WO2025230996A1

WO2025230996A1 - Methods for serial biological assays

Info

Publication number: WO2025230996A1
Application number: PCT/US2025/026829
Authority: WO
Inventors: Pier Federico GHERARDINI; Tarun Kumar Khurana; Yir-Shyuan WU; Seyedsina Moeinzadeh; Frank CHARBONIER
Original assignee: Cellanome Inc
Current assignee: Cellanome Inc
Priority date: 2024-04-30
Filing date: 2025-04-29
Publication date: 2025-11-06
Anticipated expiration: 2026-10-30

Abstract

Described herein are devices, systems, and methods for performing serial biological assays using selectively degradable hydrogels. Aspects of the present disclosure provide a method for analyzing an analyte that includes inputting the analyte and a biological material into a fluidic device and detecting an interaction between the analyte and the biological material. The analyte and the biological material may initially be separated by a hydrogel polymer wall, and may later be combined by degrading the hydrogel polymer wall.

Description

METHODS FOR SERIAL BIOLOGICAL ASSAYS

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/640,756, filed April 30, 2024, which application is incorporated herein by reference in its entirety for all purposes.

BACKGROUND

[0002] CAR T cells can undergo exhaustion in vivo, and serial killing assays are thought to better recapitulate this phenomenon in vitro. Instead of looking at the ability of a T cell to kill a single target cell, it is more relevant to look at the ability of the cell to kill multiple cells in series without undergoing exhaustion.

SUMMARY

[0003] Described herein is a method of implementing an assay for serial killing by T cells (or other cells with killing activity such as NK) using targeted formation and degradation of hydrogel chambers on a fluidic device.

[0004] In an aspect, provided herein is a method for analyzing an analyte, the method comprising: (a) inputting the analyte and one or more biological materials into a fluidic device, wherein the fluidic device comprises a hydrogel chamber comprising: (i) at least a portion of the analyte, and (ii) at least a portion of a first biological material of the one or more biological materials, wherein the analyte and the first biological material are physically separated by a first hydrogel polymer wall; (b) degrading at least a portion of the first hydrogel polymer wall; and (c) detecting an interaction between the analyte and the first biological material.

[0005] In one aspect, the hydrogel polymer wall surrounds the analyte. In another aspect, in (a), the inputting of the analyte and the inputting of the one or more biological materials into the fluidic device occurs separately. In an additional aspect, the hydrogel polymer wall prevents an interaction between the analyte and the one or more biological materials. In a further aspect, the hydrogel polymer wall is a first hydrogel polymer wall, wherein the biological material is a first biological material, and wherein the method further comprises: (d) generating a second hydrogel polymer wall, wherein the analyte and a second biological material of the one or more biological materials are physically separated by the second hydrogel polymer wall. In an additional aspect, the second hydrogel polymer wall surrounds the analyte. In a particular aspect, the hydrogel chamber is a first hydrogel chamber, and wherein the method further comprises: (e) degrading the first hydrogel chamber, and (f) forming a second hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material. In another aspect, the method further comprises: (g) degrading at least a portion of the second hydrogel polymer wall. In certain aspects, further comprising: (h) detecting an interaction between the analyte and the second biological material. In further aspects, the method further comprises: (i) generating a third hydrogel polymer wall, wherein the analyte and a third biological material are physically separated by the third hydrogel polymer wall. In another aspect, the third hydrogel polymer wall surrounds the analyte. In some aspects, the method further comprises: (j) degrading the second hydrogel chamber, and (k) forming a third hydrogel chamber around at least a portion of the analyte and at least a portion of the third biological material. In some aspects, the method further comprises: (1) degrading at least a portion of the third hydrogel polymer wall.

[0006] In some aspects, the method further comprises: (m) detecting an interaction between the analyte and the third biological material. In additional aspects, the hydrogel chamber is a first hydrogel chamber, wherein the hydrogel polymer wall is a first hydrogel polymer wall, wherein the biological material is a first biological material, wherein the analyte is physically separated from a second biological material of the one or more biological materials by a second hydrogel polymer wall, and wherein the first hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, and (iii) the second biological material. In certain aspects, the method further comprises, subsequent to (c): (n) degrading at least a portion of the second hydrogel polymer wall, and (o) detecting an interaction between the analyte and the second biological material. In further aspects, the analyte is physically separated from a third biological material of the one or more biological materials by a third hydrogel polymer wall, and wherein the first hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, (iii) the second biological material, and (iv) the third biological material. In particular aspects, the method further comprises, subsequent to (n): (p) degrading at least a portion of the third hydrogel polymer wall, and (q) detecting an interaction between the analyte and the third biological material.

[0007] In one aspect, the interaction comprises a killing of the biological material by the analyte or vice versa. In another aspect, the interaction comprises activation of the biological material by the analyte or vice versa. In a particular aspect, detecting the activation comprises counting a number of proliferated cells from the biological material. In an additional aspect, detecting the activation comprises determining a presence of a surface antigen on a cell of the biological material. In a further aspect, the analyte is selected from a plurality of analytes in the fluidic device prior to (a). In a certain aspect, the analyte is a cell. In another aspect, the analyte is an antigen targeting cell. In a particular aspect, the analyte is a CD8+ T cell or NK cell. In a further aspect, the analyte is a genetically engineered cell. In a select aspect, the analyte is a CAR T cell.

[0008] In a further aspect, the biological material is a cell. In one aspect, the biological material is an antigen presenting cell. In another aspect, the biological material is a cancer cell. In a particular aspect, the biological material is an antibody or antigen binding fragment thereof. In a select aspect, the antibody or antibody binding fragment thereof is coupled to a bead, and wherein the bead cannot diffuse through the hydrogel polymer wall. In a certain aspect, the analyte, the biological material, or both, is coupled to the hydrogel polymer wall.

[0009] In some aspects, the fluidic device comprises a flow cell. In further aspects, the method further comprises obtaining one or more genetic materials from the analyte. In certain aspects, the method further comprises amplifying the one or more genetic materials. In additional aspects, the amplifying occurs in the fluidic device. In select aspects, the method further comprises sequencing the one or more genetic materials. In particular aspects, the sequencing occurs in the fluidic device. In certain aspects, the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In further aspects, the RNA comprise messenger RNA (mRNA) or microRNA (miRNA).

[0010] In some aspects, the interaction comprises suppression of the biological material by the analyte or vice versa. In additional aspects, the interaction comprises binding or physical contact between the biological material and the analyte. In further aspects, the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel. In certain aspects, the degrading in (b) comprises exposing the hydrogel polymer wall to UV light. In further aspects, the degrading in (b) comprises selectively exposing the hydrogel polymer wall to UV light in a presence of photoinitiator and not exposing the hydrogel chamber to the UV light.

[0011] In some aspects, the method further comprises imaging the analyte, the biological material, the hydrogel chamber, the fluidic device, or any combination thereof. In additional aspects, the fluidic device comprises a channel with an inlet and an outlet, wherein the channel comprises a first surface and a second surface disposed opposite one another across the channel, wherein a polymer matrix wall extends between the first surface and the second surface, thereby forming the hydrogel chamber. In further aspects, the hydrogel chamber and the hydrogel polymer wall comprise the same material. In additional aspects, the hydrogel chamber is cured for a first duration of time, and wherein the hydrogel polymer wall is cured for a second duration of time. In one aspect, the first duration of time is longer than the second duration of time. In another aspect, the second duration of time is longer than the first duration of time. In select aspects, the hydrogel chamber and the hydrogel polymer wall have different kinetics of degradation. In certain aspects, the hydrogel chamber and the hydrogel polymer wall comprise different materials. In particular aspects, the hydrogel chamber is made of a first material, wherein the first material degrades upon exposure to a first stimulus, wherein the hydrogel polymer wall is made of a second material, wherein the second material degrades upon exposure to a second stimulus, and wherein the first stimulus and second stimulus are different. In one such aspect, the first stimulus comprises light, and wherein the second stimulus comprises a degradation reagent. In another aspect, the first stimulus comprises a degradation reagent, and wherein the second stimulus comprises light. In a particular aspect, the first stimulus comprises a first degradation reagent, and wherein the second stimulus comprises a second degradation reagent different from the first degradation reagent. In an additional aspect, the first stimulus comprises light in a first wavelength range, and wherein the second stimulus comprises a light in a second wavelength range different from the first wavelength range. In a certain aspect, the first stimulus comprises UV light selectively applied to the hydrogel polymer wall in a presence of photoinitiator, and wherein the second stimulus comprises UV light selectively applied to the hydrogel chamber in the presence of photoinitiator.

[0012] In an additional aspect, the method further comprises forming the hydrogel polymer wall around at least a portion of the analyte; and forming the hydrogel chamber around at least a portion of the analyte and at least a portion of the biological material of the one or more biological materials, wherein the inputting of the analyte into the fluidic device occurs before the inputting of the biological material into the fluidic device, and wherein the forming the hydrogel chamber is after the inputting of the biological material.

[0013] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, where only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the systems, devices, and methods described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles described herein are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0016] FIG. 1A depicts a schematic example of a biological interaction assay using a hydrogel chamber and a hydrogel polymer wall.

[0017] FIG. IB depicts a schematic example of a serial killing assay using selectively degradable hydrogels.

[0018] FIG. 1C depicts a schematic example of a serial killing assay using optically cleavable hydrogels.

[0019] FIG. 2 depicts a schematic example of a serial antibody screening assay using degradable hydrogels.

[0020] FIG. 3 shows a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.

[0021] FIG. 4 shows a portion of a system as provided herein including an energy source, according to some embodiments.

[0022] FIG. 5 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0023] FIG. 6A is a top-down image of a portion of a fluidic device that includes chambers and cells.

[0024] FIG. 6B is a top-down image of the portion of the fluidic device shown in FIG. 6A following degradation of multiple chambers.

DETAILED DESCRIPTION

Introduction [0025] In aspects, the present disclosure provides methods for releasing biological materials to an analyte at controlled times. The analyte and biological materials may be enclosed in separate polymeric chambers or compartments of a multi-compartment polymeric chamber within a fluidic device. The polymeric chambers or compartments of the multi-compartment polymeric chamber can be degraded to allow the biological materials to come into contact with the analyte. Multiple biological materials can be released to the analyte in this fashion. The timing and sequence of biological material release can be controlled to analyze the individual and cumulative effects of the biological materials on the analyte. Polymeric chamber synthesis and degradation can be performed using light, allowing highly multiplexed, parallelized spatial and temporal control over analyte-biological material interactions. The timing and/or sequence of biological material release can be varied between analytes within a fluidic device to generate complex, time-dependent datasets from individual automated assays. The timing and/or sequence of biological material release can also be determined in real time during an assay. For example, a second cellular biological material may be released to an analyte upon the death of a first cellular biological material (e.g., as evidenced by an increase in fluorescence as disclosed elsewhere herein) as determined by an imaging system as disclosed herein. The presently disclosed methods thus improve upon the control and multiplexing capabilities of conventional well plate assays which can: (1) require cells to be maintained in large volumes, within which volumes cells can require large amounts of time to come into contact, thereby limiting user control over the timing of cellcell interactions; (2) require that cells be added to individual wells by pipette, wherein the number of cells added to a well is based on a Poisson statistics, and cannot be strictly controlled by a user; and (3) provide limited real-time control over assays.

[0026] In the methods and systems disclosed herein, a polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of one or more biological components in a fluidic device to isolate selected biological components. A hydrogel matrix may be selectively generated to surround a component. One or more hydrogel or polymer matrix walls can be used to physically separate one or more biological components from one another. Upon degradation of the hydrogel or polymer matrix walls, two or more biological components can interact. These interactions can be monitored and analyzed.

[0027] In some cases, the methods described herein encompass the incorporation of an analyte and one or more biological materials into a hydrogel chamber within the device. For example, in some cases, a method includes a step of forming a hydrogel chamber or wall that physically separates an analyte from a biological material. In another example, a method includes a step of forming a hydrogel polymer wall within the device that surrounds the analyte, and then inputting the biological material into the device that is next surrounded by forming a hydrogel chamber in a way that surrounds both the biological material and the analyte. In some cases, the analyte and a biological material are initially physically separated by a hydrogel polymer wall. In some cases, degradation of the polymer wall results in an interaction between the analyte and biological material. The interaction can be monitored and detected. In some cases, imaging of the fluidic device or the hydrogel chamber can be used to determine if the analyte has effects on the biological material (or vice versa). The systems and methods described herein can allow for a detailed and targeted approach to analyte analysis, offering an understanding of the interaction between analytes and biological materials in a controlled environment.

[0028] Provided herein is a method for analyzing an analyte. The method may comprise: (a) inputting an analyte and one or more biological materials into a fluidic device. In some embodiments the fluidic device comprises a hydrogel chamber. In some embodiments the hydrogel chamber comprises: (i) at least a portion of the analyte and (ii) at least a portion of a first biological material of the one or more biological materials. For example, the analyte and the one or more biological materials can be input into the fluidic device, and then the hydrogel chamber can be formed so as to enclose (i) at least a portion of the analyte and (ii) at least a portion of the first biological material of the one or more biological materials. In some embodiments, the analyte and the first biological material are physically separated by a first hydrogel polymer wall. In some embodiments, the method further comprises (b) degrading at least a portion of the first hydrogel polymer wall. In some embodiments, the method further comprises (c) detecting an interaction between the analyte and the first biological material.

[0029] In order to compartmentalize individual components of a biological sample, a polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of an individual component in a fluidic device. The hydrogel matrix may be selectively generated to surround a component after the system detects the component or hydrogel matrices can be generated according to a predefined pattern in a fluidic device. The hydrogel matrix may allow reagents and smaller entities to pass while retaining the individual component of the biological sample in place. Because one or more individual components can be localized within a fluidic device (e.g., encapsulated) and the localized components be exposed to one or more reagents and/or washing solutions during and/or in between analyses, multiple assays can be performed within the compartments (e.g., simultaneously, substantially simultaneously, serially, etc.). [0030] Different assays may be performed in different locations of the fluidic device, for example, to test effects of different treatment conditions. Additionally, because components are not generally mixed and combined, low concentrations of components (e.g., due to dilution) can be prevented. For example, when analyzing genomic material, an amplification step can be avoided due to the preservation of the genetic material in each compartment. By having two or more components within a compartment, interactions between components can be studied as well. The polymer matrix can be degradable “on demand” allowing for controlled localization and release mechanisms. The solutions provided herein can retain spatial information of the components and generate data on a cellular, proteomic, transcriptomic, or genomic level. Since spatial information is retained, the data can be associated (e.g., linked) with phenotypic data. Further, the solutions provided herein can retain spatial information of the components and link data (e.g., phenotypic data) on a cellular, proteomic, transcriptomic, or genomic level.

[0031] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0032] Whenever the term “at least” precedes the first numerical value in a series of two or more numerical values, the term “at least” applies to each of the numerical values in that series of numerical values. For example, at least 1, 2, or 3 is equivalent to at least 1, at least 2, or at least 3.

[0033] Whenever the term “less than” precedes the first numerical value in a series of two or more numerical values, the term “less than” applies to each of the numerical values in that series of numerical values. For example, less than 3, 2, or 1 is equivalent to less than 3, less than 2, or less than 1.

[0034] The terms “coupled to,” “connected to,” and “in communication with,” as used herein, generally refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluid, biological, and thermal interaction. Two components may be coupled to each other even though they are not in direct contact with each other.

[0035] The terms “polypeptide” and “peptide,” as used interchangeably herein, generally refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (e.g., an amino acid analogue). The polypeptide can be linear or branched. The polypeptide can include modified amino acids. The polypeptide may be interrupted by non-amino acids. A polypeptide can occur as a single chain or an associated chain. The polypeptide may include a plurality of amino acids. The polypeptide may have a secondary and tertiary structure (e.g., the polypeptide may be a protein). In some examples, the polypeptide can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1,000, 10,000, or more amino acids. The polypeptide may be a fragment of a larger polymer. In some examples, the polypeptide can be a fragment of a larger polypeptide, such as a fragment of a protein.

[0036] The term “amino acid,” as used herein, generally refers to a naturally occurring or non- naturally occurring amino acid (e.g., an amino acid analogue). The non-naturally occurring amino acid may be an engineered or synthesized amino acid.

[0037] The term “sample,” as used herein, generally refers to a chemical or biological sample containing a biological component. The biological component may comprise a cell, a nucleic acid, a microbiome, a protein, a combination of cells, a metabolite, a combination thereof, or any other suitable component of a biological sample. For example, a sample can be a biological sample including one or more cells. For another example, a sample can be a biological sample including one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In some instances, the sample may be derived from a homogenized tissue sample (e.g., brain homogenate, liver homogenate, or kidney homogenate). In certain embodiments, the sample may include a specific type of cell (e.g., a neuronal cell, muscle cell, liver cell, or kidney cell,). The sample may comprise or be acquired from a diseased cell or tissue (e.g., a tumor cell or a necrotic cell), In some embodiments, the sample may include or may be from a disease-associated inclusion (e.g., a plaque, a biofilm, a tumor, or a non-cancerous growth). In certain embodiments, the sample may include or may be obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In various embodiments, the sample can include circulating tumor cells. In some cases, the sample may include or may be an environmental sample (e.g., soil, waste, or ambient air), industrial sample (e.g., samples from any industrial processes), or a food sample (e.g., dairy product, vegetable product, or meat product). The sample may be processed prior to loading into a microfluidic device. For example, the sample may be processed to purify a certain cell type or polypeptide and/or to include reagents.

[0038] As used herein, the term “polymer matrix” generally refers to a phase material (e.g., continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term “polymer matrix” may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. A polymer matrix may comprise one or more polymer precursors in a polymerized form, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon, magnetic energy, or any other suitable energy. As used herein, the term “polymer precursor” includes monomers (that are polymerized to produce a polymer matrix), porogens, and crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices, and the like.

[0039] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix. A porogen can be dispersed with the reactants before the polymerization process of forming the polymer matrix. Porogens typically diffuse out of polymer matrices following polymerization, leaving pores in the regions that they occupied. Porogen size, concentration, hydrophobicity, and hydrophilicity can thus influence pore density and pore size in polymer matrices. Examples of a porogen can be polyethylene glycol (PEG, molecular weight from 1 kDa to 1000 kDa), 8 arm PEG, 4 arm PEG, 3 arm PEG, and combinations thereof.

[0040] A polymer matrix may be semi permeable so that cells and beads (ranging from 3 to 50 microns) are too big to pass through, but smaller reagents can pass through such as antibodies, buffering salts, cellular media, lysing agents such as DTT.

[0041] In some embodiments, as used herein, the term “local parameter” means a value of a parameter (such as, pH) in or immediately adjacent to a chamber formed by polymer matrix walls.

[0042] As used herein, the term “on demand” means an operation may be directed to individual, discrete, selected locations (e.g. a spatial location of polymer precursor solution; or a selected polymer matrix chamber). Such selection may be based on manual observation of optical signals or data collected by a detector, or such selection may be based on a computer algorithm operating on 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 at a time period prior to modulating a unit of energy to polymerize polymer precursors or degrading a chamber. For example, a subset of chambers (all formed with photo-degradable polymer matrix walls) may be pre-selected for releasing and removing their contents based on position information and the values of optical signals from an analytical assay carried out in the chambers. The pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) to degrade the polymer matrix walls of the pre-selected chambers. In another embodiment, the pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) in the presence of a photoinitiator to degrade the polymer matrix walls of the pre-selected chambers.

[0043] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of a photoinitiator includes one of lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2,2’-azobis[2-methyl-N-(2- hydroxyethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin- Y, Riboflavin, and combination thereof.

[0044] In another example, a plurality of chambers may be observed in real-time (e.g. via fluorescent microscopy) for detection of an analyte of interest and one or more chambers of the plurality of chambers is selected, in real-time, upon detection of the analyte of interest, for degradation.

[0045] As used herein, the term “analyte” generally refers to a discrete biological or chemical entity to be measured, detected, and/or distinguished using the methods and systems described herein. In some embodiments, an analyte may be a biological component as described herein. [0046] The present disclosure provides systems for compartmentalizing or isolating one or more biological components. The system can include a fluidic device containing or including one or more biological components. The fluidic device may contain or include one or more polymer precursors. In some cases, the fluidic device can comprise a first surface configured to couple or receive at least one of the one or more biological components to form a coupled biological component. The systems may also include at least one energy source, wherein the energy source is in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In various embodiments, the at least one energy source may form a polymer matrix on or adjacent to at least a portion of the one or more biological components.

[0047] In some cases, a sample may be introduced or provided to the system. In certain cases, the sample may comprise one or more biological components. In various cases, the biological components may be physically separated. In some cases, the biological components may be physically separated but in fluidic communication with one another. In certain cases, the biological components may be in chemical communication with one another. The system may be used for single-cell analysis. In some embodiments, the system may be used for single-cell analysis on a genome level. For example, the system may be used for genome sequencing. For another example, the system may be used for deoxyribonucleic acid (DNA) sequencing. The system may be used for DNA sequencing of cell-free DNA, whole genome sequencing, whole exome sequencing, targeted sequencing, or 16S sequencing. The system may be used for studying DNA tags attached to biomolecules of interest. The biomolecules may comprise proteins, metabolites, etc. In some cases, the DNA may be a nuclear DNA or a mitochondrial DNA. The system may be used for single-cell or bulk analysis on a transcriptome level. For example, the system may be used for ribonucleic acid (RNA) sequencing. For example, the system may be used for 3’ or 5’ gene expression analysis, immune repertoire study of a cell, or full-length mRNA analysis. In some embodiments, the system may be used for single-cell analysis on a proteome level. The system may be used for functional assay(s) of a biological component. The system may be used for studying surface proteins, secreted proteins, or metabolites of a biological component. In some cases, the system may be used to measure a quality of a biological component. In some cases, the measured quality may be the size or shape of a biological component. In some cases, the system may be used to study epigenomics, DNA methylation, or chromatin accessibility in a biological component. The system may be used for other suitable assays, experiments, and processes.

[0048] In certain embodiments, the system may be used for single-cell analysis on an indirect cell-cell interaction level. For example, an effect of one or more molecules produced from a first cell on a second cell can be analyzed using the system as provided herein. In various embodiments, the system may be used for analyzing direct cell-cell interactions. For example, two or more cells (e.g., a first cell and a second cell) can be in physical contact and the effect or effects of the first cell on the second cell, or vice versa, can be analyzed using the system as disclosed herein. In some embodiments, the system may be used for drug response analysis in a biological component. In certain embodiments, the system may be used for analyzing a biological component’s response to various physiological conditions (e.g., various media, temperature, mechanical stimuli, etc.). In some embodiments the analyte is selected from a plurality of analytes in the fluidic device prior to (a).

[0049] In certain embodiments, one or more polymer precursors may be added to or included with the biological sample. One or more biological samples and one or more polymer precursors may be introduced into the system (e.g., into the fluidic device of the system). The one or more biological samples and the one or more polymer precursors may be introduced into the fluidic device in any order (e.g., in parallel, sequentially, etc.). For example, the biological sample(s) may be introduced prior to the polymer precursor(s), the polymer precursor(s) may be introduced prior to the biological sample(s), the biological sample(s) and polymer precursor(s) may be introduced simultaneously (or substantially simultaneously), or in any other suitable manner or order. In some embodiments, a polymer precursor may include one or more hydrogel precursors. The one or more polymer precursors may be stored and/or introduced separately into the system. In some cases, the one or more polymer precursors may be mixed with the one or more biological components prior to introduction into the system. In various cases, the one or more polymer precursors may be mixed with the one or more biological components after introduction into the system. In various cases, a mixture of the one or more first polymer precursors and the analyte are inputted into the fluidic device where the analyte is then surrounded by a generation of a hydrogel polymer wall, and then another mixture of the one or more second polymer precursors and the biological material are inputted into the fluidic device where the analyte and the biological material are both surrounded by a generation of a hydrogel chamber. The first polymer precursors and second polymer precursors may be the same or different.

[0050] The system may comprise a fluidic device. In some embodiments, the fluidic device may include one or more polymer precursors. In other words, one or more polymer precursors may be disposed within at least a portion of the fluidic device (e.g., within at least a portion of a channel of the fluidic device). In some embodiments, the fluidic device may comprise one or more channels or chambers. In some embodiments, the fluidic device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000 channels or chambers, or any number of channels or chambers between any of the two numbers mentioned herein. In some embodiments, the fluidic device comprises more than 10,000 channels or chambers. As described herein, the fluidic device may include one or more channels. The fluidic device may also, or alternatively, include one or more chambers. The terms channel and chamber may be used interchangeably in the disclosure herein unless indicated otherwise. For example, a channel or a chamber of the fluidic device may comprise a first surface, a second surface, or more surfaces.

[0051] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” or “reaction chamber,” as opposed to a chamber that is formed from polymer matrix walls within a channel) may receive or be configured to receive a biological sample. FIG. 3 shows a schematic illustration of a portion of a channel 100 that may be disposed in at least a portion of a fluidic device of a system as provided herein. The fluidic device may comprise a channel 300. The channel 300 may comprise a first surface 301. Further, the channel 300 may comprise a second surface 302. In some embodiments, the first surface 301 and the second surface 302 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 3). In some embodiments, the first surface and second surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout the channel, for example, where chambers are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part and the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 301 may be a lower surface. In certain embodiments, the second surface 302 may be an upper surface. The terms “lower” and “upper” are not intended to be limiting and are used herein for convenience when referring to the figures. The channel 300 may receive a biological sample comprising one or more biological components 50, 51. The channel 300 may receive one or more polymer precursors. As illustrated in FIG. 3, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 301, the second surface 302, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 301 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the second surface, 302 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In some embodiments, the first surface and/or second surface can be optically transmissive so that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flowcell, and the measurement of the analyte and biological components.

[0052] In certain cases, a channel may have a rectangular, circular, semi-circular, oval crosssection, or other suitably shaped cross-section. Accordingly, the channel may have a single, internal surface. In some cases, a channel may have a triangular, square, rectangular, polygonal, or other cross-section. Accordingly, the channel may have three or more internal surfaces. One or more of the internal surfaces may be couple or receive, or be configured to couple or receive, the one or more biological components.

[0053] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may be functionalized, for example, with a coating (e.g., a surface coating). In some embodiments, the surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or more moieties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer.

[0054] With continued reference to FIG. 3, in some cases, the first surface 301, the second surface 302, or the first surface 301 and the second surface 302 may be functionalized with an adherent substrate. In particular cases, a bottom surface-with respect to gravity (e.g., 301 as shown in FIG. 3)-of the fluidic device, includes an adherent substrate. As used herein, an “adherent substrate” can denote a substrate that promotes cell adherence. A cell (50, 51) may have a higher affinity for an adherent substrate than for a fluidic device surface (301, 302) on which the adherent substrate is disposed. Many cells readily adhere to constituents of extracellular matrices (ECMs) such as glycosaminoglycans and fibronectin. An adherent substrate can include an ECM biomolecule or a material that includes an ECM biomolecule. In some cases, the cell adherent support is selected from actinin, collagen, fibrinogen, fibronectin, gelatin, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, poly-l-lysine (PLL), poly-d-lysine (PDL), poly-l-omithine, talin, VCAM-1, vinculin, vitronectin, a cell adherent peptide, or a combination thereof. As a further example, the cell adherent peptide can include a sequence recognized by one or more integrins, for example GFOGER (wherein ‘O’ denotes hydroxyproline), YIGSR, LRE, GRGDS, CKKQRFRHRNRKG, KRSR, VPGIG, MNYYSNS, CSVTCG, GFRGDGQ, HAV, FLPASGL, or a combination thereof. An adherent cell can be inputted into a fluidic device and then the cell can settle onto the adherent substrate (601C) of the bottom surface of the fluidic device. After a period of time, the adherent cell can transition to an adherent state where the adherent cell couples to the adherent substrate. In many instances, the transition to the adherent state can be characterized by a change in shape from a generally spherical shape to a non-spherical shape. For example, an adherent cell in the adherent state can be elongated and may include one or more protrusions.

[0055] In some cases, an effector cell is an adherent cell. Examples of adherent effector cells that may be utilized in the presently disclosed methods include macrophages, fibroblasts, and dendritic cells. In some cases, an effector cell is a suspension cell. Examples of suspension cells that may be utilized in the presently disclosed methods include CD4+ T cells, CD8+ T cells, and B cells. In some cases, a target cell is an adherent cell.

[0056] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 500 nm² to about 100,000 pm² and preferably 500 nm² to about 5000 pm². In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes between any of the two numbers mentioned herein. In some cases, a barcode (or a portion/subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0057] In some embodiments, the fluidic device may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA or RNA sequencing). In some embodiments, the fluidic device may be a microfluidic device. In certain embodiments, the fluidic device may be a nanofluidic device.

[0058] The system disclosed herein may comprise one or more energy sources. The energy source may be in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In some cases, the energy source can be used to form one or more polymer matrices in the fluidic device (e.g., on or adjacent to a surface of a channel or chamber of the fluidic device). In some embodiments, the energy source may comprise a light generating device, a heat generating device, an electrochemical reaction generating device, an electrode, or a microwave device. A polymer matrix may be formed in a channel of the fluidic device. The energy source may direct or transfer energy to a predetermined position in the fluidic device. The energy may cause or activate the one or more polymer precursors to form a polymer matrix (e.g., to polymerize) in the predetermined position.

[0059] In some embodiments, the polymer matrix may comprise a hydrogel. In some embodiments, the hydrogel may be porous enough, or have pores of a suitable size, to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) through the polymer matrix, while the hydrogel may not allow movement or transfer of the biological component (e.g., DNA, RNA, a protein, a cell, etc.) through the polymer matrix. In some embodiments, the pores may have a diameter from 5 nm to 100 nm. In some embodiments, the pores may have a diameter from 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, the pores may have a diameter larger than 100 nm. In some embodiments, the pores may have a diameter smaller than 5 nm. The reagent may comprise an enzyme or a primer having a size of less than 50 base pairs (bp). A primer may comprise a single-stranded DNA (ssDNA). In some embodiments, a primer may have a size from 5 bp to 50 bp. In some embodiments, a primer may have a size from 5 bp to 10 bp, 10 bp to 20 bp, from 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, a primer may have a size of more than 50 bp. In certain cases, a primer may have a size of less than 5 bp. A reagent may comprise a lysozyme, a proteinase K, hexamers (e.g., random hexamers), a polymerase, a transposase, a ligase, a catalyzing enzyme, a deoxyribonuclease, a deoxyribonuclease inhibitor, a ribonuclease, a ribonuclease inhibitor, DNA oligos, deoxynucleotide triphosphates, buffers, detergents, salts, divalent cations, or any other suitable reagent.

[0060] FIG. 4 shows a portion of a system as provided herein including an energy source 203. The embodiment of FIG. 4 may include components that resemble components of FIG. 3 in some respects. For example, the embodiment of FIG. 4 includes a channel 200 that may resemble the channel 300 of FIG. 3. With continued reference to FIG. 4, the channel 200 of the system may include a first surface 201 and a second surface 202. In some embodiments, the energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise one or more non-emitting portions (e.g., a non-emitting portion 204). The non-emitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. In certain embodiments, the emitting portion 205 can emit energy to the fluidic device. In some embodiments, the fluidic channel may be coupled to on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the fluidic channel. In some embodiments, the emitting portion 205 may be coupled to an objective (e.g., a microscope objective or lens), where the objective may be moved to different portions of the fluidic device. The objective may provide a shape (e.g., virtual mask) to allow light to form a pattern on the fluidic device, in order to form a polymer matrix similar or complementary to the pattern. In various embodiments, the one or more polymer precursors in the fluidic device or mixed with the biological sample can absorb emitted energy 206. In some embodiments, the emitted energy 206 can form, or be sufficient to form, a polymer matrix from the one or more polymer precursors. For example, a portion of the one or more polymer precursors within the channel 200 of the fluidic device may be activated by the emitted energy and a polymerization reaction may be initiated to form a polymer matrix.

[0061] The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and/or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on at least a portion of a surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission. [0062] In some embodiments, a first polymer matrix 208 and/or a second polymer matrix 209 can be formed on or adjacent to a biological component 50. In certain embodiments, the first polymer matrix 208 and the second polymer matrix 209 can form an analysis chamber or compartment 220 that separates (e.g., physically separates) the biological component 50 from other biological components (e.g., biological components 51, 52, or 53) in the fluidic device. Stated another way, the polymer matrix may compartmentalize the channel (e.g., channel 200). In various embodiments, the polymer matrix may partially surround a biological component. For example, a polymer structure fully surrounding a biological component may form a closed structure (e.g., a hollow cylinder-shaped polymeric structure) or a partially open structure (e.g., a crescent-shaped polymeric structure). In some embodiments, two or more polymer matrices may be formed adjacent to a biological component forming a compartment separating the biological component from other biological components. In certain embodiments, the polymer matrix may comprise or form a wall (e.g., a polymer matrix wall).

[0063] With continued reference to FIG. 4, in some cases, the energy source 203 can, or be configured to, form or produce one or more emitting portions 205 and one or more non-emitting portions 204. The systems disclosed herein may further include a spatial energy modulating element to direct energy from the energy source to one or more targeted portions of the fluidic device. For example, the spatial energy modulating element may be configured to selectively direct the energy from the energy source to form a polymer matrix in a discrete area of the fluidic device. In some embodiments, the discrete area is chosen based on the location of a biological component. In some embodiments, the area of the discrete area is less than the area of the fluidic device. In some embodiments, a biological component is captured within the discrete area. In some embodiments, the size and shape of the discrete area is adjustable according to the size, shape, or other properties of the biological component. In some embodiments, an algorithm is used to determine the shape and size of the discrete area. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. The spatial energy modulating element may be configured to selectively direct the energy by, for example, inhibiting or preventing energy from being directed to one or more portions other than the one or more targeted portions of the fluidic device. In some embodiments, the spatial energy modulating element may comprise a physical mask. In some cases, the spatial energy modulating element may comprise a virtual mask. In some cases, the spatial energy modulating element may be a spatial light modulator (SLM). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquidcrystal based.

[0064] In some embodiments, the first surface 201 or the second surface 202 may comprise a detector that detects, or is configured to detect, one or more locations of one or more biological components in the fluidic device (e.g., in the channel 200). In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In some embodiments, the detector may be a microscope objective for imaging the fluidic device. In various embodiments, a mask may be generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 203 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. The mask may inhibit or prevent the energy source 203 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the imaging is bright-field imaging, phase-contrast imaging, or fluorescence imaging, or any combination thereof. In some embodiments, the camera may be coupled to, connected to, or in communication with the energy source 203. For example, the camera (not shown) may be in electrical communication with the energy source 203. In some embodiments, the energy source 203 may comprise the camera. In various embodiments, the energy source 203 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0065] In some embodiments, an algorithm is used to determine where a biological component or analyte is located based on the imaging. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. In some embodiments, the objective is coupled to an energy source to emit energy to the predetermined portion in the fluidic channel.

Degradable hydrogels for serial killing assays

[0066] Degradable hydrogels can be utilized to facilitate controlled cell interactions, allowing a step-wise approach for evaluating the potency of a potential cytotoxic cell over a series of cell populations. This can provide a precise and tunable way to scrutinize the serial killing capacity of different analytes in a biomimetic environment, allowing advancements in the capacity to design and conduct complex biological assays with implications for numerous fields, including biomedical research, pharmacology, and oncology.

[0067] Provided herein are methods for facilitating a controlled interaction of one or more biological components. In some cases, the method comprises (a) inputting an analyte and one or more biological materials into a fluidic device. In some embodiments, the fluidic device comprises a hydrogel chamber. In some embodiments, the hydrogel chamber comprises: (i) at least a portion of the analyte and (ii) at least a portion of a first biological material of the one or more biological materials. In some embodiments, the analyte and the first biological material are physically separated by a first hydrogel polymer wall. In some embodiments, the method further comprises (b) degrading at least a portion of the first hydrogel polymer wall. In some embodiments, the method further comprises (c) detecting an interaction between the analyte and the first biological material. In (a), the inputting of the analyte and the inputting of the one or more biological materials into the fluidic device can occur separately. In some cases, the analyte is surrounded by the first hydrogel polymer wall prior to the inputting of the one or more biological materials into the fluidic device, and then both the analyte and the one or more biological materials are contained in a hydrogel chamber. In some cases, the first hydrogel polymer wall prevents an interaction between the analyte and the one or more biological materials. Encapsulating the analyte prior to introduction of the one or more biological materials can prevent unwanted or uncontrolled interactions between the analyte and the biological materials.

[0068] In some cases, the method comprises (a) detecting an interaction between the analyte and a first biological material of the one or more biological materials in a fluidic device. In some embodiments, the method further comprises (b) forming a hydrogel polymer wall that physically separates the analyte from a second biological material of the one or more biological materials. [0069] In some cases, the method comprises (a) providing a fluidic device comprising the analyte and one or more biological materials. In some embodiments the fluidic device comprises a hydrogel chamber. In some embodiments the fluidic device comprises (i) at least a portion of the analyte and (ii) at least a portion of a first biological material of the one or more biological materials. In some embodiments, the method further comprises (b) imaging the hydrogel chamber to determine whether the analyte kills the first biological material.

[0070] In some embodiments, the method comprises (d) inputting a second biological material into the fluidic device. In some embodiments, the analyte and the second biological material are physically separated by a second hydrogel polymer wall. In some embodiments, the method further comprises (e) degrading the hydrogel chamber and (f) forming a second hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material. In some embodiments, the method further comprises (g) degrading at least a portion of the second hydrogel polymer wall. In some embodiments, the method further comprises (h) detecting an interaction between the analyte and the second biological material. In some embodiments, the method further comprises repeating these steps for additional materials of the one or more biological materials. In some cases, the steps are repeated until the analyte is exhausted. In some cases, an exhausted analyte will no longer kill target cells or biological materials.

[0071] Optically cleavable hydrogels can be used in serial killing assays. In some cases, optically cleavable hydrogels respond to light wavelengths, allowing controlled degradation. This can enable systematic testing of an agent's cytotoxic impact on distinct cell groups. This approach can be used to measure cellular response and can be used in applications such as cell biology, pharmacology, and oncology.

[0072] In some embodiments, an analyte and a first biological material are surrounded by a hydrogel chamber. Within the hydrogel chamber, the analyte and the first biological material can be physically separated by a first hydrogel polymer wall. In some embodiments, the method further comprises degrading at least a portion of the first polymer wall, thereby resulting in an interaction (or lack thereof) between the analyte and the first biological material. In some cases, a second polymer wall can be formed around the analyte. The analyte and a second biological material can be physically separated by the second polymer wall. In some embodiments, the method further comprises detecting an interaction between the analyte and the second biological material. In some embodiments, the method further comprises repeating the steps for additional materials of the one or more biological materials. [0073] In some embodiments, the analyte is a cell. In some embodiments, the analyte is an antigen targeting cell. In some embodiments, the analyte is an immune cell such as a CD8+ T cell orNK cell. Further examples of immune cells that are utilizable as analytes in the presently disclosed methods include B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine induced killer (CIK) cells, granulocytes (e.g., basophil granulocytes, eosinophil granulocytes, neutrophil granulocytes, hypersegmented neutrophils), monocytes, macrophages, mast cells, phagocytes, plasma cells, thrombocytes, megakaryocytes, and dendritic cells. In some embodiments, the analyte is an effector cell. Examples of effector cells consistent with the presently disclosed methods include cytotoxic T cells, plasma cells, phagocytes, natural killer T cells, and T helper cells.

[0074] In some embodiments, the analyte is a genetically engineered cell. In some embodiments, the analyte is a CART cell. As used herein, the terms “CAR T cell” and “chimeric antigen receptor T cell” can refer to T cells that are engineered to express one or more chimeric antigen receptors. Chimeric antigen receptors are transmembrane receptor proteins that typically include an extracellular antigen recognition domain, a transmembrane domain, a hinge domain connecting the antigen recognition domain to a transmembrane domain, and an intracellular signaling (‘costimulatory’) domain. Chimeric antigen receptor domains may be non-naturally occurring or may be derived from a naturally occurring proteins. Many chimeric antigen receptors include combinations of domains from naturally occurring proteins. For example, many chimeric antigen receptors include extracellular antigen recognition domains derived from naturally occurring antibody or T cell receptor Fab regions, hinge regions derived from naturally occurring CD28 or CD8a proteins, transmembrane domains derived from naturally occurring CD3-(^, CD4, CD8, or CD28 proteins, and intracellular signaling domains derived from naturally occurring CD3, CD27, CD28, CD134, or CD137 proteins. Chimeric antigen receptors may thus be designed to bind to one or more target antigens by selecting an extracellular domain that bind to the one or more target antigens, and to transduce a desired intracellular selected signal or collection of signals upon binding to the specified target antigen or antigens by selecting an intracellular domain that generates the desired signal or collection of signals. For example, a CD28 costimulatory domain may promote cell growth and proliferation, while CD27 costimulatory domains may polarize cells towards memory phenotypes.

[0075] An effector cell may be clonally expanded in a fluidic device to generate a plurality of copies of the effector cell. Effector cell expansion may be promoted by inputting one or more signaling molecules and or transcription factors such as IL-2, IL-4, MAPK3, or CD59 into the fluidic device. The copies of the effector cell may be adhered to a surface of the fluidic device or may be suspended in media within the fluidic device. Target cells may then be introduced into the fluidic device, and one or more disclosed methods may be performed sequentially or in parallel on at least a subset of the copies of the effector cell. At least a portion of the effector cells may optionally be enclosed in chambers before the target cells are introduced into the fluidic device.

[0076] In some embodiments, the first biological material is a cell. The first biological material may be a target cell (e.g., the analyte may be an effector cell, and the first biological material may be a cell that is targeted by the effector cell). Examples of target cells consistent with the present disclosure include cancer cells, virally infected cells (e.g., HIV infected cells or SARS- CoV-2 infected cells), bacterial cells (e.g., Streptococcus, Staphylococcus, Pseudomonas, or Chlamydophila cells), and fungal cells (e.g., Cryptococcus, Candida, or Aspergillus cells). Examples of cancer cells consistent with the present disclosure include adrenocortical carcinoma cells (e.g., NCI-H295R cells), transitional carcinoma cells (e.g., HT-1376 cells), osteosarcoma cells (e.g., MG-63 cells), leukemic cells (e.g., AML-193 cells), myeloma cells (e.g., JJN3 cells), glioblastoma cells (e.g., DBM2 cells), cervical cancer cells (e.g., HeLa cells), colon cancer cells (e.g., Caco-2 cells), gastric cancer cells (e.g., KATO III cells), liver cancer cells (e.g., Hep G2 cells), lung cancer cells (e.g., A549 cells), lymphoma cancer cells (e.g., BJAB cells), breast cancer cells (e.g., BT-20 cells), ovarian cancer cells (e.g., A2780 cells), pancreatic cancer cells (e.g., BxPC-3 cells), prostate cancer cells (e.g., 22Rvl cells), and renal cancer cells (Caki-1 cells), skin cancer cells (A-375 cells). In some embodiments, the first biological material is an antigen presenting cell. In some embodiments, the first biological material is a cancer cell. In some embodiments, the first biological material is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antibody binding fragment thereof is coupled to a bead. In some embodiments, the analyte is a CAR T cell and biological material is a cancer cell and the interaction is the killing of the cancer cell by the CAR T cell. A particular CAR T cell can be subjected to multiple killing tests in a serial manner with multiple cancer cells. After one or more killing tests, the CAR T cell can transition to an exhausted state where it can no longer kill cancer cells. In various embodiments, the identification of particular CAR T cells that are capable of serially killing a number of cancer cells above a predetermined threshold or that serially killed a largest number of cancer cells of a cohort of cancer cell is advantageous for screening or characterizing CAR T cells. Moreover, the identified CAR T cells can then have its genetic information analyzed or mRNA analyzed for determining a cohort or type of CAR T cells suitable for various disease states such as cancer.

[0077] As disclosed elsewhere herein, certain disclosed methods measure effector cell (‘analyte’) killing of target cells (‘biological materials’). As used herein, the term “killing” may be used interchangeably with the term “cytotoxicity”. Cell killing may be measured by coenclosing an effector cell in a chamber with one or more target cells, and then counting dead cells, viable cells, or a combination thereof from among the one or more target cells. Such a method may include synthesizing a chamber that co-encloses an effector cell with multiple subchambers, each of the sub-chambers enclosing one or more target cells; sequentially degrading the sub-chambers to release the one or more target cells to the effector cell, and detecting living or dead cells from among the one or more target cells. The detecting may be performed by combining the one or more target cells with a vital dye (e.g., inputting a vital dye into the fluidic device in which the effector cell and target cell are co-enclosed within a chamber or pretreating the one or more target cells with the vital dye before inputting the one or more target cells into a fluidic device), wherein the vital dye generates an optical signal in response to a characteristic of a living target cell or of a dead target cell. Examples of vital dyes consistent with the present disclosure include SytoxAADvanced, APC-Annexin V, FITC-Annexin V, propidium iodide, 7- Aminoactinomycin D, trypan blue, and erythrosine.

[0078] In certain methods disclosed herein, cell killing may be measured by enclosing and a target cell in separate chambers, and incubating the target cell with a vital dye to determine whether the effector cell released one or more soluble factors that diffused to and then killed the target cell. Such methods can further include measuring soluble factor secretion by the effector cell, for example by: (i) positioning a capture surface with capture antibodies configured to bind to one or more soluble factors near the effector cell (e.g., within the chamber enclosing the effector cell or in an additional chamber near the chamber enclosing the effector cell), (ii) coupling detection antibodies to soluble factors coupled to the capture antibodies on the capture surface, and (iii) detecting the detection antibodies. Examples of soluble factors that may be measured in such a method include cytokines, chemokines, complement proteins, granzymes, perforins, and combinations thereof.

Assays

[0079] A disclosed method can include one or more forms of cellular analysis. Such analyses may be performed on an analyte (e.g., an effector cell), a biological material (e.g., a target cell), or a combination thereof. Analysis may be performed on a single cell or collection of cells that are enclosed within a chamber within a fluidic device. Accordingly, cells within an individual chamber can be tracked independently of other cells that are present in the fluidic device. Two or more forms of analysis can be performed sequentially or in tandem. Examples of cellular characteristics that can be measured in a disclosed method include cytotoxicity, proliferation rate, activation status, cellular identity, purity, gene expression profile, transcriptome, surface marker expression, gRNA expression, soluble factor secretion, activation status, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), morphology, subcellular localization, intracellular protein expression, or a combination thereof. In certain methods, one or more characteristics of an analyte (e.g., an effector cell) are measured prior to, during, or after a measurement of an interaction between the analyte and a biological material. Similarly, one or more characteristics of a biological material (e.g., a target cell) can be measured prior to, during, or after a measurement of an interaction between the biological material and an analyte. For example, following a set of cell killing measurements on an effector cell, the effector cell can be subjected to one or more assays that assess the effector cell’s proliferation rate, activation status, cellular identity, gene expression profile, transcriptome, surface marker expression, gRNA expression, soluble factor secretion, activation status, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), morphology, subcellular localization, intracellular protein expression, or a combination thereof.

[0080] A method disclosed herein can include detecting a guide ribonucleic acid (gRNA) associated with a genetic modification of a cell. For example, following a series of cell killing measurements on an effector cell, a genetic modification of the effector cell can be detected by detecting gRNA associated with the genetic modification. The genetic modification can then be associated with the effector cell’s killing activity. The cell can be transiently or stably transfected with a nucleic acid encoding a gRNA specific for a particular genomic sequence. The guide RNA can be coupled to a barcode, an exogenous messenger RNA (e.g., a selection marker), a capture sequence (e.g., a polyA tail), or a combination thereof. The cell can express a Cas protein that can utilize the gRNA. Alternatively, a Cas protein can be delivered to the cell, for example in a chitosan particle or liposome that is configured for uptake by the cell. Cell growth, movement, or other characteristic or characteristics can then be correlated with a genomic edit imparted by a particular gRNA sequence. In one such method, the cells can be lysed to release guide RNA, the guide RNA can optionally be captured on a nucleic acid barcode, and then be used as a template for generating a cDNA molecule comprising a complement of the guide RNA sequence, and optionally additional sequences coupled to the guide RNA such as the exogenous mRNA, the barcode, or a combination thereof. The cDNA molecule can be coupled to a spatial location tag corresponding to a unique location within the channel of the fluidic device.

[0081] In some aspects, a method includes determining a proliferation rate of a cell. It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. Proliferation rate can be determined by counting cells at least partially enclosed by the one or more chambers generated during an assay. For example, one or more cells can be counted periodically (e.g., with fluorescence or brightfield imaging) following at least partial enclosure within one or more chambers to determine a rate of change in the number of cells. Separate proliferation rates can be determined for each cell or collection of cells enclosed by a unique chamber or collection of chambers. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Example intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. After counts are recorded for each chamber, further assays may be conducted on the clonal populations within the chambers to identify the cell types, for example, by an assessment of cell surface proteins, cell protein secretions, transcriptome, or the like.

[0082] In some aspects, a method includes detecting a soluble factor secreted by a cell. Soluble factor analysis can include disposing a capture surface (e.g., a bead) comprising an affinity reagent (e.g., an aptamer or an antibody) that binds the soluble factor adjacent to the cell, and detecting the soluble factor bound to the capture surface. Disposing the capture surface adj acent to the cell can include enclosing or at least partially enclosing the capture surface with the cell within one or more chambers, and optionally removing non-enclosed capture surfaces from the fluidic device that contains the cell. In an embodiment, the capture surface is a bead. As used herein, the term “bead” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide-based 30 to 10000 pm particle. However, further capture surfaces such as nanotubes, nucleic acid nanostructures, and antibody Fc domains may be used. The capture surface affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof.

[0083] Soluble factor detection may also be performed with a bispecific binding agent capable of simultaneously binding to a cell and to a soluble factor secreted by the cell. The bispecific binding agent can be coupled to a target cell of interest and then used to capture soluble factors secreted by the cell. In this way, the bispecific binding agent may couple the soluble factor to the surface of the cell. The soluble factor may then be detected, for example by coupling a detectable binding agent such as a fluorescent antibody to the soluble factor coupled to the surface of the cell, and measuring the detectable binding agent.

[0084] Examples of soluble factors include a cytokine, an immune active molecule, an interleukin, an interferon, a colony stimulating factor, a tumor necrosis factor, or a granzyme. The cytokine may be interferon-y (IFN-y) and interferon-a (IFN-a), an interleukins such as interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin- 6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL-10), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin-21 (IL-21), or interleukin-23 (IL-23), a colony stimulating factor (CSFs) such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or a tumor necrosis factors (TNF) such as TNF-a or TNF-p. In another embodiment, the secreted factor comprises an effector molecule such as a granzyme.

[0085] A soluble factor bound to a capture surface (e.g., an affinity reagent of a capture surface) can be detected by contacting the soluble factor bound to said capture surface with a labeled ‘detection’ antibody configured to bind to the soluble factor, and detecting the labeled antibody. Multiple soluble factors can be detected in a single assay by providing a capture surface or plurality of capture surfaces that comprise a plurality of affinity reagents configured to bind the plurality of soluble factors, contacting the plurality of soluble factors bound to the capture surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of soluble factors, and detecting a plurality of labels coupled to the plurality of antibodies.

[0086] In some aspects, a method includes sequencing at least a portion of a transcriptome of a cell. A chamber may co-enclose a cell with nucleic acid barcodes within a fluidic device. The nucleic acid barcodes may be coupled to one or more surfaces of the fluidic device. mRNA may be released from the one or more cells (e.g., by lysing the one or more cells) and captured on the nucleic acid barcodes. The nucleic acid barcodes may be extended using at least a portion of the captured mRNA as templates. Similarly, mRNA may be extended using at least a portion of an oligonucleotide barcode as a template. Extended nucleic acid barcodes, extended mRNA molecules, or an additional nucleic acid extended using at least a portion of an extended nucleic acid barcode or extended mRNA molecule as a template may be eluted from the fluidic device and sequenced. Examples of sequencing methods consistent with the present disclosure include nanopore sequencing, pyrosequencing, sequencing-by-hybridization, sequencing-by-ligation, sequencing-by-synthesis, single-molecule sequencing, digital gene expression, next generation sequencing, shotgun sequencing, Sanger sequencing, ion torrent sequencing, as well as other next-generation-sequencing methods known in the art.

[0087] The nucleic acid barcodes may contain spatial barcode sequences that are uniquely associated with the cell and/or the one or more chambers. Accordingly, extended nucleic acid barcodes (which contain the spatial barcodes) and mRNA extended using the nucleic acid barcodes as templates (which contain complements of the spatial barcodes) may be associated with a cell or a chamber. The nucleic acid barcodes may also contain unique molecular identifiers to facilitate mRNA quantitation by normalizing sequencing counts of extended nucleic acid barcodes and/or mRNA extended using the nucleic acid barcodes as templates. For example, during sequencing, the number of instances of each mRNA sequence may be determined based on the number of unique molecular identifier sequences associated with that mRNA sequence.

[0088] In some cases, mRNA and/or nucleic acid barcode extension involves reverse transcription. Reverse transcription reagents may comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murine leukemia virus (MMLV)), dNTPs, optional RNase inhibitor, buffer.

[0089] The sequencing step may be carried out at the sites of the captured mRNAs (in situ) or cDNAs may include a spatial barcode and be eluted and sequenced on a separate sequencing instrument (“external” sequencing). For in situ sequencing, further steps may include (i) amplifying the complementary DNAs, e.g. by bridge amplification, or like method, (ii) sequencing the amplified complementary DNAs, e.g. by a sequencing-by-synthesis technique, and (iii) determining relative expression of the mRNAs for the cells of each of the chambers. For external sequencing, further steps may include (i) providing oligonucleotide barcodes that include spatial barcode sequences, (ii) synthesizing cDNAs that include the spatial barcodes, and (iii) eluting and sequencing the cDNAs and correlating each cDNA with a chamber location by its spatial barcode.

[0090] In some aspects, a method includes measuring a surface marker expressed by a cell. Such a method may include combining a cell with a detection antibody (e.g., inputting the detection antibody into a fluidic device that contains the cell), wherein the detection antibody couples to the surface marker on the surface of the cell, and detecting a detectable moiety coupled to the detection antibody. In certain aspects, the detectable moiety is an optically detectable moiety such as a fluorophore or a dye.

[0091] In some aspects, a method includes measuring activation of one or more cells. Cellular activation can be detected using numerous assays disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation or changes in proliferation, changes in morphology, change in cytotoxicity, or a combination thereof. As nonlimiting examples, these methods are broadly amenable to detecting activation caused by an interaction between an analyte and a biological material.

Porosity of hydrogel chambers and polymer walls

[0092] In some embodiments, the hydrogel polymer wall prohibits the interaction between the analyte and the biological material. In some embodiments, hydrogel porosity is selected to permit passage of selected reagents while at the same time preventing the passage of other reagents or objects, such as, a cell. In some embodiments, hydrogel porosity is selected to prevent the passage of biological cells but to permit the passage of reagents, including proteins, such as polymerases. In some embodiments, such reagents permeable to a polymer matrix wall comprise secreted cellular proteins (e.g., cytokines), lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, deoxynucleotide triphosphates, buffers, cell culture media, or divalent cations. In some embodiments, the at least one polymer matrix comprises pores that are sized to allow diffusion of a reagent through the at least one polymer matrix but are too small to allow DNA or RNA for analysis to traverse the pores (having a size of greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs). In some embodiments, crosslinking 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 the hydrogel structures may be regulated or tuned and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids (e.g., of greater than about 300 base pairs), but to allow smaller materials, such as reagents, or smaller sized nucleic acids (e.g., of less than about 50 base pairs), such as primers, to pass through the pores, thereby passing in and out of the hydrogel structures. In some embodiments, the hydrogels can have any pore size having a diameter sufficient to allow diffusion of the above-listed reagents through the structure while retaining the nucleic acid molecules greater than 500 nucleotides or base pairs in length. In some embodiments, the hydrogel structure can be swollen when the hydrogel is hydrated. The sizes of the pores can then change depending on the water content in the hydrogel of the hydrogel structure. In some embodiments, the pores have a diameter of from about 10 nm to about 100 nm. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like, by routine experimentation. In some embodiments, the average diameter of pores of a polymer matrix wall prevent passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater; or having a molecular weight of 50 kDa or greater; or having a molecular weight of 75 kDa or greater; or having a molecular weight of 100 kDa or greater; or having a molecular weight of 150 kDa or greater. In some cases, the porosity of the polymer matrix can be selectively altered.

[0093] In some embodiments, the analyte, the first biological material, or both, is coupled to the hydrogel polymer wall. In some embodiments, the fluidic device comprises a flow cell. In some embodiments the method further comprises obtaining one or more genetic materials from the analyte. In some embodiments, the method further comprises amplifying the one or more genetic materials. In some embodiments, the amplifying occurs in the fluidic device. In some embodiments, the method further comprises sequencing the one or more genetic materials. In some embodiments, the sequencing occurs in the fluidic device. In some embodiments, the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA comprise messenger RNA (mRNA) or microRNA (miRNA). In some embodiments, the interaction comprises a killing, activation, suppression or binding of the biological material by the analyte. In some embodiments, the interaction comprises a killing, activation, suppression or binding of the first biological material by the analyte. In some embodiments, the interaction comprises a killing, activation, suppression or binding of the second biological material by the analyte. In some embodiments, the killing interaction can be determined where one cell continues to be viable and the other cell is dead where the dead cell is identified by the absorption of a dye (e.g., Sytox™ dead cell stain) that is capable of permeating through a cell membrane readily when it is dead. In some embodiments, the activation can be determined when one of the interacting cell proliferates and reproduces into multiple new cells or has an increased ability (e.g., rate) to kill target cells. In some embodiments, the suppression can be determined when the cell does not reproduce into multiple new cells or slows down the rate of secreting proteins such as, for example, cytokines.

Hydrogel Compositions

[0094] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel. In some embodiments, the degrading comprises exposing the hydrogel polymer wall to UV light. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are made of different materials. In some embodiments, the degrading in (b) does not degrade the hydrogel chamber. In some embodiments, the method further comprises degrading the hydrogel chamber. In some embodiments, the method further comprises imaging the analyte, the first biological material, the hydrogel chamber, the fluidic device, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a cPEG monomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (I):

[0095] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (II):

(II).

[0096] In some embodiments, n is between about 0 to about 100, or optionally n is between about 5 to about 50. In some embodiments, x is between 1 to 10. In some embodiments, x is between 3 to 10. In some cases, x is greater than 3. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (III):

(III).

[0097] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IV):

[0098] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (V):

[0099] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VI):

[0100] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VII):

[0101] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VIII): (vni)

[0102] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IX):

[0103] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (X):

(X).

[0104] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XI):

(XI). [0105] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XII):

(XII).

[0106] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XIII):

(XIII).

[0107] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer. In some embodiments, the oligomeric domain comprises four or more arms. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a degradable functional group. In some embodiments, said degradable function group comprises disulfide, Beta-thioether ester, Amidomethylol and vicinal diol, Vicinal diol, Alginate backbone, Dextran backbone, Chitosan backbone, Hyaluronic acid backbone, Chondroitin sulfate backbone, or Carboxy methyl cellulose backbone, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a hydrogel macromonomer. In some embodiments, the hydrogel macromonomer comprises cPEG, cSEL-BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL-hyaluronic acid, cSEL- chondroitin sulfate, or cSEL-cellulose, or a combination thereof. In some embodiments, the degradation unit is degraded by inputting a degradation reagent into the fluidic device. In some embodiments, the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises at least one beta-thioether ester. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a PEG-macromonomer containing beta-thioether esters. In some embodiments, the beta-thioether ester is formed by reacting an acrylate with a thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Michael donor. In some embodiments, the Michael donor is PEG-thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain. In some embodiments, the Michael acceptor chain comprises PEG-acrylamide, PEG-vinyl sulfone, PEG-maleimide, or PEG-carbonyl acrylic, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by cleavage of disulfide bonds. In some embodiments, the disulfide bonds are cleavable by one or more reducing agents. In some embodiments, the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more arms each comprising one or more amides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photocleavable 4-arm PEG-macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is photodegradable via an ortho-nitrobenzyl moiety. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Coumarin-based photodegradable macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a 4-arm PEG-acrylamide comprising one or more disulfides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation. In some embodiment, the hydrogel cages degrade using light and a photoinitiator. In some embodiments, the hydrogel chamber or the hydrogel polymer wall enables hydrogel formation. In some embodiments, the hydrogel enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation. In some embodiments, upon exposure to light, photogenerated radicals initial multiple fragmentation and disulfide exchange reactions, thereby permitting and promoting photodeformation, photowelding and photodegradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, one or more polymer precursors enable formation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the hydrogel exhibits a chemical or physical change in response to an external stimulus. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the method further comprises controlling a network degradation of the hydrogel chamber or the hydrogel polymer wall by concentration of a photoinitaitor infused into the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the first hydrogel polymer wall comprises a shape configured to contain the first biological material. In some embodiments, the fluidic device comprises a top layer, a bottom layer, and a spacer layer. In some embodiments, the spacer layer includes a cut-out region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region. In some embodiments, the hydrogel chamber is at least partly formed by the top layer and the bottom layer. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are the same material. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are different materials.

[0108] In some embodiments, the hydrogel chamber is made of a first material that degrades upon exposure to a first stimulus. In some cases, a hydrogel polymer wall is made of a second material that degrades upon exposure to a second stimulus. The first stimulus and second stimulus can be different. In some cases, the first stimulus comprises light, and the second stimulus comprises a degradation reagent. In some cases, the first stimulus comprises a degradation reagent, and the second stimulus comprises light. In some cases, the first stimulus comprises a first degradation reagent, and the second stimulus comprises a second degradation reagent different from the first degradation reagent. In some cases, the first stimulus comprises light in a first wavelength range, and the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

Computer Systems

[0109] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 5 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0110] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and/or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and/or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server. [OHl] The CPU 1505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0112] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).

[0113] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additional data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0114] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0115] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0116] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion. [0117] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0118] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0119] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface. [0120] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, identify a biological component, detect a barcode, generate a spatial modulating element (e.g., a mask), provide energy from an energy source, detect or measure a local parameter using a sensor, etc.

EXAMPLES

[0121] The following illustrative examples are representative of embodiments of the devices and methods described herein and are not meant to be limiting in any way.

Example 1. Biological interaction assay using a hydrogel chamber and a hydrogel polymer wall.

[0122] One purpose of this embodiment is to expose an analyte to one or more biological materials and detect an interaction between the analyte and the biological material once the hydrogel polymer wall is degraded (FIG. 1A). For example, in Step 1, the analyte (101) (which can be a CD8 T cell or an NK cell in some cases) and the first biological material (102), which can be an antigen presenting cell such as a cancer cell from a cancer cell line, are enclosed by a hydrogel chamber (104) and separated by a first hydrogel polymer wall (103). While FIG. 1A depicts the first biological material (102) as a single cell, the first biological material may include a plurality of cells, for example about 2 to about 30 cells. The plurality of cells can include copies of a single type of cell (e.g., cancer cells derived from a single cancer cell line) or multiple types of cells (e.g., different types of cancer cells). Similarly, while FIG. 1A depicts the first hydrogel polymer wall (103) as partitioning the hydrogel chamber (104) into two approximately equal subregions, the first hydrogel chamber wall may divide the hydrogel chamber (104) into subregions with unequal areas. In Step 2, the hydrogel polymer wall is degraded, allowing the analyte (101) and the first biological material (102) to interact. The hydrogel polymer wall (103) can be photo-degradable, and light may be used to degrade the hydrogel polymer wall (103). In some cases, the hydrogel polymer wall (103) is degraded upon exposure to a degradation agent. In this example, upon degradation of the hydrogel polymer wall (103), the analyte (101) and the biological material (102) interact. As shown in Step 2 of FIG. 1A, the interaction may result in a killing of the first biological material (102) by the analyte (101), thereby leaving the analyte (101) enclosed in the hydrogel chamber (104).

Example 2. Serial killing assay using orthogonal degradable hydrogels.

[0123] One purpose of this embodiment is to serially expose an analyte (105) to one or more biological components (106 and 110) in a controlled manner. The analyte (105) may be a T cell or an NK cell. The one or more biological components (106 and 110) may be cancer cells (FIG. IB) For example, in Step 1, the analyte (105) can be separated by a first hydrogel polymer wall (107) from a first biological material (106). As shown in FIG. IB, the first hydrogel polymer wall (107) can be in the form of a chamber wall, and may surround the analyte (105). With reference to Step 1, it is worthwhile to note that the analyte (105) can be initially contained by the first hydrogel wall (107) and then the first biological material (106) and polymer precursors can be inputted into the fluidic device so that the first biological material (106) is proximate to the first hydrogel wall (107). By introducing the analyte (105) and the first biological material (106) from separate reservoirs into the fluidic device at different timepoints, the interaction time between the analyte (105) and the first biological material (106) is more controlled. Alternatively, the analyte (105), the biological material (106), and the polymer precursors can be inputted into the fluidic device at the same time and then form the first hydrogel wall (107) for Step 1. In Step 2, the analyte (105), the first hydrogel polymer wall (107) and the first biological material (106) can be enclosed in a first hydrogel chamber (108). With continued reference to FIG. IB Step 2, the analyte (105) may be enclosed in a chamber formed from the first hydrogel polymer wall (107), the first biological material (106) may be outside of the chamber formed from the first hydrogel polymer wall (107). In such configurations of Step 2 of FIG. IB, the first hydrogel chamber may enclose: (i) the first hydrogel polymer wall (107), (ii) the analyte (105) inside of the chamber formed from the first hydrogel polymer wall (107), and (iii) the first biological material (106). In Step 3, the first hydrogel polymer wall (107) can be selectively degraded, allowing the analyte (105) and the first biological material (106) to interact. The hydrogel polymer wall (107) can be photo- degradable, and light may be used to selectively degrade the hydrogel polymer wall (107) and not the hydrogel chamber (108). In some cases, the hydrogel polymer wall (107) is degraded upon exposure to a degradation agent. As shown in Step 4, the interaction may result in a killing of the first biological material (106) by the analyte (105), thereby leaving the analyte (105) enclosed in the hydrogel chamber (108). In some embodiments, the killed first biological material can be washed out of the hydrogel chamber (108). In Step 5, a second hydrogel polymer wall (109) can be formed around the analyte (105) first for retention allowing for another biological material (110) to subsequently be introduced into the fluidic device proximate to the hydrogel chamber (108) . In Step 6, the first hydrogel chamber (108) can be degraded. In Step 7, the analyte (105), the second hydrogel polymer wall (109), and a second biological material (110) can be enclosed in a second hydrogel chamber (111). In Step 8, the second hydrogel polymer wall (109) can be degraded allowing the analyte (105) and second biological material (110) to interact. As shown in Step 9, the interaction may result in a killing of the second biological material (110) by the analyte (105), thereby leaving the analyte (105) enclosed in the second hydrogel chamber (111). This process can be repeated serially n times with n number of biological components. In some cases, the process is repeated until the analyte (105) becomes exhausted. This process can be used to identify a particular analyte cell capable of killing numerous biological components before transitioning to an exhausted state and then subsequently characterizing the mRNA of the analyte cell and/or guide oligonucleotides of the analyte cell indicating a category or class of genetic modification to the analyte cell. The process in this sample can be used to characterize the ability of a single CAR-T to kill a predetermined threshold or maximum number of cancer cells.

Example 3. Serial killing assay using optically cleavable hydrogels.

[0124] One purpose of this embodiment is to serially expose an analyte (112) to multiple biological materials using optically cleavable hydrogels (FIG. 1C). For example, in Step 1, an analyte (112) which can be a T cell or an NK cell is separated by a first hydrogel polymer wall (115) from a first biological material (114). In some cases, the first biological material is a cancer cell. The analyte (112), the first hydrogel polymer wall (115), and the first biological material (114) can be enclosed in a hydrogel chamber (113). The analyte (112) can be contained by generating the first hydrogel polymer wall (115) first, and then hydrogel chamber (113) can be generated in such a way that a plurality biological components are each separately contained within hydrogel chamber (113). In Step 2, a portion of the first hydrogel polymer wall (115) can be selectively cleaved by light (such as to not disturb other hydrogel structures). In Step 3, the analyte (112) and the first biological material (114) can interact. As shown in Step 4, the interaction can result in a killing of the first biological material (114). In Step 5, a second hydrogel polymer wall (117) that separates a second biological material (116) from the analyte (112) can have at least a portion be selectively cleaved by light. The first biological material (114) and second biological material (116) can initially be separated by a polymer structure (119). Different biological materials can compartmentalized and separated from other biological materials by polymer structures similar to (119). In Step 6, the cleaving of the portion of the second hydrogel polymer wall (117) can allow the analyte (112) and the second biological material (116) to interact. As shown in Step 7, the interaction may result in a killing of the second biological material (116). This process can be repeated to observe an interaction between the analyte (112) and additional biological materials in a sequential manner (by degrading additional polymer walls separating the analyte and additional biological materials). In some embodiments, multiple portions of the hydrogel polymer wall (e.g., 115 and 117) can be selectively cleaved by light at the same time to allow interaction of the analyte with multiple biological materials (e.g., 114 and 116). For example, two or more polymer walls separating the analyte and biological materials can be simultaneously cleaved in order to control the ratio of interacting CAR T cells to interacting cancer cells. In some embodiments, functional assays may be performed on the analyte or biological materials to determine which biological materials will be selected to interact with the analyte. For example, the functional assay may be a measure of secreted proteins or surface receptors on the analyte or on the biological materials. In some embodiments, only the biological materials having at least a predetermined threshold amount of cytokines will be exposed to the analyte.

Example 4. Serial antibody screening assay using orthogonally degradable hydrogels.

[0125] FIG. 2 depicts a method for sequentially or simultaneously releasing one or more biological materials (120, 123) to an analyte (118). The biological materials (120, 123) and analyte (118) may be enclosed by separate sub-chambers (121, 122, 124) within a larger chamber (119) (‘Step 1 ’). The sub-chamber (122) enclosing the analyte (118) can be degraded (as shown in ‘Step 2’). Then, the sub-chambers (121, 124) enclosing the biological materials can be sequentially degraded (as shown in ‘Steps 2-4’) to release biological materials (120, 123) to the analyte (118). Following sub-chamber (121, 122, 124) degradation, the analyte (118) and biological materials (120, 123) may be kept in proximity by the larger chamber (119). Interactions between the analyte (118) and the biological materials (120, 123) may be measured by one or more assays disclosed herein.

[0126] A fluidic device may include a plurality of instances of the configuration shown in ‘ Step 1’ of FIG. 2 (the larger chamber (119), the sub-chambers (121, 122, 124), the analyte (118), and the biological materials (120, 13)). The method of FIG. 2 may be performed on each of the instances of the separate instances of the ‘Step 1’ configuration to generate replicates. The method may vary between instances of the ‘Step 1’ configuration, for example by delaying for different amounts of times between sub-chamber degradation steps (‘Steps 2-4’), or by omitting a sub-chamber degradation step (e.g., not degrading sub-chamber 121). Such an assay may thus generate a plurality of replicates and/or treatment groups from parallel assays in a single fluidic device. As an example, when a fluidic device contains multiple instances of the configuration shown in ‘Step 1’ of FIG. 2, the length of time in between ‘Steps 1-4’ may be varied across instances of the configuration.

[0127] One purpose of this embodiment is to screen antibodies for cell activation using orthogonally degradable hydrogels (FIG. 2). One or more of the biological materials can be in the form of an antibody coupled to a bead. The bead may include one or more antibodies or signaling molecules that activate the analyte (118), polarize the analyte (118), prime the analyte (118) for expansion, suppress an activity of the analyte (118), or a combination thereof. Examples of such beads include: (i) beads with anti-CD3 and anti-CD28 antibodies for T cell activation, (ii) beads with a toll-like receptor agonist (e.g., TLR1/2 agonist Pam3CSK4, TLR2/6 agonist Pam2CGDPKHPKSF, TLR4 agonist LPS -EK, TLR5 agonist FL A- ST, and the like) or antagonist (e.g. pan-TLR antagonist chloroquine diphosphate, TLR1/2 agonist CU- T12-9, and the like) for immune cell activity modulation, and (iii) beads with an immune checkpoint inhibitor (e.g., ipilumumab, tremelimumab, nivolumab, pemrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, dostarlimab, relatimab, and the like).

[0128] With continued reference to FIG. 2, the one or more of the biological materials can be a cell or a plurality of cells. As shown in FIG. 2, in some embodiments, the analyte (118) is a cell and the biological materials (120, 123) are each a single cell. However, in other cases, the analyte (118) includes multiple cells, one or more of the biological materials (120, 123) include multiple cells, or a combination thereof.

[0129] As an example of the method of FIG. 2, in Step 1, the analyte (118) can be separated from a first biological material (120) by a first hydrogel polymer wall (121) and a hydrogel polymer wall (122) surrounding the analyte (118). The analyte (118) can also be separated from a second biological material (123) by a second hydrogel polymer wall (124) and the hydrogel polymer wall surrounding the analyte (122). The analyte (118), the hydrogel polymer walls surrounding the analyte (122), the first biological material (120), the first hydrogel polymer wall (121), the second biological material (123), and the second hydrogel polymer wall (124) can be enclosed in a hydrogel chamber (119). Hydrogel chamber (119) may also include additional biological materials that can be physically separated from the analyte (118) by additional polymer walls. In Step 2, the first hydrogel polymer wall (121) and the hydrogel polymer wall surrounding the analyte (122) can be degraded, thereby allowing the analyte (118) and the first biological material (120) to interact. In Step 3, the second hydrogel polymer wall (124) can be degraded, thereby allowing the analyte (118) and the second biological material (123) to interact. In Step 4, a third hydrogel polymer wall is degraded allowing the analyte and a third biological material to interact. This can be repeated serially for any number of biological materials separated by any number of hydrogel polymer walls.

[0130] With continued reference to FIG. 2, biological materials (120, 123) co-enclosed with an analyte (118) may be identical or different. In some cases, a hydrogel chamber (119) coencloses multiple types of biological materials (120, 123) with an analyte (118). For example, an effector cell (analyte, 118) can be co-enclosed with two or more types of target cells (biological materials 120, 123), such as two or more different types of cancer cells. Individual target cells (biological materials 120, 123) can be sequentially released to the analyte to measure the effector cell’s (analyte, 118) killing capacity against each of the individual target cells. Such an assay may be used to identify effector cells (analyte, 118) that are capable of killing multiple types of target cells (biological materials 120, 123).

[0131] In another example of the method of FIG. 2, the analyte (118) may be an effector cell, and the biological materials (120, 123) can include a combination of healthy cells and cancer cells. The biological materials (120, 123) may be sequentially released to the effector cell (analyte, 118) to assess the effector cell’s killing capacity against each healthy cell and cancer cell (biological materials 120, 123). Such an assay may be used to identify effector cells (analyte, 118) that have killing capacity against diseased cells, but which do not cross-react with healthy cells, and therefore pose low risks for autoimmune activity.

Example 5: Light-mediated degradation of sub-chambers to release cells into a common chamber

[0132] This example covers the selective degradation of sub-chambers within a larger chamber to release cells into a common space within the larger chamber. Cells were mixed with a polymer precursor and then input into a fluidic device. Light was then projected into the fluidic device to photopolymerize the polymer precursor. As shown in FIG. 6A, the light generated a large chamber (601) and multiple sub-chambers (602, 603, 604) within the large chamber (601). Each sub-chamber (602, 603, 604) enclosed a cell (an example cell is indicated by the label 605). Next, as shown in FIG. 6B, two of the sub-chambers (603, 604) were photodegraded by irradiating the sub-chambers (603, 604) in the presence of a photoinitiator. As shown in FIG. 6B, the sub-chambers (603, 604) were fully degraded, thereby releasing the cells enclosed in the sub-chambers within the larger chamber (601). As further shown in FIG. 6B, sub- chamber degradation did not lyse any of the cells enclosed within the larger chamber (601). The cells were subsequently stained with the sytox orange and annexin-v to measure cell death. No color change was detected following either staining procedure, suggesting that sub-chamber photodegradation did not kill cells in the fluidic device.

Example Embodiments

[0133] The embodiments listed below are example embodiments of the systems and methods described herein, and do not limit the description above:

[0134] Embodiment 1. A method for analyzing an analyte, the method comprising: (a) inputting the analyte and one or more biological materials into a fluidic device, wherein the fluidic device comprises a hydrogel chamber comprising: i) at least a portion of the analyte, and ii) at least a portion of a first biological material of the one or more biological materials, wherein the analyte and the first biological material are physically separated by a first hydrogel polymer wall; (b) degrading at least a portion of the first hydrogel polymer wall; and (c) detecting an interaction between the analyte and the first biological material.

[0135] Embodiment 2. The embodiment of embodiment 1, wherein the first hydrogel polymer wall surrounds the analyte.

[0136] Embodiment 3. The method of embodiment 2, wherein in (a), the inputting of the analyte and the inputting of the one or more biological materials into the fluidic device occurs separately.

[0137] Embodiment 4. The method of embodiment 3, wherein the first hydrogel polymer wall prevents an interaction between the analyte and the one or more biological materials.

[0138] Embodiment 5. The method of any one of embodiments 1-4, further comprising: (d) generating a second hydrogel polymer wall, wherein the analyte and a second biological material are physically separated by the second hydrogel polymer wall.

[0139] Embodiment 6. The method of embodiment 5, wherein the second hydrogel polymer wall surrounds the analyte.

[0140] Embodiment 7. The method of embodiment 5 or 6, further comprising: (e) degrading the hydrogel chamber, and (f) forming a second hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material.

[0141] Embodiment 8. The method of any one of embodiments 5-7, further comprising: (g) degrading at least a portion of the second hydrogel polymer wall.

[0142] Embodiment 9. The method of embodiment 8, further comprising: (h) detecting an interaction between the analyte and the second biological material. [0143] Embodiment 10. The method of embodiment 9, further comprising: (i) generating a third hydrogel polymer wall, wherein the analyte and a third biological material are physically separated by the third hydrogel polymer wall.

[0144] Embodiment 11. The method of embodiment 10, wherein the third hydrogel polymer wall surrounds the analyte.

[0145] Embodiment 12. The method of embodiment 10 or 11, further comprising: (j) degrading the second hydrogel chamber, and (f) forming a third hydrogel chamber around at least a portion of the analyte and at least a portion of the third biological material.

[0146] Embodiment 13. The method of any one of embodiments 10-12, further comprising: (k) degrading at least a portion of the third hydrogel polymer wall.

[0147] Embodiment 14. The method of embodiment 13, further comprising: (1) detecting an interaction between the analyte and the third biological material.

[0148] Embodiment 15. The method of embodiment 1, wherein the analyte is physically separated from a second biological material by a second hydrogel polymer wall, and wherein the hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, and (iii) the second biological material.

[0149] Embodiment 16. The method of embodiment 15, further comprising, subsequent to (c): (m) degrading at least a portion of the second hydrogel polymer wall, and (n) detecting an interaction between the analyte and the second biological material.

[0150] Embodiment 17. The method of embodiment 16, wherein the analyte is physically separated from a third biological material by a third hydrogel polymer wall, and wherein the hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, (iii) the second biological material, and (iv) the third biological material.

[0151] Embodiment 18. The method of embodiment 17, further comprising, subsequent to (e): (f) degrading at least a portion of the third hydrogel polymer wall, and (g) detecting an interaction between the analyte and the third biological material.

[0152] Embodiment 19. A method for analyzing an analyte, the method comprising: a) detecting an interaction between the analyte and a first biological material of one or more biological materials in a fluidic device; and b) forming a hydrogel polymer wall that physically separates the analyte from a second biological material of the one or more biological materials. [0153] Embodiment 20. The method of any one of embodiments 1-19, wherein the analyte is selected from a plurality of analytes in the fluidic device prior to (a).

[0154] Embodiment 21. The method of any one of embodiments 1-20, wherein the analyte is a cell. [0155] Embodiment 22. The method of any one of embodiments 1-21, wherein the analyte is an antigen targeting cell.

[0156] Embodiment 23. The method of any one of embodiments 1-22, wherein the analyte is a CD8+ T cell or NK cell.

[0157] Embodiment 24. The method of any one of embodiments 1-23, wherein the analyte is a genetically engineered cell.

[0158] Embodiment 25. The method of any one of embodiments 1-24, wherein the analyte is a CAR T cell.

[0159] Embodiment 26. The method of any one of embodiments 1-25, wherein the first biological material is a cell.

[0160] Embodiment 27. The method of any one of embodiments 1-26, wherein the first biological material is an antigen presenting cell.

[0161] Embodiment 28. The method of any one of embodiments 1-27, wherein the first biological material is a cancer cell.

[0162] Embodiment 29. The method of any one of embodiments 1-28, wherein the first biological material is an antibody or antigen binding fragment thereof.

[0163] Embodiment 30. The method of embodiment 29, wherein the antibody or antibody binding fragment thereof is coupled to a bead, and wherein the bead cannot diffuse through the hydrogel polymer wall.

[0164] Embodiment 31. The method of any one of embodiments 1-30, wherein the analyte, the first biological material, or both, is coupled to the hydrogel polymer wall.

[0165] Embodiment 32. The method of any one of embodiments 1-31, the fluidic device comprises a flow cell.

[0166] Embodiment 33. The method of any one of embodiments 1-32, wherein the method further comprises obtaining one or more genetic materials from the analyte.

[0167] Embodiment 34. The method of embodiment 33, wherein the method further comprises amplifying the one or more genetic materials.

[0168] Embodiment 35. The method of embodiment 34, wherein the amplifying occurs in the fluidic device.

[0169] Embodiment 36. The method of any one of embodiments 33-35, wherein the method further comprises sequencing the one or more genetic materials.

[0170] Embodiment 37. The method of embodiment 36, wherein the sequencing occurs in the fluidic device. [0171] Embodiment 38. The method of any one of embodiments 33-36, wherein the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0172] Embodiment 39. The method of embodiment 38, wherein the RNA comprise messenger RNA (mRNA) or microRNA (miRNA).

[0173] Embodiment 40. The method of any one of embodiments 1-39, wherein the interaction comprises a killing of the first biological material by the analyte or vice versa.

[0174] Embodiment 41. The method of any one of embodiments 1-39, wherein the interaction comprises activation of the first biological material by the analyte or vice versa.

[0175] Embodiment 42. The method of embodiment 41, wherein detecting the activation comprises counting a number of proliferated cells from the first biological material.

[0176] Embodiment 43. The method of embodiment 41, wherein detecting the activation comprises determining a presence of a surface antigen on a cell of the first biological material. [0177] Embodiment 44. The method of any one of embodiments 1-39, wherein the interaction comprises suppression of the first biological material by the analyte or vice versa.

[0178] Embodiment 45. The method of any one of embodiments 1-39, wherein the interaction comprises binding or physical contact between the first biological material and the analyte.

[0179] Embodiment 46. The method of any one of embodiments 1-45, wherein the first hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel.

[0180] Embodiment 47. The method of any one of embodiments 1-46, wherein the degrading in (b) comprises exposing the first hydrogel polymer wall to UV light.

[0181] Embodiment 48. The method of any one of embodiments 1-47, wherein the degrading in (b) comprises selectively exposing the first hydrogel polymer wall to UV light in a presence of photoinitiator and not exposing the first hydrogel chamber to the UV light.

[0182] Embodiment 49. The method of any one of embodiments 1-48, further comprising imaging the analyte, the first biological material, the hydrogel chamber, the fluidic device, or any combination thereof.

[0183] Embodiment 50. The method of any one of embodiments 1-49, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a cPEG monomer. [0184] Embodiment 51. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (I):

[0185] Embodiment 52. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (II):

(II).

[0186] Embodiment 53. The method of embodiment 52, wherein n is between about 0 to about 100, or optionally n is between about 5 to about 50.

[0187] Embodiment 54. The method of embodiment 52, wherein x is between 3 and 10.

[0188] Embodiment 55. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (III):

(III).

[0189] Embodiment 56. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IV):

[0190] Embodiment 57. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (V):

[0191] Embodiment 58. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VI):

[0192] Embodiment 59. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VII):

[0193] Embodiment 60. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VIII): [0194] Embodiment 61. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure

[0195] Embodiment 62. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (X): (X).

[0196] Embodiment 63. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XI): (XI).

[0197] Embodiment 64. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XII):

(XII).

[0198] Embodiment 65. The method of any one of embodiments 1-50, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XIII):

(XIII).

[0199] Embodiment 66. The method of any one of embodiments 1-49, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer.

[0200] Embodiment 67. The method of embodiment 66, wherein the oligomeric domain comprises four or more arms. [0201] Embodiment 68. The method of embodiment 66, wherein the oligomeric domain comprises 3 arms to 7 arms.

[0202] Embodiment 69. The method of any one of embodiments 1-68, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a degradable functional group.

[0203] Embodiment 70. The method of embodiment 69, wherein said degradable function group comprises disulfide, beta-thioether ester, amidomethylol and vicinal diol, vicinal diol, alginate backbone, dextran backbone, chitosan backbone, hyaluronic acid backbone, chondroitin sulfate backbone, or carboxy methyl cellulose backbone, or a combination thereof.

[0204] Embodiment 71. The method of any one of embodiments 1-70, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a polymerized form of a hydrogel macromonomer.

[0205] Embodiment 72. The method of embodiment 71, wherein the hydrogel macromonomer comprises cPEG, cSEL-BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL-hyaluronic acid, cSEL-chondroitin sulfate, or cSEL- cellulose, or a combination thereof.

[0206] Embodiment 73. The method of any one of embodiments 1-72, wherein the hydrogel chamber or the first hydrogel polymer wall are degradable upon interaction with a degradation reagent.

[0207] Embodiment 74. The method of embodiment 73, wherein the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof.

[0208] Embodiment 75. The method of any one of embodiments 1-74, wherein the hydrogel chamber or the first hydrogel polymer wall comprises at least one beta-thioether ester.

[0209] Embodiment 76. The method of any one of embodiments 1-75, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a PEG-macromonomer containing beta-thioether esters.

[0210] Embodiment 77. The method of embodiment 75 or 76, wherein the beta-thioether ester is formed by reacting an acrylate with a thiol.

[0211] Embodiment 78. The method of any one of embodiments 1-77, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a Michael donor.

[0212] Embodiment 79. The method of embodiment 78, wherein the Michael donor is PEG- thiol. [0213] Embodiment 80. The method of any one of embodiments 1-79, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm.

[0214] Embodiment 81. The method of any one of embodiments 1-80, wherein the hydrogel chamber or the first hydrogel polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain.

[0215] Embodiment 82. The method of embodiment 81, wherein the Michael acceptor chain comprises PEG-acrylamide, PEG-vinyl sulfone, PEG-maleimide, or PEG-carbonyl acrylic, or any combination thereof.

[0216] Embodiment 83. The method of any one of embodiments 1-82, wherein the hydrogel chamber or the first hydrogel polymer wall is degradable by cleavage of disulfide bonds.

[0217] Embodiment 84. The method of embodiment 83, wherein the disulfide bonds are cleavable by one or more reducing agents.

[0218] Embodiment 85. The method of embodiment 84, wherein the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof.

[0219] Embodiment 86. The method of any one of embodiments 1-85, wherein the hydrogel chamber or the first hydrogel polymer wall comprises one or more arms each comprising one or more amides.

[0220] Embodiment 87. The method of any one of embodiments 1-86, wherein the hydrogel chamber or the first hydrogel polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate.

[0221] Embodiment 88. The method of any one of embodiments 1-87, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a photocleavable 4-arm PEG- macromonomer.

[0222] Embodiment 89. The method of any one of embodiments 1-88, wherein the hydrogel chamber or the first hydrogel polymer wall is photodegradable via an ortho-nitrobenzyl moiety.

[0223] Embodiment 90. The method of any one of embodiments 1-89, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a Coumarin-based photodegradable macromonomer.

[0224] Embodiment 91. The method of any one of embodiments 1-90, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a 4-arm PEG-acrylamide comprising one or more disulfides. [0225] Embodiment 92. The method of any one of embodiments 1-91, wherein the hydrogel chamber or the first hydrogel polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation, wherein the hydrogel cages degrade using light and a photoinitiator. [0226] Embodiment 93. The method of any one of embodiments 1-92, wherein the hydrogel chamber or the first hydrogel polymer wall enables hydrogel formation, and wherein the hydrogel enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation.

[0227] Embodiment 94. The method of any one of embodiments [0025]-93, wherein upon exposure to light, photogenerated radicals initial multiple fragmentation and disulfide exchange reactions, thereby permitting and promoting photodeformation, photowelding and photodegradation of the hydrogel chamber or the first hydrogel polymer wall.

[0228] Embodiment 95. The method of any one of embodiments 1-94, wherein the inputting comprises inputting one or more polymer precursors into the fluidic device to enable formation of the hydrogel chamber or the first hydrogel polymer wall, wherein the hydrogel exhibits a chemical or physical change in response to an external stimulus.

[0229] Embodiment 96. The method of any one of embodiments 1-95, wherein the hydrogel chamber or the first hydrogel polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel chamber or the first hydrogel polymer wall.

[0230] Embodiment 97. The method of any one of embodiments 1-96, wherein the method further comprises controlling a network degradation of the hydrogel chamber or the first hydrogel polymer wall by inputting a concentration of a photoinitiator into the fluidic device, wherein the photoinitiator is infused into a polymeric portion of the hydrogel chamber or the hydrogel polymer wall.

[0231] Embodiment 98. The method of any one of embodiments 1-97, wherein the first hydrogel polymer wall comprises a shape configured to contain the first biological material. [0232] Embodiment 99. The method of any one of embodiments 1-98, wherein the fluidic device comprises a top layer, a bottom layer, and a spacer layer, wherein the spacer layer includes a cut-out region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region, and wherein the hydrogel chamber is at least partly formed by the top layer and the bottom layer.

[0233] Embodiment 100. The method of any one of embodiments 1-99, wherein the fluidic device comprises a channel with an inlet and an outlet, wherein the channel comprises a first surface and a second surface disposed opposite one another across the channel, wherein a polymer matrix wall extends between the first surface and the second surface, thereby forming the hydrogel chamber.

[0234] Embodiment 101. The method of any one of embodiments 1-100, wherein the hydrogel chamber and the first hydrogel polymer wall are the same material.

[0235] Embodiment 102. The method of any one of embodiments 1-101, wherein the hydrogel chamber is cured for a first duration of time, and wherein the first hydrogel polymer wall is cured for a second duration of time.

[0236] Embodiment 103. The method of embodiment 102, wherein the first duration of time is longer than the second duration of time.

[0237] Embodiment 104. The method of embodiment 102, wherein the second duration of time is longer than the first duration of time.

[0238] Embodiment 105. The method of any one of embodiments 102-104, wherein the hydrogel chamber and the first hydrogel polymer wall have different kinetics of degradation. [0239] Embodiment 106. The method of any one of embodiments 1-100, wherein the hydrogel chamber and the first hydrogel polymer wall are different materials.

[0240] Embodiment 107. The method of embodiment 106, wherein the hydrogel chamber is made of a first material, wherein the first material degrades upon exposure to a first stimulus, wherein the first hydrogel polymer wall is made of a second material, wherein the second material degrades upon exposure to a second stimulus, and wherein the first stimulus and second stimulus are different.

[0241] Embodiment 108. The method of embodiment 107, wherein the first stimulus comprises light, and wherein the second stimulus comprises a degradation reagent.

[0242] Embodiment 109. The method of embodiment 107, wherein the first stimulus comprises a degradation reagent, and wherein the second stimulus comprises light.

[0243] Embodiment 110. The method of embodiment 107, wherein the first stimulus comprises a first degradation reagent, and wherein the second stimulus comprises a second degradation reagent different from the first degradation reagent.

[0244] Embodiment 111. The method of embodiment 107, wherein the first stimulus comprises light in a first wavelength range, and wherein the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

[0245] Embodiment 112. The method of embodiment 107, wherein the first stimulus comprises UV light selectively applied to the first hydrogel polymer wall in a presence of photoinitiator, and wherein the second stimulus comprises UV light selectively applied to the hydrogel chamber in the presence of photoinitiator. [0246] Embodiment 113. A flow cell for analyzing an analyte, the flow cell comprising a hydrogel polymer structure comprising at least two chambers, wherein the at least two chambers are physically separated by a degradable polymer wall, wherein a first chamber of the at least two chambers comprises the analyte, and wherein a second chamber of the at least two chambers comprises a first biological material.

[0247] Embodiment 114. The flow cell of embodiment 113, wherein the second chamber comprises the first chamber.

[0248] Embodiment 115. The flow cell of embodiment 113, wherein the second chamber and the first chamber comprise a shared wall, and wherein the shared wall is the degradable polymer wall.

[0249] Embodiment 116. The flow cell of any one of embodiments 113-115, wherein the hydrogel polymer structure or the degradable polymer wall comprises an optically cleavable hydrogel.

[0250] Embodiment 117. The flow cell of embodiment 116, wherein the optically cleavable hydrogel is configured to be degraded by UV light.

[0251] Embodiment 118. The flow cell of any one of embodiments 113-117, wherein the analyte is a cell.

[0252] Embodiment 119. The flow cell of any one of embodiments 113-118, wherein the analyte is an antigen targeting cell.

[0253] Embodiment 120. The flow cell of any one of embodiments 113-119, wherein the analyte is a CD8+ T cell orNK cell.

[0254] Embodiment 121. The flow cell of any one of embodiments 113-120, wherein the analyte is a genetically engineered cell.

[0255] Embodiment 122. The flow cell of any one of embodiments 113-121, wherein the analyte is a CAR T cell.

[0256] Embodiment 123. The flow cell of any one of embodiments 113-122, wherein the first biological material is a cell.

[0257] Embodiment 124. The flow cell of any one of embodiments 113-123, wherein the first biological material is an antigen presenting cell.

[0258] Embodiment 125. The flow cell of any one of embodiments 113-124, wherein the first biological material is a cancer cell.

[0259] Embodiment 126. The flow cell of any one of embodiments 113-135, wherein the first biological material is an antibody or antigen binding fragment thereof. [0260] Embodiment 127. The flow cell of embodiment 126, wherein the antibody or antibody binding fragment thereof is coupled to a bead, wherein the bead cannot diffuse through the hydrogel polymer wall.

[0261] Embodiment 128. The flow cell of any one of embodiments 113-127, wherein the analyte, the first biological material, or both, is coupled to the degradable polymer wall.

[0262] Embodiment 129. The flow cell of embodiment 128, wherein degradation of the degradable polymer wall releases the analyte, the first biological material, or both.

[0263] Embodiment 130. The flow cell of any one of embodiments 113-129, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a cPEG monomer.

[0264] Embodiment 131. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (I):

[0265] Embodiment 132. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (II):

(II).

[0266] Embodiment 133. The flow cell of embodiment 132, wherein n is between about 0 to about 100, or optionally n is between about 5 to about 50.

[0267] Embodiment 134. The flow cell of embodiment 132, wherein x is between 3 and 10.

[0268] Embodiment 135. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (III):

(III).

[0269] Embodiment 136. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (IV): [0270] Embodiment 137. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (V):

[0271] Embodiment 138. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (VI):

[0272] Embodiment 139. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (VII): (VII).

[0273] Embodiment 140. The flow cell of any one of embodiments 113-130, wherein the hydrogel comprises a polymerized form of a monomer including a structure (VIII):

[0274] Embodiment 141. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (IX):

[0275] Embodiment 142. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form

[0276] Embodiment 143. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form

[0277] Embodiment 144. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (XII):

(XII).

[0278] Embodiment 145. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer including a structure (XIII):

(XIII).

[0279] Embodiment 146. The flow cell of any one of embodiments 113-130, wherein the hydrogel polymer structure or the degradable polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer.

[0280] Embodiment 147. The flow cell of embodiment 146, wherein the oligomeric domain comprises four or more arms.

[0281] Embodiment 148. The flow cell of any one of embodiments 113-147, wherein the hydrogel polymer structure or the degradable polymer wall comprises a degradable functional group.

[0282] Embodiment 149. The flow cell of embodiment 148, where the degradable function group comprises disulfide, Beta-thioether ester, Amidomethylol and vicinal diol, Vicinal diol, Alginate backbone, Dextran backbone, Chitosan backbone, Hyaluronic acid backbone, Chondroitin sulfate backbone, or Carboxy methyl cellulose backbone, or a combination thereof.

[0283] Embodiment 150. The flow cell of any one of embodiments 113-149, wherein the hydrogel polymer structure or the degradable polymer wall comprises a hydrogel macromonomer. [0284] Embodiment 151. The flow cell of embodiment 150, wherein the hydrogel macromonomer comprises cPEG, cSEL-BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL-hyaluronic acid, cSEL-chondroitin sulfate, or cSEL- cellulose, or a combination thereof.

[0285] Embodiment 152. The flow cell of any one of embodiments 113-151, wherein the hydrogel polymer structure or the degradable polymer wall are degradable upon interaction with a degradation reagent.

[0286] Embodiment 153. The flow cell of embodiment 152, wherein the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof.

[0287] Embodiment 154. The flow cell of any one of embodiments 113-153, wherein the hydrogel polymer structure or the degradable polymer wall comprises at least one betathioether ester.

[0288] Embodiment 155. The flow cell of any one of embodiments 113-154, wherein the hydrogel polymer structure or the degradable polymer wall comprises a PEG-macromonomer containing beta-thioether esters.

[0289] Embodiment 156. The flow cell of embodiment 154 or 155, wherein the beta- thioether ester is formed by reacting an acrylate with a thiol.

[0290] Embodiment 157. The flow cell of any one of embodiments 113-156, wherein the hydrogel polymer structure or the degradable polymer wall comprises a Michael donor.

[0291] Embodiment 158. The flow cell of embodiment 157, wherein the Michael donor is PEG-thiol.

[0292] Embodiment 159. The flow cell of any one of embodiments 113-158, wherein the hydrogel polymer structure or the degradable polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm.

[0293] Embodiment 160. The flow cell of any one of embodiments 113-159, wherein the hydrogel polymer structure or the degradable polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain.

[0294] Embodiment 161. The flow cell of embodiment 160, wherein the Michael acceptor chain comprises PEG-acrylamide, PEG-vinyl sulfone, PEG-maleimide, or PEG-carbonyl acrylic, or any combination thereof. [0295] Embodiment 162. The flow cell of any one of embodiments 113-161, wherein the hydrogel polymer structure or the degradable polymer wall is degradable by cleavage of disulfide bonds.

[0296] Embodiment 163. The flow cell of embodiment 162, wherein the disulfide bonds are cleavable by one or more reducing agents.

[0297] Embodiment 164. The flow cell of embodiment 163, wherein the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof.

[0298] Embodiment 165. The flow cell of any one of embodiments 113-164, wherein the hydrogel polymer structure or the degradable polymer wall comprises one or more arms each comprising one or more amides.

[0299] Embodiment 166. The flow cell of any one of embodiments 113-165, wherein the hydrogel polymer structure or the degradable polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate.

[0300] Embodiment 167. The flow cell of any one of embodiments 113-166, wherein the hydrogel polymer structure or the degradable polymer wall comprises a vicinal diol functionality that can be cleaved via oxidation of one or more hydroxyls.

[0301] Embodiment 168. The flow cell of any one of embodiments 113-167, wherein the hydrogel polymer structure or the degradable polymer wall is used to formulate a stable hydrogel.

[0302] Embodiment 169. The flow cell of any one of embodiments 113-168, wherein the hydrogel polymer structure or the degradable polymer wall comprises a photocleavable 4-arm PEG-macromonomer.

[0303] Embodiment 170. The flow cell of any one of embodiments 113-169, wherein the hydrogel polymer structure or the degradable polymer wall is photodegradable via an orthonitrobenzyl moiety.

[0304] Embodiment 171. The flow cell of any one of embodiments 113-170, wherein the hydrogel polymer structure or the degradable polymer wall comprises a Coumarin-based photodegradable macromonomer.

[0305] Embodiment 172. The flow cell of any one of embodiments 113-171, wherein the hydrogel polymer structure or the degradable polymer wall comprises a 4-arm PEG- acrylamide comprising one or more disulfides.

[0306] Embodiment 173. The flow cell of any one of embodiments 113-172, wherein the hydrogel polymer structure or the degradable polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation, wherein the hydrogel cages degrade using light and a photoinitiator.

[0307] Embodiment 174. The flow cell of any one of embodiments 113-173, wherein the hydrogel polymer structure or the degradable polymer wall enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation.

[0308] Embodiment 175. The flow cell of any one of embodiments 113-174, wherein the hydrogel polymer structure or the degradable polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel polymer structure or the degradable polymer wall.

[0309] Embodiment 176. The flow cell of any one of embodiments 113-175, wherein the hydrogel polymer structure or the degradable polymer wall are photopolymerized via photocrosslinking of polymer precursors by UV light.

[0310] Embodiment 177. The flow cell of any one of embodiments 113-176, wherein the flow cell comprises a top layer, a bottom layer, and a spacer layer, wherein the spacer layer includes a cut-out region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region, and wherein the hydrogel polymer structure is at least partly formed by the top layer and the bottom layer.

[0311] Embodiment 178. The flow cell of any one of embodiments 113-177, wherein the flow cell comprises a channel with an inlet and an outlet, wherein the channel comprises a first surface and a second surface disposed opposite one another across the channel, wherein a polymer matrix wall extends between the first surface and the second surface, thereby forming the hydrogel polymer structure.

[0312] Embodiment 179. The flow cell of any one of embodiments 113-178, wherein the hydrogel polymer structure and the degradable polymer wall are the same material.

[0313] Embodiment 180. The flow cell of any one of embodiments 113-179, wherein the hydrogel chamber is cured for a first duration of time, and wherein the first hydrogel polymer wall is cured for a second duration of time.

[0314] Embodiment 181. The flow cell of embodiment 180, wherein the first duration of time is longer than the second duration of time.

[0315] Embodiment 182. The flow cell of embodiment 180, wherein the second duration of time is longer than the first duration of time. [0316] Embodiment 183. The flow cell of any one of embodiments 180-182, wherein the hydrogel chamber and the first hydrogel polymer wall have different kinetics of degradation. [0317] Embodiment 184. The flow cell of any one of embodiments 113-178, wherein the hydrogel polymer structure and the degradable polymer wall are different materials.

[0318] Embodiment 185. The flow cell of embodiment 184, wherein the hydrogel polymer structure is made of a first material, wherein the first material degrades upon exposure to a first stimulus, wherein the degradable polymer wall is made of a second material, wherein the second material degrades upon exposure to a second stimulus, and wherein the first stimulus and second stimulus are different.

[0319] Embodiment 186. The flow cell of embodiment 185, wherein the first stimulus comprises light, and wherein the second stimulus comprises a degradation reagent.

[0320] Embodiment 187. The flow cell of embodiment 185, wherein the first stimulus comprises a degradation reagent, and wherein the second stimulus comprises light.

[0321] Embodiment 188. The flow cell of embodiment 185, wherein the first stimulus comprises a first degradation reagent, and wherein the second stimulus comprises a second degradation reagent different from the first degradation reagent.

[0322] Embodiment 189. The flow cell of embodiment 185, wherein the first stimulus comprises light in a first wavelength range, and wherein the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

[0323] Embodiment 190. The method of claim 3, further comprising forming the first polymer wall around at least a portion of the analyte; and forming the first hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material, wherein the inputting of the analyte into the fluidic device occurs before the inputting of the first biological materials into the fluidic device, wherein the forming the first hydrogel chamber is after the inputting of the first biological material.

[0324] While preferred embodiments of the present invention have been shown and described herein, it will be obvious 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 within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for analyzing an analyte, the method comprising:

(a) inputting the analyte and one or more biological materials into a fluidic device, wherein the fluidic device comprises a hydrogel chamber comprising: i. at least a portion of the analyte, and ii. at least a portion of a biological material of the one or more biological materials, wherein the analyte and the biological material are physically separated by a hydrogel polymer wall;

(b) degrading at least a portion of the hydrogel polymer wall; and

(c) detecting an interaction between the analyte and the biological material.

2. The method of claim 1, wherein the hydrogel polymer wall surrounds the analyte.

3. The method of claim 2, wherein in (a), the inputting of the analyte and the inputting of the one or more biological materials into the fluidic device occurs separately.

4. The method of claim 3, wherein the hydrogel polymer wall prevents an interaction between the analyte and the one or more biological materials.

5. The method of claim 1, wherein the hydrogel polymer wall is a first hydrogel polymer wall, wherein the biological material is a first biological material, and wherein the method further comprises:

(d) generating a second hydrogel polymer wall, wherein the analyte and a second biological material of the one or more biological materials are physically separated by the second hydrogel polymer wall.

6. The method of 5, wherein the second hydrogel polymer wall surrounds the analyte.

7. The method of claim 5, wherein the hydrogel chamber is a first hydrogel chamber, and wherein the method further comprises:

(e) degrading the first hydrogel chamber, and

(f) forming a second hydrogel chamber around at least a portion of the analyte and at least a portion of the second biological material.

8. The method of claim 5, further comprising: (g) degrading at least a portion of the second hydrogel polymer wall.

9. The method of claim 8, further comprising: (h) detecting an interaction between the analyte and the second biological material.

10. The method of claim 9, further comprising: (i) generating a third hydrogel polymer wall, wherein the analyte and a third biological material are physically separated by the third hydrogel polymer wall.

11. The method of 10, wherein the third hydrogel polymer wall surrounds the analyte.

12. The method of claim 10, further comprising:

(j) degrading the second hydrogel chamber, and

(k) forming a third hydrogel chamber around at least a portion of the analyte and at least a portion of the third biological material.

13. The method of claim 10, further comprising: (1) degrading at least a portion of the third hydrogel polymer wall.

14. The method of claim 13, further comprising: (m) detecting an interaction between the analyte and the third biological material.

15. The method of claim 1, wherein the hydrogel chamber is a first hydrogel chamber, wherein the hydrogel polymer wall is a first hydrogel polymer wall, wherein the biological material is a first biological material, wherein the analyte is physically separated from a second biological material of the one or more biological materials by a second hydrogel polymer wall, and wherein the first hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, and (iii) the second biological material.

16. The method of claim 15, further comprising, subsequent to (c):

(n) degrading at least a portion of the second hydrogel polymer wall, and

(o) detecting an interaction between the analyte and the second biological material.

17. The method of claim 16, wherein the analyte is physically separated from a third biological material of the one or more biological materials by a third hydrogel polymer wall, and wherein the first hydrogel chamber comprises: (i) the analyte, (ii) the first biological material, (iii) the second biological material, and (iv) the third biological material.

18. The method of claim 17, further comprising, subsequent to (n):

(p) degrading at least a portion of the third hydrogel polymer wall, and

(q) detecting an interaction between the analyte and the third biological material.

19. The method of claim 1, wherein the interaction comprises a killing of the biological material by the analyte or vice versa.

20. The method of claim 1, wherein the interaction comprises activation of the biological material by the analyte or vice versa.

21. The method of claim 20, wherein detecting the activation comprises counting a number of proliferated cells from the biological material.

22. The method of claim 20, wherein detecting the activation comprises determining a presence of a surface antigen on a cell of the biological material.

23. The method of claim 1, wherein the analyte is selected from a plurality of analytes in the fluidic device prior to (a).

24. The method of claim 1, wherein the analyte is a cell.

25. The method of claim 1, wherein the analyte is an antigen targeting cell.

26. The method of claim 1, wherein the analyte is a CD8+ T cell or NK cell.

27. The method of claim 1, wherein the analyte is a genetically engineered cell.

28. The method of claim 1, wherein the analyte is a CAR T cell.

29. The method of claim 1, wherein the biological material is a cell.

30. The method of claim 1, wherein the biological material is an antigen presenting cell.

31. The method of claim 1, wherein the biological material is a cancer cell.

32. The method of claim 1, wherein the biological material is an antibody or antigen binding fragment thereof.

33. The method of claim 32, wherein the antibody or antibody binding fragment thereof is coupled to a bead, and wherein the bead cannot diffuse through the hydrogel polymer wall.

34. The method of claim 1, wherein the analyte, the biological material, or both, is coupled to the hydrogel polymer wall.

35. The method of claim 1, the fluidic device comprises a flow cell.

36. The method of claim 1, wherein the method further comprises obtaining one or more genetic materials from the analyte.

37. The method of claim 36, wherein the method further comprises amplifying the one or more genetic materials.

38. The method of claim 37, wherein the amplifying occurs in the fluidic device.

39. The method of claim 36, wherein the method further comprises sequencing the one or more genetic materials.

40. The method of claim 39, wherein the sequencing occurs in the fluidic device.

41. The method of claim 37, wherein the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

42. The method of claim 41, wherein the RNA comprise messenger RNA (mRNA) or microRNA (miRNA).

43. The method of claim 1, wherein the interaction comprises suppression of the biological material by the analyte or vice versa.

44. The method of claim 1, wherein the interaction comprises binding or physical contact between the biological material and the analyte.

45. The method of claim 1, wherein the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel.

46. The method of claim 1, wherein the degrading in (b) comprises exposing the hydrogel polymer wall to UV light.

47. The method of claim 1, wherein the degrading in (b) comprises selectively exposing the hydrogel polymer wall to UV light in a presence of photoinitiator and not exposing the hydrogel chamber to the UV light.

48. The method of claim 1, further comprising imaging the analyte, the biological material, the hydrogel chamber, the fluidic device, or any combination thereof.

49. The method of claim 1, wherein the fluidic device comprises a channel with an inlet and an outlet, wherein the channel comprises a first surface and a second surface disposed opposite one another across the channel, wherein a polymer matrix wall extends between the first surface and the second surface, thereby forming the hydrogel chamber.

50. The method of claim 1, wherein the hydrogel chamber and the hydrogel polymer wall comprise the same material.

51. The method of claim 1, wherein the hydrogel chamber is cured for a first duration of time, and wherein the hydrogel polymer wall is cured for a second duration of time.

52. The method of claim 51, wherein the first duration of time is longer than the second duration of time.

53. The method of claim 51, wherein the second duration of time is longer than the first duration of time.

54. The method of claim 1, wherein the hydrogel chamber and the hydrogel polymer wall have different kinetics of degradation.

55. The method of claim 1, wherein the hydrogel chamber and the hydrogel polymer wall comprise different materials.

56. The method of claim 55, wherein the hydrogel chamber is made of a first material, wherein the first material degrades upon exposure to a first stimulus, wherein the hydrogel polymer wall is made of a second material, wherein the second material

-n - degrades upon exposure to a second stimulus, and wherein the first stimulus and second stimulus are different.

57. The method of claim 56, wherein the first stimulus comprises light, and wherein the second stimulus comprises a degradation reagent.

58. The method of claim 56, wherein the first stimulus comprises a degradation reagent, and wherein the second stimulus comprises light.

59. The method of claim 56, wherein the first stimulus comprises a first degradation reagent, and wherein the second stimulus comprises a second degradation reagent different from the first degradation reagent.

60. The method of claim 56, wherein the first stimulus comprises light in a first wavelength range, and wherein the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

61. The method of claim 56, wherein the first stimulus comprises UV light selectively applied to the hydrogel polymer wall in a presence of photoinitiator, and wherein the second stimulus comprises UV light selectively applied to the hydrogel chamber in the presence of photoinitiator.

62. The method of claim 3, further comprising forming the hydrogel polymer wall around at least a portion of the analyte; and forming the hydrogel chamber around at least a portion of the analyte and at least a portion of the biological material of the one or more biological materials, wherein the inputting of the analyte into the fluidic device occurs before the inputting of the biological material into the fluidic device, and wherein the forming the hydrogel chamber is after the inputting of the biological material.