Cell sorting and selection using degradable hydrogels

Abstract

Described herein are systems and methods for sorting and selecting cells by immobilizing or encapsulating cells in a degradable hydrogel chamber, including a method of sorting cells comprising: (a) providing a fluidic device comprising: (i) a channel comprising a surface, (ii) a spatial energy modulating element in optical communication with the surface, and (iii) a detector in optical communication with the surface and operatively associated with the spatial energy modulation element; (b) loading cells and one or more polymer precursors to the channel such that the cells are disposed on or near the surface; (c) immobilizing one or more cells selected based on one or more optical signals from the one or more cells by synthesizing one or more gel structures encapsulating each of the one or more cells; and (d) removing unselected cells from the channel.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 350,813, filed on June 9, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Due to its high information content, image-based cell analysis and sorting has attracted great interest in many fields including cancer diagnosis, drug discovery, immunocytochemistry, phenotypic-genotypic analysis, etc., for example, 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 (2017). 1994). As LaBelle points out, current image-based sorting systems broadly follow three approaches: (i) imaging during flow through a microfluidic system, (ii) imaging after capture or containment in a microfluidic system, and (iii) imaging in a microwell array. These three approaches have significant tradeoffs between important performance parameters such as throughput, scalability, and power. For example, imaging during flow requires complex high-speed optics and processing. Microfluidic capture and containment avoids the imaging challenges of flow systems but reduces throughput and flexibility. Microwell array systems allow excellent image collection but have low throughput. Summary of the invention

[0004] In view of the above, image-based selection and sorting will be advanced by the availability of image-based technologies that have the advantages of microarray-based systems but are easily scalable and have flexible containment and sorting capabilities.

[0005] In one aspect, a method for selecting or sorting cells using a degradable gel structure is described herein. In one aspect, a method for sorting cells is described herein, the method comprising: (a) providing a fluid device, the fluid device comprising: (i) a channel including a surface, (ii) a spatial energy modulation element optically connected to the surface, and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector identifying the cell and determining the position of the cell on the surface; (b) loading cells and one or more polymer precursors to the channel so that the cells are arranged on the surface or arranged near the surface; (c) by projecting light into the channel with a spatial energy modulation element so that the projected light causes one or more polymer precursors to crosslink to form a gel structure, one or more gel structures are synthesized and one or more cells selected based on one or more optical signals are fixed, the one or more optical signals are from the one or more cells, the one or more gel structures encapsulate each of the one or more cells, wherein the position of the gel structure in the channel is determined by the position of the cell encapsulated by the gel structure identified by the detector; and (d) removing unselected cells from the channel. In some cases, the method also includes repeating steps (b) to (c). In some embodiments, step (b) to step (c) are repeated until the fixed cells reach a predetermined density on the surface. In some embodiments, the method also includes degrading the gel structure of the fixed cells and eluting the selected cells from the channel. In some embodiments, one or more optical signals indicate cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell mobility, cell replication rate or protein secretion. In some specific embodiments, one or more optical signals include a series of images, from which cell mobility can be determined. For example, from a series of surface images, cell position can be recorded as a function of time, so that mobility or speed can be determined. In other specific embodiments, one or more optical signals include a series of images, from which cell-cell interactions, such as cell killing, can be determined or quantified. In some embodiments, the gel structure includes a photochemically degradable linkage. In some embodiments, the gel structure includes an enzymatically degradable linkage. In some embodiments, the gel structure includes a chemically degradable linkage, such as a disulfide bond. In some embodiments, the gel structure includes a hydrogel chamber, and the hydrogel chamber each has an interior. In some specific embodiments, cells are captured or contained in such interiors. In some embodiments, the hydrogel chambers each have a toroidal-like shape.In some embodiments, the gel structure comprises a solid mass that encapsulates the cells.

[0006] In another aspect, the present invention describes a method for measuring cells from multiple samples, the method comprising: (a) providing a fluid device comprising: (i) a channel comprising a surface, (ii) a spatial energy modulation element optically connected to the surface, and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector identifying the cell and determining the position of the cell on the surface; (b) loading the channel with cells of the sample and one or more polymer precursors so that the cells are arranged on the surface or near the surface; (c) synthesizing one or more gel structures by projecting light into the channel using the spatial energy modulation element so that the projected light causes the one or more polymer precursors to cross-link to form a gel structure, the one or more gel structures encapsulating each of the one or more cells of the sample, wherein the position of the synthesized gel structure in the channel is determined by the position of the cell encapsulated by the synthesized gel structure identified by the detector; (d) repeating steps (b) and (c) for each sample in the multiple samples; and (e) measuring the cells in the channel to obtain a measurement result for each of the cells encapsulated by the gel structure. In some embodiments, the method further comprises determining the assay result for each of the samples by correlating the assay result with the location of cells of the samples.In some embodiments, the gel structure is a hydrogel chamber.

[0007] In another aspect, a method for sorting cells is provided herein, the method comprising: (a) providing a fluidic device comprising: (i) a channel comprising a surface, one or more polymer precursors and one or more cells, (ii) a spatial energy modulation element optically connected to the surface, and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element; (b) identifying the location of a subset of one or more cells using the detector, wherein the subset of one or more cells is selected based on one or more optical signals from the subset of one or more cells; (c) control system Spatial energy modulation element Based on the location of one or more subsets of cells identified by the detector Energy is projected into the channel such that the projected energy causes one or more polymer precursors to form one or more gel structures that encapsulate one or more subsets of cells, and wherein one or more cells that are not in the one or more subsets of cells are not encapsulated by the one or more gel structures; and (d) one or more cells that are not in the one or more subsets of cells are removed from the channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A-1B Embodiments of the systems and methods described herein for selecting and sorting cells are shown.

[0009] Figure 2A device for detecting and encapsulating desired cells in a gel structure is shown.

[0010] Figure 3 An embodiment is shown in which cells are captured on the surface at regularly spaced locations.

[0011] Figure 4A-4B An embodiment for sorting cells that secrete a selected protein at high levels is shown.

[0012] Figure 5A-Figure 5B An embodiment for sorting cells with a high growth rate is shown.

[0013] Figure 6A-6B An embodiment for sorting cells with high mobility is shown.

[0014] Figure 7A-7B An embodiment for assay multiplexing is shown. Figure 7C An embodiment is shown in which cells are encapsulated by a hydrogel chamber comprising two different orthogonal degradable hydrogels.

[0015] Figure 8A-8B Apparatus for implementing embodiments of the systems and methods described herein is shown.

[0016] Figure 9A-9B Shown is a multichannel flow cell for use with embodiments of the systems and methods described herein.

[0017] Figure 10A-10B Data related to the use of two orthogonal degradable hydrogels to select and sort specific cells based on optical properties are shown. DETAILED DESCRIPTION

[0018] Unless otherwise stated, the practice of the systems and methods described herein can adopt the conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant technology), cell biology and biochemistry within the technical scope of the art. Such conventional techniques include, but are not limited to, preparing synthetic polynucleotides, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, etc. By referring to the examples below this article, suitable techniques can be specifically described. However, other equivalent routine procedures can also be used, of course. Such conventional techniques and descriptions can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Volumes I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Renault and Duchateau, ed., Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer, ed., Protein Engineering Handbook (Wiley-VCH, 2009); etc. Guidance for selecting materials and components to perform particular functions can be found in available treatises and references on scientific instrumentation, including but not limited to Moore et al., Building Scientific Apparatus, 3rd ed. (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd ed. (Academic Press, 2013); and similar references.

[0019] Methods and systems described herein can use degradable gel to select, sort and separate cells, especially based on optically measurable properties. In one aspect, single cells with desired properties are fixed by synthesizing a degradable gel structure that encapsulates or encapsulates cells of interest, and sorting, selection and separation are achieved. In some embodiments, whenever a desired number of cells with desired properties are accumulated (and unselected cells are removed), the encapsulation or encapsulation gel structure can be degraded to release the selected cells. The following will describe the equipment for performing such procedures in more detail. In short, in some embodiments, cells combined with polymer precursors are loaded into a flow cell channel so that cells are arranged on the surface and detected (possibly after the determination step), and then the selected cells (for example, based on the determination results) are encapsulated in a degradable gel structure synthesized by light energy to fix the selected cells on the surface. (In some embodiments, cells and polymer precursors can be loaded into the channel in order, for example, cells can be loaded and arranged on the channel surface, and then polymer precursors are loaded). Remove unselected cells, then degrade the gel structure of the selected cells and recycle the selected cells. Sorting and selection can be based on a variety of properties measured from images and / or assay results, including but not limited to size, morphology, organelle identity, motility, mobility, surface protein expression, secreted protein profiles, cell-cell interactions, and the like.

[0020] In another aspect, sorting, selection, and separation are achieved by immobilizing single cells that do not possess the desired properties via a synthetic gel structure that encapsulates or encapsulates such cells but leaves cells that possess the desired properties free to elute from the flow cell.

[0021] On the other hand, multiple cell samples can be processed or subjected to the same determination in the same flow cell channel at the same time. In some embodiments of this aspect, multiple cell samples can be arranged on the same surface in sequence. After loading each sample, the position of the cell is determined optically, and the cell is fixed by synthesizing one or more gel structures of the encapsulated cell. Therefore, for each sample, a diagram of the cell and / or gel structure position is obtained, so that the determination result of the single cell is allowed to be assigned to a specific sample. After loading all samples and fixing the cell, one or more determinations can be performed on all cells on the surface simultaneously. In one aspect, depending in part on whether the cell needs to be recovered after the determination is completed, the gel structure may be degradable or may not be degradable. In addition, the gel structure synthesized for the cell of the encapsulated sample can have different compositions or geometric shapes, so that (for example) if the determination includes pulsing different doses of compounds to the cell, the gel structure composition and / or wall thickness can be configured to produce a gel structure with different diffusivities, so that the cell of different samples receives different doses of compounds.

[0022] Figure 1A-1B Embodiments of cell sorting methods using devices as described more fully below are shown. In some embodiments, such devices are fluidic devices having channels with surfaces on which cells can be arranged, imaged, and immobilized by synthesizing a gel structure for encapsulating or encapsulating the cells. Figure 1A At the top of the apparatus, cells (e.g., 102) are loaded (104) into a channel (not shown) so that they are arranged on the surface (100), after which the device's detectors record their positions and collect optical signals indicative of cell characteristics (such as, for example, size, morphology, nuclear to cytoplasmic area or volume ratio, surface protein profile, secreted protein profile, etc.). Based on such measurements, cells (e.g., 108a-108d) having values ​​of these measurements that fall within a predetermined range can be selected. Such selected cells synthesize a gel structure (shown as a circle around the selected cells 108a-108d) by photoenergy around each of them (also shown in Figure 2 The position of the gel structure is determined by the position of the cells they encapsulate, and the position of the cells is determined by optical detection. Such gel structures can have a variety of shapes and compositions. In some embodiments, the gel structure can include a solid block of gel material that contacts and encapsulates the cells. In some embodiments, such blocks can have a clear shape, such as a solid cylinder, a hemisphere, a cube, etc. In some embodiments, the gel structure can include a polymer matrix wall that surrounds and encapsulates the cells without contacting the cells, such as a hydrogel chamber described more fully below. In some embodiments, the channel can be part of a flow cell, wherein the channel includes a first surface and a second surface, and the hydrogel chamber includes a polymer matrix wall extending from the first surface to the second surface and encapsulating the cells. After the selected cells are fixed by the synthetic gel structure, the unselected cells (110) are removed from the surface (100) so as to retain only the cells (111) fixed by the gel structure. This process can be repeated so that another group of cells are loaded (112) into the channel so as to arrange them on the surface (100). The positions of the newly loaded cells are determined and recorded, and optical signals are collected to determine which newly loaded cells have the characteristics for selection. After such determination, the selected cells of the second load are fixed by synthesizing a gel structure that encapsulates each of the cells, for example (114a-114h). Unselected cells are removed (118). These steps (118) are repeated until a predetermined number of selected cells is reached or the surface (100) becomes too crowded for further selection and fixation. For sorting, the gel composition can be formulated to allow degradation by various mechanisms described below. As Figure 1BAs shown, the gel structure (120) on the surface (100) can be treated with an agent that degrades or depolymerizes the gel structure (122) and releases the immobilized cells, which can then be eluted from the flow cell (124).

[0023] As discussed below, a variety of photosynthesized gels and degradable gels can be used to implement the systems and methods described herein. The following references incorporated herein by reference provide guidance for selecting such gels to obtain desired properties (including but not limited to biocompatibility, gelation rate, degradation rate, etc.): 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); Bowman et al., U.S. Patent 9631092; 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); Sugiura et al., U.S. Patent Publication US2016 / 0177030; Shih et al., Biomacromolecules, 13 (7): 2003-2012 (2012); etc. In some embodiments, a photoinitiator is used for free radical polymerization to form a photosynthesized gel. In some embodiments, the photoinitiator includes Irgacure 2959, lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) or eosin-Y (see, for example, Choi et al., Biotechniques, 66 (1): 40-53 (2019)). In some embodiments, the hydrogel precursor includes 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 acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid-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 cross-linking agent. In some embodiments, such enzymatically degradable cross-linking agents can be degraded by esterases or peptidases. In some embodiments, the polymer precursor includes a photodegradable cross-linking agent. In some embodiments, such a photodegradable cross-linking agent includes nitrobenzyl.In some embodiments, such photodegradable cross-linking agents include coumarin moieties. In some embodiments, photodegradable hydrogels are used together with the methods described herein, for example, because the photodegradation of the hydrogel chambers can be selectively and as required, so that the specified hydrogel chambers can be degraded without affecting the unselected hydrogel chambers. In some embodiments, the hydrogel chambers are non-selectively degraded so that all the hydrogel chambers in a given channel (or other container) are degraded simultaneously. In some embodiments, such non-selective degradation is carried out with a cutting agent that specifically cuts unstable bonds in the hydrogel. For example, such cutting agents include reducing agents. In some embodiments, such non-specific degradation is carried out with enzymes that cut bonds or chemical elements in the hydrogel. For example, chemical elements include but are not limited to peptides, polysaccharides, and oligonucleotides.

[0024] Figure 2 Features of a device for immobilizing cells by means of a synthetic gel structure are shown, as well as examples of cylindrical hydrogel chambers. (Further description of such devices is in Figure 8A-8B . Cells (e.g., 201) combined with one or more polymer precursors are loaded into a channel (200) comprising a first surface (202) and a second surface (203) such that the cells are arranged on the surface (202). The first surface (202) and the second surface (203) are parallel and typically comprise surfaces of a light-transmitting material (such as glass). A detector (204) (together with an image recording system and a computer) detects and / or collects images of the cells, including their positions on the surface (202). Operationally associated with the detector (204) is a spatial energy modulation element (206) (such as a digital micromirror device (DMD)) that projects light energy into the channel (200) under program control to photopolymerize the polymer precursors to form a polymer matrix within the channel (200). Such a polymer matrix may include a solid gel block that encapsulates the cells, or it may include a more complex structure, such as a hollow cylinder or similar structure, that can encapsulate the cells without directly contacting the cells. Such hollow structures may include polymer matrix walls extending from a first surface (202) to a second surface (203) to form an enclosure (sometimes referred to herein as a "hydrogel chamber" or simply a "chamber") containing the cells. After optical analysis of the cells, a gel block or chamber (208) is synthesized to immobilize the selected cells. Figure 2 In the embodiment of the present invention, for convenience, the hydrogel chambers are shown as solid cylinders (225a-225d) in the channel (200), although they are intended to represent the hollow cylinder (210) shown in the enlarged view. The hollow cylinder or chamber (210) has a wall (221) having a thickness (216) that encapsulates the interior (211). After the hydrogel chambers encapsulating the selected cells are synthesized, the unselected cells (228) are removed.

[0025] Figure 3 It is shown that in some embodiments, cells can be arranged non-randomly on a surface (300) by providing a surface with a regular array of cell adhesion sites (302), which can be prepared by a variety of techniques, such as Zhu et al., Analytica Chimica Acta, 608 (2): 186-196 (2008) etc. In some embodiments, such sites can include antibodies that are specific to the cell surface proteins selected. In some embodiments, such sites can include integrin proteins or peptides or similar proteins. After loading (304) cells (such as 306), cells adhere to the site (302) in a regular pattern as shown. Then a hydrogel chamber (310) can be synthesized to encapsulate and fix cells, and further loading and selection can be performed afterwards. In some embodiments, such spatially regular arrangement makes imaging and synthesis of gel structures more efficient.

[0026] Figure 4A-4B , Figure 5A-Figure 5B and Figure 6A-6B Embodiments for sorting cells based on protein secretion profiles, growth rates, and mobility are shown, respectively. In other embodiments, cells may be sorted based on other (or additional) properties, such as cell-cell interactions, cell killing, antigen presentation, etc.

[0027] With respect to protein secretion, prior to loading the cells, a uniform distribution of protein capture beads (e.g., BioLegend, Inc., San Diego) is attached to the surface (450). As shown in the enlarged view (444), the uniform distribution can include beads with antibodies specific for different proteins, for example, different types of beads for each of a plurality of cytokines. In one embodiment, secretion-based sorting is performed as follows: after depositing the cells on the surface (450), the cells are positioned by a detector (445) and the hydrogel chamber is synthesized (446) by a spatial energy modulation element (447). The secreted proteins are detected by being captured by the protein capture beads and then labeled with detection antibodies, as shown by the solid dots (e.g., 457) in the hydrogel chamber (for clarity, only a single bead type is shown). In some embodiments, the porosity of the polymer matrix wall of the chamber is selected to prevent the passage of cells but allow the free passage of proteins. In other embodiments, such porosity is selected to prevent the passage of cells and proteins. In the latter embodiment, an additional step of depolymerizing the polymer matrix wall is included to provide access to the detection antibody. For example, the polymer matrix wall is selected to allow selective photodegradation. The relative amount of secreted protein from each cell is determined by counting different types of labeled beads adjacent to or adjacent to the cells in each chamber, or by integrating different fluorescent signals from beads adjacent to or adjacent to the cells in each chamber (453). Figure 4A As shown in the figure, the hydrogel chambers (460, 462, 464 and 466) include cells with the highest secretion rate (indicated by the number of filled beads). After identifying the high secretion cells, they are re-encapsulated by synthesizing (458) hydrogel chambers around them, and then the unselected cells (472) are removed. As shown, the chambers (482, 484 and 486) include cells with high secretion rates. After such removal, the chambers of high secretion rate cells can be degraded and the cells are eluted (459). Alternatively, low secretion cells can be re-encapsulated or fixed by the gel structure, and the selected high secretion cells are eluted.

[0028] Figure 5A-Figure 5BSteps of an embodiment for sorting cells based on growth rate are shown. Cells (e.g., 551) are arranged on a surface (550) where they are located by a detection system (545) and encapsulated by synthesizing (546) hydrogel chambers (549) (also shown in the top view in the figure below) using a spatial energy modulation element (547). The cells are incubated (553) under growth conditions, during which the hydrogel chambers are periodically imaged by the detection system to determine the number of cells in each chamber. In some embodiments, the number of cells is used as a measure of growth rate, so that after a given incubation period, the hydrogel chambers can be identified as including high growth rate cells or low growth rate cells based on the number of cells, such as Figure 5B As shown in the figure above, chambers (562, 564, 566 and 568) are selected to include high growth rate cells. Such hydrogel chambers (558) are then selectively degraded or equivalently depolymerized (i.e., "as needed"), followed by elution of the released high growth rate cells (560). In some embodiments of the proliferation assay, each of the synthesized hydrogel chambers has the same shape and area, for example, having a diameter selected from 0.001 to 0.01 mm 2 The range is from 0.001 to 1.0 mm 2The hydrogel chamber is a quasi-annular chamber with an internal area within the range of . In some embodiments, for each different type of cells to be measured, each hydrogel chamber synthesized has the same shape and area, for example, cytotoxic T lymphocytes can be confined to a chamber with one area, and helper T lymphocytes can be confined to a chamber with another area. After synthesizing the required number of hydrogel chambers, the cells are incubated under growth conditions for a period of time, and then (for example) the cells in each chamber are counted (220) to measure the proliferation capacity of each cell. In some embodiments, after the chamber is synthesized to encapsulate the selected cells, the unselected cells can be removed by washing steps or by other changes in reagents in the channel. In some embodiments, the cells can be stained with membrane or intracellular dyes to determine proliferation by dye dilution, so that independent measurements of cell proliferation can be obtained. Exemplary intracellular dyes for dye dilution include but are not limited to Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), etc. The required number of single cells encapsulated by the chamber depends on the statistical confidence required for the measured value. If the subpopulation of interest accounts for 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 can be greater than 100; or greater than 1000; or greater than 10,000; or the number can be in the range of 100 to 100,000; or in the range of 1000 to 100,000. After recording the counts for each chamber, the clonal population within the chamber can be further assayed to identify the cell type, e.g., by assessing cell surface proteins, cellular protein secretion, transcriptome, etc. This method is particularly useful for assessing populations of immune cells, particularly populations of engineered immune cells.

[0029] Figure 6A-6B The steps of an embodiment for sorting cells based on mobility are shown. For convenience, Fig. 6A Not included Figure 5A Abstract perspective view of the imaging and synthesis equipment in, but the steps will use the same instrumentation. Cells (e.g., 602) are loaded (604) and arranged on the surface (600), after which a detection system determines their position and tracks their movement, as shown in the tracks in the figure, which define the starting position, the ending position, and the path taken within a predetermined time interval. A measure of cell mobility can be the length of the path taken by the cell within a predetermined interval. In some embodiments, the cells with the highest mobility are encapsulated in hydrogel chambers (e.g., (612), (612) and (614)), after which the low mobility cells (616) are removed, the chambers of high motility cells (618) are degraded, and the released high mobility cells (620) are eluted.

[0030] In some embodiments, single cells from multiple samples can be assayed using gel structures, such as Figure 7A-7B As shown. Cells from each sample are sequentially loaded, arranged on the surface, and encapsulated in gel structures whose locations are associated with each sample. In some specific embodiments, the gel structure is a hydrogel chamber. Fig. 6A Same, Fig. 7A Not included Figure 5A , but the steps of this embodiment can use the same instrument. Cells from sample 1 (702) are loaded and arranged (e.g., 701) on the surface (700), after which the cells are positioned and the hydrogel chambers (e.g., 705) (704) are synthesized. Cells from sample 2 are then loaded (706) and arranged on the surface (700), after which the cells are positioned and the hydrogel chambers (e.g., 707) are synthesized. Finally, cells from sample 3 are loaded (710) and arranged on the surface (700), after which the cells are positioned and the hydrogel chambers (e.g., 711) (712) are synthesized. After the final hydrogel chambers are synthesized, all three samples can be measured simultaneously, and the measurement results can be associated with the specific samples by the position of the hydrogel chambers.

[0031] Determination can include but is not limited to measurement of viability, surface protein expression, cell motility, cell migration rate, cell replication rate, protein secretion, cytotoxicity, vector copy number of transduced cells, viral integration site, transcriptome, etc. In some embodiments, the transcriptome of the encapsulated cells is determined. In some embodiments, the protein secretion of the encapsulated cells is determined. In some embodiments, the cytokine secretion of the encapsulated cells is determined. In some embodiments, the gel structure for encapsulating or fixing the cells of a specific sample can be degradable, so that such cells can be eluted or recovered after, for example, the determination is performed. In some embodiments, the gel structure encapsulating or fixing at least two samples can be degraded using an orthogonal method so that the cells of such samples can be eluted or recovered separately. In some embodiments, such orthogonal methods include photodegradable gels and chemically degradable gels. In some embodiments, the chemically degradable gels include disulfide bonds. In some embodiments, the chemically degradable gels include pH sensitive bonds.

[0032] Figure 7C Embodiments are shown in which cells can be encapsulated by hydrogel chambers comprising two or more orthogonal degradable hydrogels. Such embodiments can be used to expose selected cells to gel-impermeable reagents, such as beads, which can be used for specific assays, for example, assays for cytokines or other secreted proteins. Figure 7CThree cells (722a, 722b, and 722c) are shown arranged on an area of ​​a surface (720) of a flow cell (not shown). The cells (722a and 722b) are selected based on optical properties (e.g., size and shape) and are encapsulated (725) by hydrogel chambers (724a and 724b, respectively), wherein the walls of each chamber have gaps (726) of predetermined locations and sizes. The cells (722c) are removed by washing. In some embodiments (such as the embodiment shown), the number and size of the gaps are selected so that they are small enough that the encapsulated cells cannot leave the chamber, but they are large enough and numerous enough that protein capture beads (e.g., (729)) can diffuse into the chamber in actionable quantities (i.e., for statistically reliable measurements) within 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 Figure 7C As shown, protein capture beads are loaded into the channel of the flow cell (728) so that they are arranged around and inside the hydrogel chambers (e.g., 724a and 724b), after which the polymer precursor is loaded into the channel again, and the gap (732) is encapsulated by photosynthesizing the hydrogel in the gap. As described more fully below, the geometry and size of the initial hydrogel chamber with the gap and the subsequent gap filling are controlled by a computer-driven spatial energy modulation element (such as a DMD). After filling the gap, the protein capture beads outside the chamber can be removed by washing (734). Then, the protein secretion profile can be determined based on the protein capture beads in the hydrogel chamber, for example, using the protocol of the bead manufacturer.

[0033] In the figures, for convenience, the hydrogel chambers are shown as being placed in isolation without being connected to adjacent chambers, and are shown as having a cylindrical or toroidal shape; however, the spatial energy modulation element can synthesize chambers of different shapes and sizes, as may be useful for a particular application. In some embodiments of the proliferation assay, each hydrogel chamber synthesized has the same shape and area, for example, having a diameter selected from .001 to .01 mm 2 The inner area of ​​the range is annular.

[0034] As used herein, "channel" means a container capable of accommodating a fluid (which may be static or mobile) and having at least one surface on which beads can be arranged and a synthetic chamber. In some embodiments, a channel may have a first surface and / or a second surface on which a synthetic chamber may be arranged and / or on which beads or particles may be arranged. As used herein, mentioning "surface" without mentioning "first" or "second" is intended to include a first surface or a second surface (if there are two surfaces in a fluid device (e.g., including a flow cell)). In some embodiments, a channel may limit the flow of a fluid passing therethrough from an inlet to an outlet. In other embodiments, a channel may contain a non-flowable volume of fluid that may be removed, substituted, or added through an opening or an inlet; that is, in some embodiments, a channel may be a hole or a pore-like structure.

[0035] Systems and Instruments

[0036] exist Fig. 8A A system for performing the above method is shown in FIG. A flow cell (800) is a component of a fluidic device that provides one or more channels and liquid handling components under programmable control to deliver beads and reagents to the 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. Fig. 8A In the abstract view of the flow cell (800), the inlet, outlet and other features of the channel are not shown. On the first surface (814) of channel 2 (804), a plurality of beads (e.g., (818)) are each encapsulated by a hydrogel chamber (e.g., (816)). In some embodiments, the porosity of the polymer matrix wall of the hydrogel chamber is selected to be impermeable to the beads, but permeable to reagents used to form the spatial barcode. Thus, reagents can be introduced into the interior of the hydrogel chamber and removed from the interior of the hydrogel chamber by flowing (820) the reagents through the channel, but the beads remain inside. The enlarged view (812) below of the channel segment (810) shows an optical system (821) for causing the hydrogel chamber to perform photosynthesis at the location of the beads in the channel. One of ordinary skill in the art will recognize that a system having the same Fig. 8A and Figure 8B Optical systems of different configurations can perform these functions. In some embodiments, one or more DMD-objective subsystems for synthesizing hydrogel structures can be used to increase the synthesis speed by synthesizing multiple structures simultaneously.

[0037] return Fig. 8AFor making the hydrogel chamber perform photosynthesis, the light source (822) generates a beam (823) of light of appropriate wavelength (e.g., UV light), which passes through an appropriate photomask or beam shaping or beam pointing (Galvo) system to shape the beam so as to synthesize the desired one or more structures in the channel. In some embodiments, a digital micromirror device (DMD) (824) is used, and in other embodiments, a physical photomask can be used. The chamber position, shape, and polymer matrix wall thickness are determined at least in part based on the bead position information determined by the image collected by the detector (832). The reflected light from the DMD (824) is shaped using conventional optical devices (e.g., collimating optical devices (828)) and is directed through an objective lens system (834) into the channel 2 section (810). The objective lens (834) and the flow cell (800) move relative to each other in the xy direction (836) to make the chamber perform photosynthesis at any position in any channel. In some embodiments, the flow cell (800) moves while the optical system (821) is stationary. In some embodiments, the objective lens (834) can also direct the light beam (827) from the light source (829) to the target (such as cells) on the first surface (814) and collect the optical signal (such as fluorescent signal) from the measurement performed on the first surface (814). Alternatively, the optical system (821) can be used as shown in FIG. Figure 8B The optical signal is collected by a separate objective lens as shown. Figure 8B The information collected by the counterparts in the embodiments, particularly the cell positions in their corresponding channels, is used by a computer (838) and / or an auxiliary controller to guide the DMD (824) and a translation device that controls the relative positions of the objective lens (834) and the flow cell (800) to synthesize hydrogel chambers of appropriate shape and size at appropriate locations.

[0038] Figure 8B An alternative optical system is shown in which the detection portion (850) of the optical system moves (872) independently of the movement (868) of the combining 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 interconnected optical elements, such as a dichroic mirror (862). Fig. 8A As in the embodiment of the present invention, the detector (856) is operatively associated with the computer (864) and the synthesis portion (852) of the optical system to provide bead position information to the synthesis portion (852). The computer (864) and (838) can also be operatively associated with a platform 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 opposite side of the first surface (864) from the detection portion (850). Fig. 8ALike the embodiment of , it includes conventional components objective lens (874), reflector (876), collimating optics (880), DMD (882) and light source (878).

[0039] In some embodiments, the systems described herein include: (i) a channel comprising a surface; (ii) a spatial energy modulation element optically connected to the surface; and (iii) a detector optically connected to the surface and operably associated with the spatial energy modulation element, the detector identifying cells and determining their locations on the surface. It should be understood that the term "detector" as used herein may include, but is not limited to: a microscope element that collects and optionally magnifies an image of a portion of the channel; and an image analysis element that includes information for identifying cells and associated location information. A computer element uses such information generated by the detector as well as user input to generate commands for other elements (such as the spatial energy modulation element) to perform a variety of functions, including but not limited to synthesis chambers, "on demand" degradation of chambers, selective photodegradation chambers, and the like. The configuration of such embodiments is described above Figure 8A-8B In some embodiments, the channel of the fluidic device further comprises a second surface (e.g. Figure 2 ), wherein the first surface and the second surface are arranged relative to each other across the channel, and wherein the polymer matrix wall of the chamber extends from the first surface to the second surface to form a chamber each having an interior. In some embodiments, the chambers in the channel each encapsulate a single cell. In some embodiments, the first wall and the second wall are both made of a light-transmitting material (such as glass, plastic, etc.) and are positioned so that the first surface and the second surface are substantially parallel to each other. The vertical distance between the first surface and the second surface can be in the range of 10 μm to 500 μm or in the range of 50 μm to 250 μm.

[0040] In some embodiments, multiple channels can be Figure 9A-9B1 to 6. In some embodiments, the plurality of channels may be arranged together in a flow channel. In some embodiments, the plurality of channels may be in the range of 2 to 12, or 2 to 8, or 2 to 6, or in the range of 2 to 4. The flow cell (900) is shown in a cross-sectional view and a top view. The flow cell (900) has: a bottom wall or first wall (906), the bottom wall or first wall (906) having a first surface (905); a top wall or second wall (902), the top wall or second wall (902) having a second surface (901); and a spacer (904) sealingly sandwiched between the bottom wall or first wall (906) and the top wall or second wall (902), the longitudinal holes of the spacer (904) forming channels 1-channel 6, one of which is represented by (908) in the cross-sectional view and by (912) in the top view. In some embodiments, the thickness of the spacer (904) can be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, which determines the internal height of the channel. The top wall (902) includes an inlet (914) and an outlet (916), which are used to load and remove reagents and beads from channel 1-channel 6 individually or collectively. In some embodiments, at least one of the walls (902) and (906) is made of a light-transmitting material (such as glass, plastic, etc.). The flow cell (900) can be operationally associated with a fluid device that delivers reagents and beads to any one of channel 1-channel 6 under program control. Guidelines for specific designs (including fluid handling and valve adjustment of such fluid systems) can be found in U.S. Patents 8921073, 8173080 and 8900828, etc., which are incorporated herein by reference. Fig. 9B A channel of a flow cell (900) is shown having randomly distributed (not to scale) hydrogel chambers having a quasi-ring-shaped cross-section, such as (920), on a first surface thereof.

[0041] As described above, any of the first surface, second surface, or polymer matrix wall of the chamber may include capture elements and other functional groups for performing various operations including, but not limited to, capturing beads, capturing cells, capturing analytes (such as mRNA, secreted proteins, intracellular proteins, or genomic sequences), capturing components of analytical reagents (such as oligonucleotide tags from antibodies), etc. Derivatized surfaces for such purposes are well known to those skilled in the art, as evidenced by the following references: Integrated DNA Technologies brochure (cited above); Hermanson (cited above); etc.

[0042] As described above, in some embodiments, the fluid device of the method includes a detector or is operationally associated with a detector, which can share the optical path of the spatial energy modulation element, or can be arranged near the second wall or arranged near the first wall opposite to the spatial energy modulation element in an embodiment (such as a groove) having only a first wall and a first surface. The detector is positioned so that it can detect optical signals from cells in the channel or detect optical signals near cells in the channel, for example, the cells are distributed on the first surface in the chamber. In some embodiments, the first wall and the second wall each include a light-transmitting material, for example, so that the spatial energy modulation element can project light energy into the interior of the channel, and so that the detector can detect optical signals (such as fluorescent emission or reflected light from biological components). In some embodiments, the projected energy 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 can have a complex cross-section, thereby allowing (in various embodiments) to synthesize multiple chambers simultaneously. Light-transmitting materials include but are not limited to glass, quartz, plastics and similar materials.

[0043] Spatial energy modulation elements that can use light energy to achieve polymerization can include physical photomasks or virtual photomasks, such as digital micromirror devices (DMDs). The following references are incorporated by reference to provide guidance in selecting and operating DMDs to achieve photopolymerization gels: Chung et al., U.S. Patent 10464307; Hribar et al., U.S. Patent 10351819; Das et al., U.S. Patent 9561622; Huang et al., Biomicrofluidics, 5:034109 (2011); etc.

[0044] Hydrogel chamber

[0045] Function. A variety of photosynthesizable gels can be used in conjunction with the systems and methods described herein. In some embodiments, hydrogels are used, particularly because they are compatible with living cells and are widely used in preparing gels with desired properties (including but not limited to porosity (which largely determines the contents and substances passing through the gel (or polymer matrix) wall), degradability, mechanical strength, ease and speed of synthesis, etc.). In some embodiments, the gel or hydrogel is both photosynthesizable and photodegradable. In some embodiments, the gel degradation mechanism is compatible with living cells. In some embodiments, the synthesis of gel chambers or gel walls can be used directly to select the size of cells, either independently or in combination with imaging. Hydrogel chambers or other hydrogel structures (such as barriers or walls) can be synthesized in channels with gaps in polymer matrix walls of predetermined sizes, so that smaller cells can pass through the gaps, but larger cells are blocked and retained on one side of the barrier or wall.

[0046] Porosity. In some embodiments, the hydrogel porosity is selected to allow the selected reagent to pass through while preventing other reagents or objects (such as cells) from passing through. In some embodiments, the hydrogel porosity is selected to prevent biological cells from passing through, but allows reagents (including proteins, such as polymerases) to pass through. In some embodiments, such reagents that can penetrate the polymer matrix wall include lysozyme, proteinase K, random hexamer, polymerase, transposase, ligase, deoxynucleotide triphosphate, buffer, cell culture medium or divalent cations. In some embodiments, at least one polymer matrix includes a pore, which is sized to allow the reagent to diffuse through at least one polymer matrix, but is too small to allow the DNA or RNA for analysis to cross the pore (size greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, the polymer chains of the cross-linked hydrogel structure form a hydrogel matrix (i.e., a porous hydrogel matrix) with pores. In some versions, the size of the pores in the hydrogel structure can be regulated or tuned, and can be formulated to encapsulate sufficiently large genetic material (such as cells or nucleic acids) (e.g., genetic material greater than about 300 base pairs), but allow smaller substances (such as reagents) or nucleic acids of smaller size (e.g., less than about 50 base pairs) (such as primers) to pass through the pores, thereby entering and exiting the hydrogel structure. In some embodiments, the hydrogel can have a diameter sufficient to allow the reagents listed above to diffuse through the structure while retaining any pore size of nucleic acid molecules greater than 500 nucleotides or base pairs in length. In some embodiments, when the hydrogel is hydrated, the hydrogel structure can expand. Then, the size of the pores can be changed depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, the diameter of the pores is from about 10nm to about 100nm. In some embodiments, the pore size of the hydrogel structure is tuned by routine experiments by changing the ratio of the concentration of the polymer precursor to the concentration of the cross-linking agent, changing pH, salt concentration, temperature, light intensity, etc. In some embodiments, the average diameter of the pores of the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater, a molecular weight of 50 kDa or greater, a molecular weight of 75 kDa or greater, a molecular weight of 100 kDa or greater, or a molecular weight of 150 kDa or greater.

[0047] In some embodiments, the length of the DNA or RNA retained can be sequenced using conventional synthetic sequencing techniques. For example, such DNA or RNA comprises at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the average diameter of the pore can be 5nm to 100nm. In some embodiments, the average diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the average diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. Reagents can include enzymes or primers with a size less than 50 base pairs (bp). Primers can include single-stranded DNA (ssDNA). In some embodiments, the size of primers can be 5bp to 50bp. In some embodiments, the size of the primer can be 5bp to 10bp, 10bp to 20bp, 20bp to 30bp, 30bp to 40bp or 40bp to 50bp. In some embodiments, the size of the primer can exceed 50bp. In some cases, the size of the primer can be less than 5bp. In some embodiments, the diameter of the pore can be 5nm to 100nm. In some embodiments, the diameter of the pore can be 5nm to 10nm, 10nm to 20nm, 20nm to 30nm, 30nm to 40nm, 50nm to 60nm, 60nm to 70nm, 70nm to 80nm, 80nm to 90nm, 90nm to 100nm. In some embodiments, the diameter of the pore can be greater than 100nm. In some embodiments, the average diameter of the pore can be less than 5nm. The pore size of the polymer matrix can be from about 5 nanometers (nm) to about 100nm.The pore size of the polymer matrix can be 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, nm, about 10 nm to about 80 nm, about 10 nm to about 90 nm, about 10 nm to about 100 nm, about 10 nm to about 110 nm, about 20 nm to about 30 nm, about 20 nm to about 40 nm, about 20 nm to about 50 nm, about 20 nm to about 60 nm, about 20 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm to about 70 nm, about 20 nm to about 80 nm, about 20 nm to about 90 nm, about 20 nm to about 100 nm, about 20 nm to about 110 nm, about 30 nm to about 40 nm, about 30 nm to about 50 nm, about 30 nm to about 60 nm, about 30 nm about 70nm, about 30nm to about 80nm, about 30nm to about 90nm, about 30nm to about 100nm, about 30nm to about 110nm, about 40nm to about 50nm, about 40nm to about 60nm, about 40nm to about 70nm, about 40nm to about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 40nm to about 110nm, about 50nm to about 60nm, about 50nm to about 70nm, about 50nm to about 80nm, about 50nm to about 90nm, about 50nm to about 100nm The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm, about 60nm to about 110nm, about 70nm to about 80nm, about 70nm to about 90nm, about 70nm to about 100nm, about 70nm to about 110nm, about 80nm to about 90nm, about 80nm to about 100nm, about 80nm to about 110nm, about 90nm to about 100nm, about 90nm to about 100nm, or about 100nm to about 110nm. The pore size of the polymer matrix can be about 5nm, about 10nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm, about 100nm or about 110nm. The pore size of the polymer matrix may be 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 pore size of the polymer matrix may be 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.

[0048] Size and shape of hydrogel chambers . In some embodiments, the polymer matrix wall of the chamber inhibits the passage of predetermined components, such as mammalian cells, bacterial cells, genomic DNA, larger polynucleotides (e.g., mRNA greater than 200 ribonucleotides, greater than 300 ribonucleotides, or 500 ribonucleotides), etc. In some embodiments, the polymer matrix wall extends from the first surface to the second surface (parallel to the first surface) to form a chamber in the channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the size of the interior of the chamber is designed to encapsulate cells, such as mammalian cells. For example, such chambers may include a cylindrical shell or a polygonal shell, which includes an interior space or an interior and a polymer matrix wall. In some embodiments, such chambers have a cross-section of a ring-like shape. As used herein, the term "ring-like cross-section" means a cross-section that is topologically equivalent to a ring. In some embodiments, the interior space or interior of the chamber has an inner diameter of 1 μm to 500 μm and a volume ranging from 1 picoliter to 200 nanoliters or 100 picoliters to 100 nanoliters or 100 picoliters to 10 nanoliters. In some embodiments, the thickness of the polymer matrix wall is at least 1 μm (micrometer). In some embodiments, the value of the height of the chamber with a quasi-annular cross section ranges from 10 μm to 500 μm or ranges from 50 μm to 250 μm. In some embodiments, the aspect ratio (i.e., height / width) of the polymer matrix wall with a quasi-annular cross section is 1 or less. In some embodiments, the aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability of resistance (such as reagent flow through the channel), washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix is ​​degradable. In some embodiments, the degradation of at least one polymer matrix is ​​"as needed". In some embodiments, the chamber in the channel is discontinuous. In some embodiments, the chamber in the channel can be adjacent to an adjacent chamber. In some embodiments, the chamber can share a polymer matrix wall with each other. In some embodiments, the chamber can be synthetic, and its slit or other orifice is large enough to allow certain components (such as beads) to pass through, but small enough to prevent other components (such as cells) from passing through.

[0049] Hydrogel compositions. As mentioned above, hydrogel compositions can vary greatly, and hydrogels can be formed by a variety of methods. 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 acid-co-glycolic acid)-b-poly (ethylene glycol)-b-poly (lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) and poly (vinyl alcohol). In some embodiments, hydrogels are formed by light-induced free radical crosslinking. In some embodiments, hydrogels are formed by light-induced thiol-ene reactions.

[0050] Hydrogel degradation. In some embodiments, the hydrogel chamber is usually degradable or depolymerizable in the channel or "as needed". The usually degradable hydrogel chamber is degraded by treating with a degradation agent or a depolymerizing agent that is equivalently exposed to all chambers in the channel. The depolymerizing agent may include, but is not limited to, heat, light, and / or chemical depolymerizing agents (sometimes also referred to as cutting agents or degradation agents). In some embodiments, polymer precursors that allow photocrosslinking and photodegradation can be used according to the degradation required, for example, different wavelengths are used for crosslinking and degradation to achieve. For example, Eosin Y can be used for free radical polymerization at a defined zone using a 500nm wavelength, after which 380nm irradiation can be used to cut the crosslinking agent. In other embodiments, a light cage hydrogel cutting agent 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 produce a hydrogel, and then UV light can be used to generate local acidic conditions, which in turn degrade the hydrogel. In some embodiments, at least one polymer matrix can be degraded by at least one of the following: (i) contacting at least one polymer matrix with a cleavage agent; (ii) heating at least one polymer matrix to at least 90°C; or (iii) exposing at least one polymer matrix to a certain wavelength of light that cuts a photo-cleavable cross-linking agent, which cross-links the polymers of at least one polymer matrix. In some embodiments, at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage agent degrades the hydrogel. In some embodiments, the cleavage agent includes a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage agent, or a combination thereof. In some embodiments, the cleavage agent includes 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 coupling functional groups to the polymer matrix or hydrogel. Some non-limiting examples of functional groups can include capture reagents (e.g., picolinaldehyde (PCA)), acrylamide, agarose, biotin, streptavidin, strep-tag II, linkers, functional groups comprising aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEGs, thiols, olefins, alkynes, azides, or combinations thereof. In some cases, the functionalized polymer matrix can be used to capture biomolecules within the polymer matrix compartments formed adjacent to (e.g., around or on) the biological component. The biomolecules can be produced by the biological component (e.g., from the secretome of the cell). 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 reagents, molecular sensors, or any molecules of interest (e.g., antibodies).

[0051] Photosynthesis. In some embodiments, generating a polymer matrix in a channel or hole of a fluid device includes exposing one or more 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 of 350nm to 800nm. In some embodiments, the light generating device generates light of 350nm to 600nm. In some embodiments, the light generating device generates light of 350nm to 450nm. In some embodiments, the light generating device generates UV light. In some embodiments, generating a polymer matrix in 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 laser beam manipulated using a galvanometer. In some embodiments, the SLM is based on liquid crystal.

[0052] Although the invention has been described with reference to several specific example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the invention. The invention is applicable to various sensor embodiments and other subject matter in addition to those discussed above.

[0053] Example: Isolation of NK cells expressing CD56 from a mixed NK / Jurkat population

[0054] In this example, NK cells with high expression of CD56 were isolated from a mixed NK / Jurkat cell population. Fig. 10A As shown. A mixed population of NK cells and Jurkat cells is loaded into a flow cell so that the cells settle on the surface (1000), as shown in the field of view (1001), after which a hydrogel chamber is synthesized around four cells, as indicated by the use of cPEG (described more fully below). The cells are stained with an anti-CD56 PE-labeled antibody (1002), and cells expressing high levels of CD56 are identified by fluorescence intensity (1004). Highly expressing CD56 cells are again encapsulated by a second orthogonal degradable hydrogel cSEL (1008, as described more fully below), after which all cPEG hydrogels (1006) are degraded using glutathione (GSH) (described below). Cells not encapsulated by the cSEL chamber are removed by washing (1010). High CD56 expressing cells are identified (1011), followed by (i) encapsulation (1012) of them in another cPEG chamber (1013), (ii) degradation of the cSEL hydrogel, and (iii) removal of other cells by washing (1014) to leave high CD56 expressing cells, such as (1016).

[0055] The materials and methods used in the examples are as follows:

[0056] 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 (from Gibco) supplemented with 10% FBS and 1X antibiotic-antimycotic (from Gibco).

[0057] cPEG chamber synthesis: cPEG macromer (1018, Fig. 10B ) was dissolved in PBS to prepare cPEG gel stock solution. To prepare 40 μL precursor solution, 6 μL cPEG stock solution was mixed with 2 μL polyethylene glycol (PEG, 20 kDa) PBS solution, 4 μL phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium (LAP) PBS solution, 4 μL cell suspension and 24 μL PBS. The gel solution was loaded on the flow cell and the single cell was caged using the Polygon 1000DMD system (Mightex). The uncrosslinked polymer solution was then washed with PBS.

[0058] CD56-PE staining: The antibody solution was prepared by adding 5 μL of CD56-PE stock solution to 100 μL of PBS. The antibody solution was then loaded on the flow cell, and the flow cell was incubated at 37°C. The antibody solution was then washed with PBS, and the flow cell was incubated at 37°C. Bright field and green fluorescence images of the cells were taken at 10X magnification using 10ms and 300ms exposure times, respectively.

[0059] cSEL room synthesis: cSEL macromer (1020, Fig. 10B ) was dissolved in PBS to prepare the cSEL gel stock solution. To prepare 40 μL of the precursor solution, 20 μL of the cSEL stock solution was mixed with 4 μL of phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium (LAP) in PBS and 16 μL of PBS. The gel solution was loaded on the flow cell and the selected cells were caged using the DMD and illumination parameters mentioned above. The uncrosslinked polymer solution was then washed with PBS.

[0060] cPEG gel cleavage: Prepare 10 mM L-glutathione (GSH) solution in PBS and adjust the pH to 8.0 using sodium hydroxide. Load the GSH solution on the flow cell and incubate the flow cell at room temperature for 5 minutes to cleave the cPEG gel. Then wash the flow cell three times with PBS.

[0061] cPEG secondary compartment synthesis: cPEG macromer (1018, Fig. 10B) was dissolved in PBS to prepare cPEG gel stock solution. Then, to prepare 40 μL precursor solution, 6 μL cPEG stock solution was mixed with 2 μL polyethylene glycol (PEG, 20 kDa) PBS solution, 4 μL phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium (LAP) PBS solution and 28 μL PBS. The gel solution was loaded on the flow cell and the selected cells were caged using the aforementioned DMD.

[0062] cSEL gel lysis: Alginate lyase was dissolved in PBS. The lyase solution was loaded on the flow cell and the flow cell was incubated at room temperature for 5 minutes to lyse the cSEL gel. The flow cell was then washed three times with PBS to remove the lyase solution.

[0063] In some embodiments, the above-described method for sorting cells by multiple optical criteria can be performed in steps including: (a) synthesizing one or more first gel structures that fix one or more cells disposed on the surface of a channel and selected by a first optical property; (b) synthesizing one or more second gel structures that encapsulate one or more cells fixed by the first gel structure, wherein the one or more cells encapsulated by the second gel structure are selected by a second optical property; (c) degrading the first gel structure; and (d) removing cells released by degrading the first gel structure, so that the 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 also include degrading the second gel structure and eluting the one or more cells selected by the first optical property and the second optical property from the channel. In some embodiments, the first gel structure and the second gel structure are orthogonally degradable; that is, the first gel structure can be degraded in the presence of the second gel structure without significantly affecting the second gel structure, and similarly, the second gel structure can be degraded in the presence of the first gel structure without significantly affecting the first gel structure. In some embodiments, the first gel structure and the second gel structure are orthogonally degradable by having different modes of degradability selected from: chemical degradability (e.g., by reduction of disulfide bonds), enzymatic degradability, degradability caused by temperature changes, and photodegradability. In some embodiments, multiple optical properties for selecting a subset of cells according to the above method include cell size, nuclear to cytoplasm size ratio, mobility, motility, cell killing, cell shape, optical signal intensity monotonically associated with surface protein expression, and optical signal intensity monotonically associated with protein secretion. In specific embodiments, multiple optical properties include mobility and optical signal intensity monotonically associated with surface protein expression. In specific embodiments, multiple optical properties include cell killing and optical signal intensity monotonically associated with surface protein expression. Typically, such optical signal intensity can be generated by a labeled antibody specific for a predetermined surface or secreted protein. In some embodiments, the optical intensity includes fluorescence intensity.

[0064] definition

[0065] Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics, and molecular biology used herein follow standard treatises and texts in the field, e.g., Kornberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, 6th edition (Saunders, 2007).

[0066] In some embodiments, "determination" refers to a process for detecting or measuring a cell property or property of a single cell or a cell group. Typically, the process steps of the determination include chemical, biochemical or molecular reactions (such as the cutting of a bond, specific binding of complementary components, enzyme reactions, dissolution of complementary components, etc.) or changes in physical state (such as an increase or decrease in temperature, a change in energy level, etc.), and result in the generation of a signal (or multiple signals), from which the presence, absence or size of a quantity associated with the cell can be inferred. The properties of the signal generated by the determination may vary greatly, and may include, but are not limited to, electrical signals, optical signals, chemical signals or material signals. Material signals include the generation of materials containing information that can be extracted. For example, a material signal can be an amplification of a polynucleotide whose length, quantity, composition or nucleotide sequence indicates a cell property. For example, a barcode oligonucleotide can be a material signal. The characteristics or properties of the cells detected or measured may vary widely and include, but are not limited to, cytotoxicity, viability, ability to proliferate under selected conditions, size, shape, motility, type and profile of cell surface or membrane proteins, type and profile of secreted proteins, metabolite production, transcriptome, gene copy number, gene or allele identity, chromatin accessibility profile, vector copy number of engineered or infected cells, etc.

[0067] "Cell" refers to a biological cell that can be measured 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 measured by the methods and systems described herein. Specifically, any mammalian cell that can or has been genetically altered to be used in medicine, industry, environment or treatment 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 cells 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. Particularly interesting are cytotoxic T lymphocytes engineered for therapeutic applications (such as cancer therapy).

[0068] "Hydrogel" means a gel comprising a cross-linked hydrophilic polymer network that can absorb and retain a large amount of water (e.g., 60% to 90% of the water, or 70% to 80%) without dissolving due to the establishment of physical or chemical bonds (which can be covalent, ionic or hydrogen bonds) between the polymer chains. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and culture applications. Hydrogels can comprise natural or synthetic polymers and can 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 9631092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0069] The term "as needed" means that operations can be directed to individual, discrete, selected locations (e.g., 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 a computer algorithm that operates on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include real-time detection or detection of a certain period of time before modulating unit energy to polymerize a polymer precursor or degrading a chamber. For example, a subset of chambers (all formed with photodegradable polymer matrix walls) can be preselected based on position information and the value of an optical signal from an analytical determination performed in the chamber for release and removal of its contents. The preselected chamber can be photodegraded by selectively projecting a beam of light with appropriate wavelength characteristics (e.g., with a spatial energy modulation element) to degrade the polymer matrix wall of the preselected chamber. In another example, multiple chambers can be observed in real time (e.g., via fluorescence microscopy) to detect an analyte of interest, and one or more chambers in multiple chambers can be selected in real time after detecting the analyte of interest for degradation.

[0070] "Polymer matrix" generally refers to a phase material (e.g., continuous phase material) comprising at least one polymer. In some embodiments, polymer matrix refers to at least one polymer and 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 straight chain, branched and cross-linked polymer units. The polymer matrix can also include non-polymer species inserted into the interstitial space not occupied by the polymer chain. The inserted species can be solid, liquid or gas species. For example, the term "polymer matrix" can encompass dry hydrogels, hydrated hydrogels and hydrogels comprising glass fibers. The polymer matrix can include polymer precursors, which generally refer to one or more molecules that can trigger or initiate polymerization when activated. Polymer precursors 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 are polymerized to produce polymer matrix) and cross-linked compounds, which can include photoinitiators, other compounds necessary or useful for generating polymer matrix (especially as a polymer matrix of hydrogel), etc.

[0071] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited to the specific examples provided in the specification. Although the present invention has been described with reference to the aforementioned specification, the description and illustration of the embodiments herein are not meant to be interpreted in a limiting sense. Without departing from the present invention, those skilled in the art will now conceive of many variations, changes and substitutions. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations or relative proportions depending on various conditions and variables set forth herein. It should be understood that in practicing the present invention, various alternatives to the embodiments of the present invention described herein may be adopted. Therefore, it is contemplated that the present invention should also encompass any such alternatives, modifications, variations or equivalents. The appended claims are intended to define the scope of the present invention, and thus encompass methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for sorting cells, the method comprising: (a) providing a fluidic 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 a cell and determining a location of the cell on the surface; (b) loading the channel with cells and one or more polymer precursors such that the cells are disposed on or near the surface; (c) using the spatial energy modulation element to project light into the channel, so that the projected light causes the one or more polymer precursors to cross-link to form one or more gel structures, to synthesize the one or more gel structures to fix one or more cells selected based on one or more optical signals, the one or more optical signals coming from the one or more cells, the one or more gel structures encapsulating each of the one or more selected cells, wherein the position of the one or more gel structures in the channel is determined by the position of the cells encapsulated by the one or more gel structures identified by the detector; and (d) Removing unselected cells from the channel.

2. The method of claim 1, further comprising repeating steps (b) to (c).

3. The method of claim 2, wherein steps (b) to (c) are repeated until the fixed cells reach a predetermined density on the surface.

4. The method of claim 1, further comprising degrading the gel structure of the fixed cells and eluting the selected cells from the channel.

5. The method of claim 1, wherein the one or more optical signals are indicative of cell morphology, cell size, cell shape, organelle size, organelle shape, surface protein expression, cell motility, cell migration rate, cell replication rate, or protein secretion. The method of claim 5 , wherein the one or more optical signals are indicative of protein secretion by the cell.

7. The method of claim 6, wherein the protein secretion is cytokine secretion.

8. The method of claim 5, wherein the one or more optical signals are indicative of cell replication rate.

9. The method of claim 8, wherein the replication rate is determined by counting the progeny of the cells.

10. The method of claim 8, wherein the replication rate is determined by dilution of an intracellular dye.

11. The method of claim 5, wherein the one or more optical signals are indicative of cell mobility.

12. The method of claim 11, wherein the cell mobility is determined by a series of images from which the cell position is recorded as a function of time.

13. The method of claim 1, wherein the gel structure comprises photochemically degradable linkages.

14. The method of claim 1, wherein the gel structure comprises chemically degradable linkages.

15. The method of claim 14, wherein the chemically degradable linkage comprises a disulfide linkage.

16. A method for measuring cells from a plurality of samples, the method comprising: (a) providing a fluidic 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 a cell and determining a location of the cell on the surface; (b) loading one or more cells of a sample and one or more polymer precursors into the channel such that the one or more cells are disposed on or near the surface; (c) using the spatial energy modulation element to project light into the channel, so that the projected light causes the one or more polymer precursors to cross-link to form one or more gel structures, to synthesize the one or more gel structures, the one or more gel structures encapsulating each of the one or more cells of the sample, wherein the position of the synthesized gel structure in the channel is determined by the position of the cell encapsulated by the synthesized gel structure identified by the detector; (d) repeating steps (b) and (c) for each sample in the plurality of samples; and (e) performing an assay on the cells in the channel to obtain an assay result for each of the cells encapsulated by the gel structure.

17. The method of claim 16, further comprising determining the assay result for each of the samples by correlating the assay result with the location of the cells of the samples.

18. The method of claim 17, wherein the one or more gel structures comprise hydrogel chambers.

19. A method for sorting cells by multiple optical criteria, the method comprising: (a) synthesizing one or more first gel structures to immobilize one or more cells disposed on a surface of a channel, wherein the one or more cells are selected by a first optical property; (b) synthesizing one or more second gel structures, the one or more second gel structures encapsulating at least a portion of the one or more cells immobilized by the first gel structure, wherein the portion of the one or more cells encapsulated by the second gel structure is selected by a second optical property; (c) degrading the first gel structure; as well as (d) removing cells released by degrading the first gel structure such that a portion of the one or more cells selected by the first optical property and the second optical property remain on the surface of the channel.

20. The method of claim 19, further comprising degrading the second gel structure and eluting the one or more cells selected by the first optical property and the second optical property from the channel.

21. The method of claim 19, wherein the first gel structure and the second gel structure are orthogonally degradable.

22. The method of claim 19, wherein the first optical property and the second optical property comprise one or more of cell size, nucleolus to cytoplasm size ratio, cell shape, optical signal intensity monotonically correlated with surface protein expression, and optical signal intensity monotonically correlated with protein secretion.

23. The method of claim 19, wherein the gel structure comprises a toroidal-like chamber.

24. A method of sorting one or more cells, the method comprising: (a) providing a fluidic device comprising: (i) a channel comprising a surface, one or more polymer precursors and the 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) identifying the location of a subset of the one or more cells using the detector, wherein the subset of the one or more cells is selected based on one or more optical signals from the subset of the one or more cells; (c) controlling the spatial energy modulation element to project energy into the channel based on the position of the subset of the one or more cells identified by the detector, such that the projected energy causes the one or more polymer precursors to form one or more gel structures, the one or more gel structures encapsulating the subset of the one or more cells, and wherein one or more cells not in the subset of the one or more cells are not encapsulated by the one or more gel structures; and (d) removing from the channel the one or more cells that are not in the subset of the one or more cells.

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