cells or nucleic acids (e.g., greater than about 300 base pairs)), while smaller substances (e.g., reagents) or nucleic acids of smaller size (e.g., less than about 50 base pairs) (e.g., primers) can pass through the pores, and thus can be prepared in a manner to pass in and out through these hydrogel structures. In some embodiments, the hydrogels can have any pore size that has a diameter sufficient to allow diffusion of the reagents listed above through their structures while retaining nucleic acid molecules of a length greater than 500 nucleotides or 500 base pairs. In some embodiments, the hydrogel structure can swell when the hydrogel is hydrated.Subsequently, the size of those pores can vary depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, those pores have a diameter of about 10 nm to about 100 nm. In some embodiments, the pore size of those hydrogel structures is adjusted by varying the ratio of the polymer precursor concentration to the crosslinker concentration, various pH values, salt concentrations, temperatures, light intensities, etc. by conventional experiments. In some embodiments, the average diameter of the pores in the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kilodaltons (kDa) or more; or having a molecular weight of 50 kDa or more; or having a molecular weight of 75 kDa or more; or having a molecular weight of 100 kDa or more; or having a molecular weight of 150 kDa or more.

[0044] In some embodiments, the DNA or RNA to be retained has a length that can be sequenced using conventional sequencing-by-synthesis techniques. For example, such DNA or RNA can include at least 50 nucleotides, or in some embodiments at least 100 nucleotides. In some embodiments, those pores can have an average diameter of 5 nm to 100 nm. In some embodiments, those pores can have an average diameter of 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, those pores can have an average diameter greater than 100 nm. In some embodiments, those pores can have an average diameter less than 5 nm. The reagent can include an enzyme or a primer having a size less than 50 base pairs (bp). The primer can include single-stranded DNA (ssDNA). In some embodiments, the primer can have a size of 5 bp to 50 bp. In some embodiments, the primer can have a size of 5 bp to 10 bp, 10 bp to 20 bp, 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, the primer can have a size greater than 50 bp. In certain cases, the primer can have a size less than 5 bp. In some embodiments, those pores can have a diameter of 5 nm to 100 nm. In some embodiments, those pores can have a diameter of 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, those pores can have a diameter greater than 100 nm. In some embodiments, those pores can have an average diameter less than 5 nm. The polymer matrix can have a pore size of about 5 nanometers (nm) to about 100 nm.The polymer matrix can have a pore size of from about 5 nm to about 10 nm, from about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, from about 5 nm to about 100 nm, from about 5 nm to about 110 nm, from about 10 nm to about 20 nm, from about 10 nm to about 30 nm, from about 10 nm to about 40 nm, from about 10 nm to about 50 nm, from about 10 nm to about 60 nm, from about 10 nm to about 70 nm, from about 10 nm to about 80 nm, from about 10 nm to about 90 nm, from about 10 nm to about 100 nm, from about 10 nm to about 110 nm, from about 20 nm to about 30 nm, from about 20 nm to about 40 nm, from about 20 nm to about 50 nm, from about 20 nm to about 60 nm, from about 20 nm to about 70 nm, from about 20 nm to about 80 nm, from about 20 nm to about 90 nm, from about 20 nm to about 100 nm, from about 20 nm to about 110 nm, from about 30 nm to about 40 nm, from about 30 nm to about 50 nm, from about 30 nm to about 60 nm, from about 30 nm to about 70 nm, from about 30 nm to about 80 nm, from about 30 nm to about 90 nm, from about 30 nm to about 100 nm, from about 30 nm to about 110 nm, from about 40 nm to about 50 nm, from about 40 nm to about 60 nm, from about 40 nm to about 70 nm, from about 40 nm to about 80 nm, from about 40 nm to about 90 nm, from about 40 nm to about 100 nm, from about 40 nm to about 110 nm, from about 50 nm to about 60 nm, from about 50 nm to about 70 nm, from about 50 nm to about 80 nm, from about 50 nm to about 90 nm, from about 50 nm to about 100 nm, from about 50 nm to about 110 nm, from about 60 nm to about 70 nm, from about 60 nm to about 80 nm, from about 60 nm to about 90 nm, from about 60 nm to about 100 nm, from about 60 nm to about 110 nm, from about 70 nm to about 80 nm, from about 70 nm to about 90 nm, from about 70 nm to about 100 nm, from about 70 nm to about 110 nm, from about 80 nm to about 90 nm, from about 80 nm to about 100 nm, from about 80 nm to about 110 nm, from about 90 nm to about 100 nm, from about 90 nm to about 110 nm, or from about 100 nm to about 110 nm. The polymer matrix can have a pore size of about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, or about 110 nm.The polymer matrix can have pore sizes of at least about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm or less. The polymer matrix can have pore sizes of up to about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm or more.

[0045] (Size and Shape of the Hydrogel Chamber). In some embodiments, the polymeric matrix wall of the chamber blocks the passage of certain components (e.g., mammalian cells, bacterial cells, genomic DNA, larger polynucleotides (e.g., mRNA larger than 200 ribonucleotides, or larger than 300 ribonucleotides, or larger than 500 ribonucleotides), etc.). In some embodiments, the polymeric matrix wall extends from its first surface to a second surface (parallel to its first surface) to form a chamber within the channel. In some embodiments, the chamber has a polymeric matrix wall and an interior. In some embodiments, the interior of the chamber is sized to surround cells (e.g., mammalian cells). For example, such a chamber can include a cylindrical shell or a polygonal shell, including an internal space or interior, and a polymeric matrix wall. In some embodiments, such a chamber has an annulus-like cross-section. As used herein, the term "annulus-like cross-section" means a cross-section that is morphologically equal to an annulus. In some embodiments, the internal space or interior of the chamber has an inner diameter in the range of 1 μm to 500 μm and a volume in the range of 1 picoliter to 200 nanoliters, or 100 picoliters to 100 nanoliters, or 100 picoliters to 10 nanoliters. In some embodiments, the polymeric matrix wall has a thickness of at least 1 μm (micrometer). In some embodiments, the height of the chamber having an annulus-like cross-section has a value in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm. In some embodiments, the polymeric matrix wall having an annulus-like cross-section has an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, the aspect ratio and the thickness of the polymeric matrix wall are selected to maximize the stability of the chamber against forces (e.g., reagent flow through the channel, washing, etc.). In some embodiments, the at least one polymeric matrix wall is a hydrogel wall. In some embodiments, the at least one polymeric matrix is degradable.In some embodiments, the degradation of at least one of the polymer matrices is “on demand”. In some embodiments, the groups of chambers in the channels are not adjacent. In some embodiments, the groups of chambers in the channels can be adjacent to neighboring chambers. In some embodiments, the groups of chambers can share the polymer matrix walls with each other. In some embodiments, the chambers can be fabricated with slits or other orifices that are large enough to allow passage of certain components (e.g., beads) but small enough to prevent passage of other components (e.g., cells).

[0046] (Hydrogel composition). As noted above, the hydrogel composition can vary widely and the hydrogel can be formed in a variety of ways. Examples of biocompatible hydrogel precursors include, but are not limited to, hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, poly(ethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA), and poly(vinyl alcohol). In some embodiments, the hydrogel is formed by photoinitiated free radical crosslinking. In some embodiments, the hydrogel is formed by photoinitiated thiol-ene reaction.

[0047] (Hydrogel degradation). In some embodiments, the hydrogel chamber is degradable or depolymerizable either generally within the channel or “on demand” within the channel. A generally degradable hydrogel chamber is degraded by treatment with a degrading agent or equivalently a depolymerizing agent that is exposed to all chambers within the channel. Depolymerizing agents can include, but are not limited to, heat, light, and / or chemical depolymerization reagents (sometimes also referred to as cleavage reagents or degrading reagents). In some embodiments, on-demand degradation can be carried out using a polymer precursor that enables photocrosslinking and photodegradation, for example, using different wavelengths for crosslinking and for degradation. For example, eosin Y can be used for radical polymerization in a defined area using a 500 nm wavelength, and then irradiation at 380 nm can be used to cleave its crosslinking agent. In other embodiments, photo-caged hydrogel cleavage reagents can be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinking agent (such as an ester, etc.) can be used to generate the hydrogel, and then UV light can be used to generate local acidic conditions, and then the local acidic conditions can degrade the hydrogel. In some embodiments, the at least one polymer matrix is degradable by at least one of the following: (i) contacting the at least one polymer matrix with a cleavage reagent; (ii) heating the at least one polymer matrix to at least 90 °C; or (iii) exposing the at least one polymer matrix to light of a wavelength that cleaves a photocleavable crosslinking agent that crosslinks the polymer of the at least one polymer matrix. In some embodiments, the at least one polymer matrix includes a hydrogel. In some embodiments, the cleavage reagent degrades the hydrogel. In some embodiments, the cleavage reagent includes a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage reagent, or a combination thereof.In some embodiments, the cleavage reagent may include dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydrogel can be functionalized by linking a functional group to the polymer matrix or hydrogel. Some non-limiting examples of functional groups include capture reagents (e.g., pyridinecarboxaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linker, functional groups containing aldehyde, phosphate, silicate, ester, acid, amide, aldehyde dithiolane, PEG, thiol, alkene, alkyne, azide, or a combination thereof. In some cases, the functionalized polymer matrix can be used to capture biomolecules within a polymer matrix compartment formed in proximity to (e.g., surrounding or on) its biological component. The biomolecule can be produced by its biological component (e.g., the secretome derived from cells). The functionalized surface of the polymer matrix within the compartment can be used to capture reagents or molecules from outside the compartment. The functionalized surface can increase the surface area covered by a reagent, molecular sensor, or any molecule of interest (e.g., an antibody).

[0048] (Photosynthesis). In some embodiments, the generation of a polymer matrix within a channel or well of a fluid device involves exposing one or more of their polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In some embodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymer matrix within the fluid device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulation element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a galvanometer-guided laser beam. In some embodiments, the SLM is liquid crystal-based.

[0049] Although the present invention has been described with reference to several specific exemplary embodiments, those skilled in the art will recognize that many changes can be made thereto without departing from the spirit and scope of the present invention. The present invention is applicable to various sensor implementations and other subjects in addition to the above-described subject matter.

Examples

[0050] (Example: Sorting of CD56-expressing NK cells from a mixed NK / Jurkat population) In this example, CD56-high-expressing NK cells are sorted from a mixed NK / Jurkat cell population. Exemplary results are shown in FIG. 10A. A mixed population of NK cells and Jurkat cells is loaded into a flow cell such that the cells are placed on the surface (1000), as shown in field (1001), and then a hydrogel chamber is synthesized using cPEG (described more fully below) around the four cells shown. The cells are stained with an anti-CD56 PE-labeled antibody (1002), and cells expressing high levels of CD56 (1004) are identified by fluorescence intensity. The CD56-high-expressing cells are also surrounded by cSEL (1008, described more fully below), a second orthogonally-degradable hydrogel, and then all of their cPEG hydrogels are degraded using glutathione (GSH) (1006) as described below. Cells not surrounded by the cSEL chamber are removed by washing (1010). Those CD56-high-expressing cells are identified (1011) and then (i) they are surrounded (1012) in another cPEG chamber (1013), (ii) the cSEL hydrogel is degraded, and (iii) other cells are removed by washing (1014), leaving the CD56-high-expressing cells (e.g., (1016)).

[0051] The materials and methods used in this example were as follows:

[0052] (Cells): Jurkat cells and NK cells were cultured in separate tissue culture flasks, centrifuged at 200 rcf, and resuspended at a cell density of 10 million cells / mL in RPMI medium (obtained from Gibco) supplemented with 10% FBS and 1× antibiotic-antimycotic solution (obtained from Gibco).

[0053] (cPEG Chamber Synthesis): The cPEG gel stock solution was prepared by dissolving the cPEG macromonomer (1018, Figure 10B) in PBS. To generate 40 μL of its precursor solution, 6 μL of the cPEG stock solution was mixed with 2 μL of polyethylene glycol (PEG, 20 kDa) in PBS solution, 4 μL of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in PBS solution, 4 μL of cell suspension, and 24 μL of PBS. The gel solution was loaded into the flow cell, and a single cell group was caged using a Polygon 1000 DMD system (Mightex). Then, the uncrosslinked polymer solution was washed with PBS.

[0054] (CD56-PE Staining): The antibody solution was generated by adding 5 μL of CD56-PE stock solution to 100 μL of PBS. Then, the antibody solution was loaded into the flow cell, and the flow cell was incubated at 37 °C. Then, the antibody solution was washed with PBS, and the flow cell was incubated at 37 °C. Bright-field images and green fluorescence images of the cells were taken at 10× magnification using exposure times of 10 ms and 300 ms, respectively.

[0055] (cSEL Chamber Synthesis): The cSEL gel stock solution was prepared by dissolving the cSEL macromonomer (1020, Figure 10B) in PBS. To generate 40 μL of its precursor solution, 20 μL of the cSEL stock solution was mixed with 4 μL of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) in PBS solution, and 16 μL of PBS. The gel solution was loaded into the flow cell, and the selected cells were caged using the above DMD and irradiation parameters. Then, the uncrosslinked polymer solution was washed with PBS.

[0056] (cPEG gel dissolution): A 10 mM L - glutathione (GSH) solution in PBS was prepared and its pH was adjusted to 8.0 using sodium hydroxide. The GSH solution was loaded into a flow cell and the flow cell was incubated at room temperature for 5 minutes to dissolve the cPEG gel. Then, the flow cell was washed three times with PBS.

[0057] (cPEG secondary chamber synthesis): A cPEG gel stock solution was prepared by dissolving the cPEG macromonomer (1018, Figure 10B) in PBS. Then, to generate 40 μL of its precursor solution, 6 μL of the cPEG stock solution was mixed with 2 μL of polyethylene glycol (PEG, 20 kDa) in PBS solution, 4 μL of lithium phenyl - 2,4,6 - trimethylbenzoylphosphinate (LAP) in PBS solution, and 28 μL of PBS. The gel solution was loaded into a flow cell and the selected cells were caged using the above - mentioned DMD.

[0058] (cSEL gel dissolution): Alginase was dissolved in PBS. The lyase solution was loaded into a flow cell and the flow cell was incubated at room temperature for 5 minutes to dissolve the cSEL gel. Then, the flow cell was washed three times with PBS to remove the lyase solution.

[0059] In some embodiments, the above-described method for sorting cells by multiple optical criteria can be performed in the following steps: (a) synthesizing one or more first gel structures disposed on the surface of the channel and fixing one or more cells selected by a first optical property; (b) synthesizing one or more second gel structures surrounding the one or more cells fixed by the first gel structures, wherein the one or more cells surrounded by the second gel structures are selected by a second optical property; (c) decomposing the first gel structures; and (d) removing the cells released by the step of decomposing the first gel structures such that one or more cells selected by the first optical property and the second optical property remain on the surface of the channel. In some embodiments, such steps may further include decomposing the second gel structures and eluting one or more cells selected by the first optical property and the second optical property from the channel. In some embodiments, the first gel structures and the second gel structures are orthogonally decomposable; that is, the first gel structures can be decomposed in the presence of the second gel structures without a recognizable effect on the second gel structures, and similarly, the second gel structures can be decomposed in the presence of the first gel structures without a recognizable effect on the first gel structures. In some embodiments, the first gel structures and the second gel structures are orthogonally decomposable by having various modes of degradability selected from the following: chemical degradability (e.g., by reduction of disulfide bonds), enzymatic degradability, degradability by temperature change, and photodegradability. In some embodiments, the multiple optical criteria for selecting a subset of cells according to the above-described method include optical signal intensity monotonically related to cell size, the size ratio of nucleolus to cytoplasm, migration speed, motility, cell death, cell shape, surface protein expression, and optical signal intensity monotonically related to protein secretion.In certain embodiments, the plurality of optical properties includes an optical signal intensity that is monotonically related to a migration rate and surface protein expression. In certain embodiments, the plurality of optical properties includes an optical signal intensity that is monotonically related to cell death and surface protein expression. Typically, such optical signal intensity can be generated by a labeled antibody specific for a given surface protein or a labeled antibody specific for a given secreted protein. In some embodiments, the optical intensity includes fluorescence intensity.

[0060] (Definition) Unless specifically defined otherwise herein, the nucleic acid chemistry, biochemistry, genetics, and molecular biology terms and symbols used herein shall conform to those used in standard treatises and textbooks in the art, such as Kornberg and Baker, DNA Replication, 2nd ed. (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immunology, 6th ed. (Saunders, 2007).

[0061] In some embodiments, an “assay” refers to a process for detecting or measuring the cell characteristics or properties of a single cell population or a group of cells. Typically, the process steps of an assay include chemical reactions, biochemical reactions or molecular reactions (e.g., bond cleavage, specific binding of complementary components, enzyme reactions, degradation of complementary components, etc.) or physical state changes (e.g., increase or decrease in temperature, change in energy level, etc.), resulting in the generation of a signal (or group of signals) that can infer the presence, absence, or quantity associated with the cells. The nature of the signals generated by the assay can vary widely and can include, but is not limited to, electrical signals, optical signals, chemical signals, or substance signals. Substance signals include the generation of substances containing information that can be extracted. For example, a substance signal can be the amplification of a polynucleotide whose length, quantity, composition, or nucleotide sequence indicates cell characteristics. For example, a barcode oligonucleotide can be a substance signal. The cell characteristics or properties that can be detected or measured can vary widely and can include, but are not limited to, cytotoxicity, viability, proliferation ability under selected conditions, size, shape, motility, types and profiles of cell surface proteins, or types and profiles of cell membrane proteins, types and profiles of secreted proteins, production of metabolites, transcriptome, gene copy number, identity of genes or alleles, chromatin accessibility profile, vector copy number for engineered cells or vector copy number for infected cells, etc.

[0062] As used herein, "cell" refers to a biological cell that can be assayed by the methods and systems described herein, including but not limited to vertebrate cells, invertebrate cells, eukaryotic cells, mammalian cells, microbial cells, protozoan cells, prokaryotic cells, bacterial cells, insect cells, or fungal cells. In some embodiments, mammalian cells are assayed by the methods and systems described herein. Specifically, any mammalian cell that can be or has been genetically modified for use in medical, industrial, environmental, or therapeutic processes can be analyzed by the methods and systems described herein. In some embodiments, "cell" as used herein includes genetically modified mammalian cells. In some embodiments, "cell" includes stem cells. In some embodiments, "cell" refers to a cell modified by CRISPR Cas9 technology. In some embodiments, "cell" refers to cells of the immune system, including but not limited to cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen-presenting cells, or dendritic cells. Cytotoxic T lymphocytes engineered for therapeutic applications (e.g., cancer therapy) are of particular interest.

[0063] "Hydrogel" means a gel containing a crosslinked hydrophilic polymer network that has the ability to absorb and retain large amounts of water (e.g., 60% - 90% water, or 70% - 80%) without dissolving, due to the establishment of physical or chemical bonds (which can be covalent, ionic, or hydrogen bonds) between its polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, which makes them attractive materials for cell encapsulation applications and cell culture applications. Hydrogels may contain natural polymers or synthetic polymers, and may be reversible (i.e., degradable or depolymerizable) or irreversible. Synthetic hydrogel polymers can include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Natural hydrogel polymers can include alginate, hyaluronic acid, and collagen. The following references describe hydrogels and their biomedical uses: Drury et al., Biomaterials, 24:4337 - 4351 (2003); Garagorri et al., Acta Biomatter, 4(5):1139 - 1147 (2008); Caliari et al., Nature Methods, 13(5):405 - 414 (2016); Bowman et al., U.S. Patent No. 9631092; Koh et al., Langmuir, 18(7):2459 - 2462 (2002).

[0064] "On demand" means that the operation can be directed at individual, separate, selected locations (e.g., the spatial location of a polymer precursor solution; or a selected polymer matrix chamber). Such selection can be based on manual observation of optical signals or data collected by a detector, or such selection can be based on computer algorithm manipulation of optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include either real-time detection or detection over a certain period of time, before modulating a unit of energy to polymerize a polymer precursor or decompose a chamber. For example, a subset of chambers (all formed with a photodegradable polymer matrix wall) can be pre-selected to release and remove the contents of those chambers based on location information and the value of the optical signal from the analytical assay performed in those chambers. Those pre-selected chambers can be photodecomposed by selectively projecting a light beam of appropriate wavelength characteristics (e.g., with a spatial energy modulation element) to decompose the polymer matrix walls of those pre-selected chambers. In another example, multiple chambers can be observed in real-time (e.g., by fluorescence microscopy) for detection of a target analyte, and one or more of those multiple chambers can be selected in real-time for decomposition upon detection of that target analyte.

[0065] "Polymer matrix" generally refers to a phase material (e.g., a continuous phase material) containing at least one polymer. In some embodiments, the polymer matrix refers to at least one polymer as well as the interstitial space not occupied by the polymer. The polymer matrix can be composed of one or more types of polymers. The polymer matrix can include linear polymer units, branched polymer units, and cross-linked polymer units. The polymer matrix can also include non-polymer species inserted within the interstitial space not occupied by the polymer chains. The inserted species can be a solid species, a liquid species, or a gaseous species. For example, the term "polymer matrix" can include dry hydrogels, hydrated hydrogels, and glass fiber-containing hydrogels. The polymer matrix can include a polymer precursor, which generally refers to one or more molecules that can induce or initiate a polymer reaction upon activation. The polymer precursor can be activated by electrochemical energy, photochemical energy, photons, magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which polymerize to form the polymer matrix) and cross-linking compounds, which can include other compounds necessary or useful for forming a photopolymerization initiator, a polymer matrix (particularly a polymer matrix that is a hydrogel).

[0066] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided herein. Although the invention has been described in connection with the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Many variations, modifications, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it is understood that all aspects of the invention are not limited to the specific descriptions, configurations, or relative ratios shown herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the invention described herein may be used in practicing the invention. Accordingly, it is intended that the invention also cover any such alternatives, modifications, variations, or equivalents. It is intended that the appended claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered by the invention.

Claims

1. A method for selecting multiple cells, (a) Loading the plurality of cells into a fluid device such that the plurality of cells are positioned on the surface of the fluid device or in close proximity to the surface of the fluid device; (b) A step of introducing one or more polymer precursors into the fluid device; (c) A step of selecting a subset of cells from a plurality of cells based on one or more optical signals derived from the subset of cells; (d) A step of determining the location of a subset of cells in a fluid device using a detector, wherein the detector is in optical communication with the fluid device; (e) A step of surrounding one or more cells of a subset of cells in the fluid device by synthesizing one or more gel structures, wherein the synthesis is performed by projecting light into the fluid device with a spatial energy modulation element such that projected light causes polymerization of the one or more polymer precursors to form the one or more gel structures surrounding the one or more cells, and the position of the one or more gel structures in the fluid device is determined by the position of the subset of cells determined by the detector in (d); (f) The step of removing any unselected cells from the fluid device; (g) A step of performing an assay on the one or more cells surrounded by the one or more gel structures to obtain assay results for each of the one or more cells surrounded by the one or more gel structures; and (h) The step of relating the assay results to the location of the subset of cells determined in (d). Methods that include...

2. The method according to claim 1, further comprising a step of repeating steps (a) to (f).

3. The method according to claim 2, wherein steps (a) to (f) are repeated until one or more surrounded cells reach a predetermined density on the surface.

4. The method according to claim 1, further comprising the steps of: selecting an additional cell subset of a plurality of cells based on one or more additional optical signals derived from an additional cell subset; determining the location of the additional cell subset in a fluid device using the detector; and surrounding the one or more additional cells of the additional cell subset by synthesizing one or more additional gel structures in the fluid device surrounding one or more additional cells of the additional cell subset, wherein the synthesis is performed by projecting light into the fluid device with the spatial energy modulation element such that the projected light causes polymerization of one or more polymer precursors to form the one or more additional gel structures, and the location of the one or more additional gel structures in the fluid device is determined by the location of the additional cell subset.

5. The method according to claim 1, further comprising the step of dissolving at least a portion of the one or more gel structures surrounding the one or more cells, thereby eluting the one or more cells from the fluid device.

6. The method according to claim 1, wherein the one or more optical signals indicate cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell migration speed, cell replication speed, or protein secretion.

7. The method according to claim 1, wherein the one or more optical signals indicate protein secretion from a subset of the cells.

8. The method according to claim 7, wherein the protein secretion is cytokine secretion.

9. The method according to claim 6, wherein the cell replication rate is determined based on one or more optical signals by i) counting the offspring of one or more cells of a subset of the cells, ii) diluting an intracellular dye, or iii) a combination thereof.

10. The method according to claim 1, wherein the one or more optical signals indicate the cell migration speed.

11. The method according to claim 10, wherein the cell migration velocity is determined by a series of images in which the cell position is recorded as a function of time.

12. The method according to claim 1, wherein the one or more optical signals are collected using the detector.

13. The method according to claim 1, wherein the step of loading the plurality of cells into the fluid device in (a) is performed simultaneously with the step of introducing one or more polymer precursors into the fluid device in (b).

14. The method according to claim 1, wherein the one or more gel structures include photochemically degradable bonds.

15. The method according to claim 1, wherein the one or more gel structures include chemically decomposable bonds.

16. The method according to claim 15, wherein the chemically decomposable bond includes a disulfide bond.

17. The method according to claim 1, wherein the one or more gel structures include a hydrogel chamber with an interior.

18. The method according to claim 1, wherein the subset of cells is of a first cell type and the unselected cells are of a second cell type.

19. The method according to claim 18, wherein both the first cell type and the second cell type are immune cells.

20. A method for selecting multiple cells, (a) Loading the plurality of cells into a fluid device such that the plurality of cells are positioned on the surface of the fluid device or in close proximity to the surface of the fluid device; (b) A step of introducing one or more polymer precursors into the fluid device; (c) A step of selecting a subset of cells from a plurality of cells based on one or more optical signals derived from the subset of cells; (d) A step of determining the location of a subset of cells in a fluid device using a detector, wherein the detector is in optical communication with the fluid device; (e) A step of surrounding one or more cells of a subset of cells by synthesizing one or more gel structures in the fluid device, wherein the synthesis is performed by projecting light into the fluid device with a spatial energy modulation element such that projected light causes polymerization of the one or more polymer precursors to form the one or more gel structures surrounding the one or more cells, the position of the one or more gel structures in the fluid device is determined by the position of the subset of cells determined by the detector in (d), and the one or more gel structures include photochemically degradable bonds; and (f) A step of removing any unselected cells from the plurality of cells from the fluid device; Methods that include...

21. The method according to claim 20, further comprising the step of repeating steps (a) to (f) until one or more surrounded cells reach a predetermined density on the surface.

22. The method according to claim 21, wherein the one or more optical signals indicate cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell migration rate, cell replication rate, or protein secretion.

23. A method for selecting multiple cells, (a) Loading the plurality of cells into a fluid device such that the plurality of cells are positioned on the surface of the fluid device or in close proximity to the surface of the fluid device; (b) A step of introducing one or more polymer precursors into the fluid device; (c) A step of selecting a subset of cells from a plurality of cells based on one or more optical signals derived from the subset of cells; (d) A step of determining the location of a subset of cells in a fluid device using a detector, wherein the detector is in optical communication with the fluid device; (e) A step of surrounding one or more cells of a subset of cells in the fluid device by synthesizing one or more gel structures, wherein the synthesis is performed by projecting light into the fluid device with a spatial energy modulation element such that projected light causes polymerization of the one or more polymer precursors to form the one or more gel structures surrounding the one or more cells, and the position of the one or more gel structures in the fluid device is determined by the position of the subset of cells determined by the detector in (d); (f) A step of removing any unselected cells from the plurality of cells from the fluid device; (g) A step of selecting an additional subset of cells from a plurality of cells based on one or more additional optical signals derived from the additional cell subset; (h) Using the detector to determine the location of the additional subset of cells in the fluid device; and (i) A step of surrounding one or more additional cells of the additional cell subset by synthesizing one or more additional gel structures in a fluid device surrounding one or more additional cells of the additional cell subset, wherein the synthesis is performed by projecting light into the fluid device with a spatial energy modulation element such that the projected light causes polymerization of the one or more polymer precursors to form the one or more additional gel structures, and the position of the one or more additional gel structures in the fluid device is determined by the position of the additional cell subset. Methods that include...

24. The method according to claim 23, wherein the one or more optical signals indicate cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell migration speed, cell replication speed, or protein secretion.

25. The method according to claim 23, wherein the step of loading the plurality of cells into the fluid device in (a) is performed simultaneously with the step of introducing the one or more polymer precursors into the fluid device in (b).

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