Multiplexed cell assays for screening and quality assessment of engineered cells

Abstract

The present invention relates to a method and system for performing one or more highly multiplexed cellular assays. In some embodiments, one or more channels of a fluidics device are provided with photopolymerizable polymer precursors and cells randomly arranged on a surface, after which the positions of the cells are measured by a detector, hydrogel chambers are synthesized by photopolymerization to encapsulate individual cells, and the channels are loaded with assay reagents. An assay signal indicative of a desired cell characteristic can be generated for each of the encapsulated cells.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 322,601 (filed March 22, 2022), which is incorporated by reference in its entirety.

[0002] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein 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. In the event that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Background technology]

[0003] background Therapeutic agents based on engineered cells offer promising new approaches to treat complex diseases due to the ability of cells to sense and integrate a wide range of signals, actively migrate to specific tissue compartments, and activate context-dependent responses (e.g., Fischbach et al., Science Transl. Med., 5:1797 (2013)). Such cell-based approaches provide novel therapeutic devices that address current obstacles faced by small molecules and biologics, such as poor target specificity, undesirable tissue compartment localization, lack of personalization, and limited potential for the effects of drugs to be modified, either spatially and temporally, once the drug is administered to a patient, or in response to changing clinical manifestations. These issues may reduce the pharmaceutical usefulness of such compounds. Cytotoxic lymphocytes (CLs), such as cytotoxic T lymphocytes (CTLs) and natural killer cells (NKs), are excellent platforms for engineering cell-based therapeutic systems for several reasons: (i) cytotoxic lymphocytes possess a unique delivery cell-target cell molecular transfer system in the granzyme-perforin pathway; (ii) the T cell receptor (TCR) or related chimeric antigen receptor (CAR) endows cytotoxic lymphocytes with an exquisite level of specificity in targeting cell populations displaying cognate antigen bound to the major histocompatibility complex (MHC), or in the case of CARs, any surface antigen; (iii) activated cytotoxic lymphocytes differentially express cytokine and tissue-specific receptors, which allow selective lymphocyte homing throughout the body to target tissues; (iv) experimental and clinical protocols for lymphocyte engineering and therapeutic administration have been developed in the field of adoptive cell therapy (e.g., Restifo et al., Nature Reviews, 2010). Immunology, 12:269-281 (2012). In light of these advantages and positive clinical results, a number of cell-based therapeutics have been approved for treating a range of cancers and other disorders.

[0004] However, along with the success of cell-based therapeutics, there are significant development, manufacturing and quality assurance challenges due to the complexity of using living organisms as drugs. Testing must be performed to select the appropriate cell subpopulations to manipulate, and to ensure that there are no adverse changes in the identity, purity or operability of the manipulated subpopulations at each step of the manufacturing process (e.g., Tanna et al., Cytotherapy, 21:278-288 (2019); Wang et al., Molecular Therapy, 3:16015 (2016); Levine et al., Molecular Therapy: Methods & Clinical Development, 4:92-101 (2017)). Thus, the field of cell-based therapeutics will be advanced by the availability of cell analysis platforms, including methods and systems, for performing a wide range of highly multiplexed cell assays related to the development and manufacture of cell-based therapeutics. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Tanna et al., Cytotherapy (2019) 21:278-288 [Non-Patent Document 2] Wang et al., Molecular Therapy (2016) 3:16015 [Non-Patent Document 3] Levine et al., Molecular Therapy: Methods & Clinical Development (2017) 4:92~101 Summary of the Invention [Means for solving the problem]

[0006] The present invention relates to methods and systems for performing large-scale multiplexed single-cell assays, in particular for characterizing populations of engineered cells developed for medical or industrial applications (e.g., cell-based therapeutics). Various exemplary single-cell assays include, but are not limited to, cytotoxicity, proliferation potential, activation status, vector copy number, and insertion site analysis of transformed cells. In certain aspects, provided herein is a method of determining one or more cellular characteristics, the method comprising: (a) synthesizing one or more hydrogel chambers, where one hydrogel chamber of the one or more hydrogel chambers encapsulates a cell disposed on a surface of a channel; (b) lysing the cell such that genomic DNA of the cell is released into the hydrogel chamber; (c) amplifying the genomic DNA of the cell, thereby obtaining amplified genomic DNA; and (d) using the amplified genomic DNA to measure cellular viral copy number, viral integration site, or genomic copy number variation.

[0007] In some cases, the measuring step further comprises (i) annealing a vector-specific primer to the amplified genomic DNA, (ii) extending the vector-specific primer, thereby obtaining an extension product comprising a copy of a segment of the genomic DNA, and (iii) identifying from the segment one or more sites of integration of the vector-specific primer with the genomic DNA. In some cases, the cell is a mammalian cell, and the measuring step further comprises (i) sequencing one or more fragments of the amplified genomic DNA, thereby obtaining the sequence of the one or more fragments, and (ii) determining the genome copy number variation for the cell from the sequence of the one or more fragments. In some cases, the sequencing comprises obtaining a sequence coverage of the amplified genomic DNA of 0.25x or more. In some cases, the determination of genome copy number variation has a resolution of 3 megabases or more.

[0008] In another embodiment, a method of determining one or more cellular properties of one or more cells includes the steps of: (a) providing a fluidics device including: (i) a channel comprising a first surface, one or more cells, and one or more polymer precursors, where the one or more cells are disposed on or adjacent to the first surface; (ii) a spatial energy modulating element in optical communication with the first surface; and (iii) a detector; (b) locating the one or more cells in the channel with the detector; (c) projecting energy into the channel using the spatial energy modulating element, whereby As a result, the projected energy causes one or more polymer precursors to form polymer matrix walls of one or more chambers, where the one or more chambers at least partially encapsulate one or more cells at a location identified by the detector; (d) loading one or more assay reagents into the channels; and (e) incubating the one or more cells under assay conditions to generate a signal from the one or more chambers, the signal being indicative of one or more cellular characteristics of the cells encapsulated by the chambers.

[0009] In some cases, (i) the one or more cell characteristics include a proliferation rate, (ii) the incubating under assay conditions includes incubating under growth conditions, and (iii) the method further includes determining a proliferation rate of the one or more cells at least partially enclosed by the one or more chambers after the incubating step. In some cases, the determining proliferation rate includes counting the one or more cells at least partially enclosed by the one or more chambers. In some cases, the one or more cell characteristics further include a cell membrane protein profile, and the method further includes incubating the one or more cells with antibodies each specific for a different cell surface protein whose relative expression allows identification of the one or more cells, each such antibody having a distinct label. In some cases, the first surface includes one or more assay components or one or more capture elements for capturing one or more components of the one or more cells. In some cases, each of the antibodies has an oligonucleotide label that includes an antibody-specific barcode that can be captured by one or more capture elements.

[0010] In some cases, the method further comprises the steps of: (i) providing an oligonucleotide label, where the oligonucleotide label is attached to the antibody by a scissile linkage; (ii) loading the channel with a releasing reagent to cleave the scissile linkage, such that the oligonucleotide label is released and captured by the capture element; (iii) copying the captured oligonucleotide label to generate its complementary DNA; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide label.

[0011] In some cases, the first surface comprises one or more assay components or one or more capture elements for capturing one or more components of the one or more cells, and the one or more cellular characteristics further comprise a cellular transcriptome, and the method further comprises the steps of (i) loading a lysis reagent into the channel, such that messenger RNA of the one or more cells is released and captured by the one or more capture elements, (ii) loading the channel with a reverse transcription reagent that copies the captured messenger RNA to produce complementary DNA; and (iii) sequencing the complementary DNA.

[0012] In some cases, (i) the one or more cell characteristics further comprises a profile of proteins secreted by the one or more cells, (ii) the channel further comprises a protein capture surface comprising a protein affinity reagent that binds to proteins secreted by the one or more cells, and (iii) the method further comprises detecting proteins secreted by the one or more cells that bind to the protein capture surface adjacent to the one or more cells using an amount of a labeled protein detection antibody.

[0013] In some cases, (i) the one or more cellular characteristics include cytotoxicity, (ii) the first surface includes target cells disposed thereon, (iii) the method further includes loading effector cells into the channel such that the effector cells are disposed on or adjacent to the first surface, (iv) the incubating step includes incubating the effector cells and the target cells with a vital dye that produces an optical signal in dead cells but not in live cells, and (v) counting the dead cells in each of the one or more chambers and determining the cytotoxicity value of the effector cells enclosed by the chamber. In some cases, the first surface includes one or more assay components or one or more capture elements for capturing one or more components of the one or more cells. In some cases, the one or more cellular characteristics further include a profile of cell membrane proteins, and the method further includes incubating the one or more cells with antibodies each specific for a different cell surface protein whose relative expression allows for identification of the cells, each such antibody having a different label. In some cases, each of the antibodies has an oligonucleotide label that includes an antibody-specific barcode that can be captured by one or more capture elements. In some cases, the method further includes the steps of (i) providing an oligonucleotide label, where the oligonucleotide label is attached to the antibody by a scissile linkage; (ii) loading the channel with a releasing reagent to cleave the scissile linkage, such that the oligonucleotide label is released and captured by the capture element; (iii) copying the captured oligonucleotide label to generate its complementary DNA; and (iv) sequencing the complementary DNA to identify the captured oligonucleotide label.

[0014] In some cases, the first surface comprises one or more assay components or one or more capture elements for capturing one or more components of the one or more cells, the one or more cellular characteristics further comprise a cellular transcriptome, and the method further comprises (i) loading the channel with a lysis reagent, whereby messenger RNA of the one or more cells is released and captured by the one or more capture elements, (ii) loading the channel with a reverse transcription reagent that copies the captured messenger RNA to generate complementary DNA; and (iii) sequencing the complementary DNA. In some cases, (i) the one or more cellular characteristics further comprise a profile of proteins secreted by the one or more cells, (ii) the channel further comprises a protein capture surface comprising a protein affinity reagent that binds to the proteins secreted by the one or more cells, and (iii) the method further comprises detecting proteins secreted by the one or more cells that bind to the protein capture surface adjacent to the one or more cells using an amount of a labeled protein detection antibody.

[0015] In some cases, the one or more cell characteristics include copy numbers of one or more nucleotide sequences in the genomic DNA of the one or more cells, and the method further includes (a) lysing the one or more cells to release the genomic DNA, (b) amplifying the one or more nucleotide sequences, thereby obtaining one or more amplified nucleotide sequences, (c) capturing the one or more amplified nucleotide sequences with one or more capture elements disposed in the channel, (d) copying the captured one or more amplified nucleotide sequences to generate complementary DNAs thereof, and (e) sequencing the complementary DNAs to identify copy numbers of the one or more nucleotide sequences. In some cases, each of the one or more nucleotide sequences is a barcode. In some cases, each of the barcodes includes a unique molecular identifier. In some cases, the unique molecular identifier identifies a viral DNA integrated into the genomic DNA, and the number of distinct unique molecular identifiers identified in a single cell indicates the viral copy number of such a single cell.

[0016] In some cases, the one or more cell characteristics include copy numbers of one or more nucleotide sequences in genomic DNA of the one or more cells, and the method further includes (a) lysing the one or more cells to release the genomic DNA, (b) loading the channel with an amplification reagent that generates a signal proportional to the copy number of the one or more nucleotide sequences, and (c) amplifying the one or more nucleotide sequences and generating an optical signal proportional to the copy number of the one or more nucleotide sequences. In some cases, the amplification reagent is a quantitative PCR reagent, and the signal is an optical signal. In some cases, (i) the first surface includes bridge PCR primers, (ii) the amplification reagent includes bridge PCR reagent, and (iii) the signal is the number of clusters formed by bridge PCR. In some cases, the amplification reagent includes rolling circle amplification reagent, and the signal is the number of DNA nanoballs.

[0017] In some cases, one or more cells are randomly arranged on the first surface. In some cases, each of the one or more chambers encloses a single cell of the one or more cells. In some cases, the one or more cells include cells transduced by a vector, and the one or more cell characteristics include vector integration sites of the cells transduced by the vector, and the method further includes (a) lysing the cells transduced by the vector to release the genomic DNA of each cell into its respective chamber; (b) amplifying the released genomic DNA; (c) annealing a vector-specific primer to the amplified genomic DNA; (d) extending the vector-specific primer, thereby obtaining an extension product that includes a copy of a segment of genomic DNA; and (e) identifying one or more sites of integration of the vector-specific primer with the genomic DNA from the segment.

[0018] In some cases, the one or more cells are mammalian cells, the one or more cell characteristics include genome copy number variation, and the method further comprises (a) lysing the one or more cells to release genomic DNA of each cell into its respective chamber, (b) amplifying the released genomic DNA, (c) sequencing fragments of the amplified genomic DNA, thereby obtaining sequences of the genomic DNA fragments, and (d) determining genome copy number variation for each cell from the sequences of the genomic DNA fragments. In some cases, the sequencing comprises obtaining sequence coverage of the genomic DNA fragments of 0.25× or more, and the determination of genome copy number variation has a resolution of 3 megabases or greater.

[0019] In another aspect, provided herein is a system for measuring single cell characteristics of a population of cells, the system including: (a) one or more channels, each channel including a surface, a plurality of cells disposed on the surface, and one or more polymer precursors; (b) at least one spatial energy modulation element in optical communication with the surface of each channel; (c) at least one detector in optical communication with the surface of each channel and operably associated with the at least one spatial energy modulation element, the detector configured to detect each of the plurality of cells and determine their location at the surface of the at least one channel; and (d) a plurality of gel chambers in each channel, each gel chamber encapsulating one or more of the plurality of cells, the gel chambers being synthesized by projecting light into the channel by the at least one spatial energy modulation element, such that the projected light causes the one or more polymer precursors to form polymer matrix walls of the gel chamber, and the location of the synthesized chamber is determined at least in part by the location of the cell encapsulated by the chamber identified by the detector.

[0020] In some cases, the gel chambers are randomly arranged on at least one of the surfaces. In some cases, each of the gel chambers encapsulates a single cell. In some cases, the polymer matrix walls are sized to accommodate 3×10 6 Permeable to molecules with molecular weights less than 3 x 10 Daltons 6 In some cases, the polymer matrix wall is impermeable to molecules having a molecular weight greater than 3×10 Daltons. 5 Permeable to molecules with molecular weights less than 3 x 10 Daltons 5 In some cases, the polymer matrix wall is impermeable to molecules having a molecular weight greater than 3×10 Daltons. 4 Permeable to molecules with molecular weights less than 3 x 10 Daltons 4In some cases, the polymer matrix wall is impermeable to molecules having a molecular weight greater than 3×10 Daltons. 3 Permeable to molecules with molecular weights less than 3 x 10 Daltons 3 It is impermeable to molecules having a molecular weight above Daltons.

[0021] In some cases, the gel chamber is a degradable hydrogel chamber. In some cases, the gel chamber is hollow and encloses an area of ​​the surface. In some cases, the surface includes capture elements configured to capture nucleic acids.

[0022] In another embodiment, a method of determining one or more cellular properties of one or more cells includes the steps of: (a) providing a fluidics device comprising: (i) a channel comprising a first surface, one or more cells, and one or more polymer precursors, where the one or more cells are disposed on or adjacent to the first surface; (ii) a spatial energy modulating element in optical communication with the first surface; and (iii) a detector; (b) locating the one or more cells in the channel with the detector; (c) projecting energy into the channel using the spatial energy modulating element; Provided herein are methods comprising (a) applying a first energy beam to the one or more polymer precursors, such that the projected energy causes the one or more polymer precursors to form polymer matrix walls of the one or more chambers, where the one or more chambers at least partially encapsulate the one or more cells at a location identified by the detector; and (b) performing one or more assays on the one or more cells in the channels to determine one or more cellular characteristics selected from the group consisting of cytotoxicity, viability, proliferation rate, phenotype, vector copy number, vector integration site, transcriptome, and genome copy number variation. In some cases, the fluidics device further comprises a plurality of channels, and performing the one or more assays comprises performing a plurality of assays, where each different assay of the one or more assays is performed in a different channel of the plurality of channels. [Brief description of the drawings]

[0023] [Figure 1A] FIG. 1A diagrammatically illustrates the process steps in an exemplary cell-based therapy, e.g., autologous CAR-T cell therapy.

[0024] [Figure 1B] FIG. 1B contains a table listing assay combinations that may be performed according to the present invention.

[0025] [Figure 2A] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion. [Figure 2B] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion. [Figure 2C] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion. [Figure 2D] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion. [Figure 2E] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion. [Figure 2F] 2A-2F illustrate the steps of an exemplary assay for determining the proliferative potential of cells in a population, followed by identification of the cells by cell surface protein expression and protein secretion.

[0026] [Figure 3A] Figures 3A-3B illustrate the steps of an assay for determining the cytotoxicity of a population of cells. Subsequent assays to identify cells having desirable cytotoxicity values ​​can be performed as shown in Figures 2B-2G. [Figure 3B] Figures 3A-3B illustrate the steps of an assay for determining the cytotoxicity of a population of cells. Subsequent assays to identify cells having desirable cytotoxicity values ​​can be performed as shown in Figures 2B-2G.

[0027] [Figure 4A]4A-B illustrate an assay for determining the copy number of integrated vector in transduced cells. [Figure 4B] 4A-B illustrate an assay for determining the copy number of integrated vector in transduced cells.

[0028] [Figure 4C] 4C-4F illustrate assays to determine the copy number and insertion site of integrated vector in transduced cells. [Figure 4D] 4C-4F illustrate assays to determine the copy number and insertion site of integrated vector in transduced cells. [Figure 4E] 4C-4F illustrate assays to determine the copy number and insertion site of integrated vector in transduced cells. [Figure 4F] 4C-4F illustrate assays to determine the copy number and insertion site of integrated vector in transduced cells.

[0029] [Figure 4G] 4G-H illustrate an assay for determining genome copy number variation in a single cell. [Figure 4H] 4G-H illustrate an assay for determining genome copy number variation in a single cell.

[0030] [Figure 5A] 5A-5B diagrammatically illustrate an embodiment of a system for carrying out the methods of the present invention. [Figure 5B] 5A-5B diagrammatically illustrate an embodiment of a system for carrying out the methods of the present invention.

[0031] [Figure 6A] 6A-6B illustrate one embodiment of a flow cell containing multiple channels. [Figure 6B]6A-6B illustrate one embodiment of a flow cell containing multiple channels.

[0032] [Figure 7A] 7A-7B illustrate exemplary steps for preparing cDNA from a single cell target template (e.g., mRNA) and sequencing either at the site of the hydrogel chamber ("in situ" or "internal" sequencing) or after elution from the channel on an external sequencing instrument. [Figure 7B] 7A-7B illustrate exemplary steps for preparing cDNA from a single cell target template (e.g., mRNA) and sequencing either at the site of the hydrogel chamber ("in situ" or "internal" sequencing) or after elution from the channel on an external sequencing instrument. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description The practice of the present invention may employ conventional techniques and explanations of organic chemistry, molecular biology (including recombinant technology), cell biology and biochemistry that are within the skill of the art, unless otherwise indicated. Such conventional techniques include, but are not limited to, synthetic polynucleotide preparation, monoclonal antibodies, antibody display systems, cell and tissue culture techniques, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques may be obtained by reference to the examples herein below. However, other equivalent conventional procedures may, of course, also be used. Such prior art and explanations can be found in standard laboratory manuals, such as Genome Analysis: A Laboratory Manual Series (Vol. I-IV); PCR Primer: A Laboratory Manual; Retroviruses; and Molecular Cloning: A Laboratory Manual (all available from Cold Spring Harbor Laboratory Press); Renault and Duchateau (editors), Site-directed Insertion of Transgenes (Springer, Heidelberg, 2013); Lutz and Bornscheuer (editors), Protein Engineering Handbook (Wiley-VCH, 2009), etc. Guidance for selecting materials and components to perform specific functions can be found in available treatises and references on scientific instrumentation, including but not limited to Moore et al., Building Scientific Apparatus, 3rd Edition (Perseus Books, Cambridge, MA); Hermanson, Bioconjugate Techniques, 3rd Edition (Academic Press, 2013), etc.

[0034] The present invention relates to various methods and systems for performing one or more large-scale multiplexed assays that simultaneously evaluate multiple properties of cells, especially for the purpose of developing and quality assurance of cell-based therapeutics. In some embodiments, the present invention includes methods and systems for simultaneously performing multiple assays to determine multiple cellular properties important for cell-based therapies, including but not limited to cell-based therapies for treating cancer, e.g., using CAR-T cells, or other conditions, e.g., spinal cord injury, stroke, diabetes, etc., using, e.g., induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, etc. In some embodiments, the present invention relates to systems for simultaneously performing multiple single-cell assays designed to detect or measure selected or predetermined cellular properties of a population of cells. In some embodiments, one or more of such cellular assays rely on or utilize a hydrogel chamber (sometimes referred to herein as an "analysis chamber", or simply a "chamber", or a "hydrogel cage"). In some embodiments, such cellular assays are single-cell assays, such that one or more hydrogel chambers used in such assays each encapsulate only a single cell. In some embodiments, such single cell assays may include a single engineered cell and one or more target cells, i.e., one or more cells that may be acted upon (e.g., killed) by the engineered cell.

[0035] "Cells" that may be assayed by the methods and systems of the present invention include any biological cells, including, but not limited to, vertebrate, invertebrate, eukaryotic, mammalian, microbial, protozoan, prokaryotic, bacterial, insect, or fungal cells. In some embodiments, mammalian cells are assayed by the methods and systems of the present invention. In particular, any population of mammalian cells that may be derived, treated, modified, or genetically altered (i.e., genetically engineered) for use in medical, industrial, environmental, or remedial processes, or that have been derived, treated, modified, or genetically altered (i.e., genetically engineered), may be analyzed by the methods and systems of the present invention. In some embodiments, "cells" as used herein include genetically altered mammalian cells. In some embodiments, "cells" include stem cells that have been induced to differentiate. In some embodiments, "cells" refers to cells that have been altered by CRISPR Cas9 technology. In some embodiments, "cells" refers to cells of the immune system, including, but not limited to, cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen-presenting cells, or dendritic cells. Of particular interest are cytotoxic T lymphocytes that have been engineered for therapeutic applications, such as for cancer treatment.

[0036] As used herein, the term "assay" refers to a process for detecting or measuring cellular characteristics or properties of a single cell or a population of cells. Typically, the various process steps of an assay involve chemical, biochemical or molecular reactions (e.g., bond cleavage, specific binding of complementary components, enzymatic reactions, separation of complementary components, etc.), or changes in physical state (e.g., increase or decrease in temperature, change in energy level, etc.), resulting in the production of a signal or signals from which the presence, absence or magnitude of an amount associated with a cell can be inferred. The nature of the signal produced by an assay can vary widely and can include, but is not limited to, electrical signals, optical signals, chemical signals, or material signals. Material signals include the production of a material that contains information that can be extracted. For example, a material signal can be the amplification of a polynucleotide whose length, amount, composition, or nucleotide sequence indicates a cellular characteristic. For example, a barcode oligonucleotide can be a material signal. The cellular characteristics or properties detected or measured can vary widely and include, but are not limited to, cytotoxicity, viability, ability to grow under selected conditions, size, shape, motility, type and profile of cell surface or membrane proteins, type and profile of secreted proteins, production of metabolites, transcriptome, gene copy number, gene or allele identity, chromatin accessibility profile, vector copy number for engineered or infected cells, etc. Assays of particular interest for cell-based therapeutics include, but are not limited to, measurements of the characteristics listed in Table 1. [Table 1]

[0037] As mentioned above, CAR-T cell therapy, e.g., CAR-T cell therapy for treating cancer, is an exemplary application of the present invention. The main steps of therapeutic autologous CAR-T cell production are illustrated in FIG. 1A and described in many publications, e.g., Wang et al., Molecular Therapeutics-Oncolytics, 3:16015 (2016); Barrett et al., Annu. Rev. Med., 65:333-347 (2014). The diagram illustrates where quality assurance testing can be performed in the production process. White blood cells, including lymphocytes, are separated from other blood components of the patient (100) by apheresis (102) (e.g., Allen et al., Transfusion, 57(5):1133-1141 (2017)). Further enrichment of specific subsets of lymphocytes (104) may be performed by depleting undesirable subsets or enriching for desired subsets, for example, using magnetic beads or other techniques to remove T regulatory cells that may suppress the desired activity of the CAR-T cells. After the suitable subset is selected, the T cells are activated and transduced (106); that is, the T cells are made eligible to perform immune functions and to express the CAR. Activation can be achieved by various treatments, for example, by exposure to anti-CD3 / anti-CD28 beads. Transduction can also be achieved by various techniques, for example, by lentiviral transduction and integration into the T cell genome (e.g., Wang et al., cited above; Barrett et al., cited above). Following such transformation, the T cells are tested (108), e.g., using the present invention, to determine whether the desired properties for CAR-T cell therapy are present (e.g., a sufficient degree of cytotoxicity against the desired target cells, viability, proliferation capacity, identity, absence of contaminants, not excessive viral copy number, etc.). If the activated, transduced cells do not have acceptable therapeutic properties (109), new cells must be prepared. If the cells have acceptable therapeutic properties, the cells are expanded (110); i.e., the cells are cultured to increase their numbers.Since the conditions for expansion may alter the cells, the cells are again tested for the desired properties after expansion (112). Successfully expanded cells may be formulated (114) for infusion into the patient (100) and / or storage (116) for subsequent infusion. If subsequent infusion is required, cells removed from storage are again tested to ensure that the cells have the desired therapeutic properties. Exemplary single cell assays that may be performed using the methods and systems of the present invention are listed in Table 1 and FIG. 1B. In some embodiments, sequential assays, each obtaining additional information, may be performed on the same cell(s) in the chambers of the channel.

[0038] The development and production of CAR-T cells or therapeutic stem cells is an exemplary application of the present invention. In some embodiments, the method of the present invention for determining one or more cell characteristics (such as cell characteristics related to the development or production of CAR-T cells or therapeutic stem cells) can be carried out by the following steps: (a) providing a fluidics device including (i) one or more channels, each including a first surface and, optionally, an assay component for detecting or measuring the cell characteristic, (ii) a spatial energy modulating element in optical communication with each first surface, and (iii) a detector for identifying the location of a cell in each channel based on one or more optical signals from the cell; (b) disposing a cell on the first surface of the channel; (c) disposing one or more polymer precursors in each channel; (d) synthesizing one or more chambers in the channel, each chamber enclosing a cell, by projecting light into each channel with the spatial energy modulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, where the location of the synthesized chamber is determined in each channel by the location of the cell enclosed or enveloped by the chamber as determined by the detector; and (e) incubating the cells under assay conditions in each channel to generate a signal from the chamber of each channel indicative of a cell characteristic. In some embodiments, incubating the cells under assay conditions includes performing one or more assays on the cells in the channel to determine one or more cell characteristics selected from the group consisting of cell injury, viability, proliferation rate, phenotype, vector copy number, vector integration site, transcriptome, and genome copy number variation. In some embodiments, the polymer matrix wall of the chamber comprises a degradable hydrogel.In some embodiments, incubating includes lysing the cells and capturing one or more cellular components on the first surface with the capture agent. In some embodiments, incubating further includes degrading the degradable polymer matrix wall after such one or more cellular components are captured. As used herein, the terms "capture element" and "capture agent" are used interchangeably. In some embodiments, the capture agent (or capture element) is covalently attached to the surface of the channel. In some embodiments, the capture agent (or element) comprises an oligonucleotide having a sequence capable of hybridizing to the cellular component either directly (e.g., the polyA tail of an mRNA) or indirectly (e.g., the adapter strand of an adaptor-tagged genomic DNA fragment resulting from tagmentation). In some embodiments, the capture agent (or element) comprises one or more antibodies. In some embodiments, the capture agent (or element) comprises a spatial barcode. In some embodiments, the capture agent (or element) comprising an oligonucleotide further comprises a primer for solid-phase amplification (e.g., bridge PCR, etc.). In some embodiments, the fluidics device comprises a plurality of channels. In some embodiments, a plurality of cells is disposed on the surface of each channel, in some embodiments, such a plurality of cells is 100 or more cells, alternatively 1000 or more cells, alternatively 10,000 or more cells, alternatively 100 to 10. 6 It includes a number of cells in the range of cells.

[0039] In some embodiments, the steps of disposing the cells and loading the polymer precursor are performed by mixing the cells and the polymer precursor outside the fluidics device and then loading the mixture of cells and the polymer precursor into the channel. In some embodiments, the cells are delivered to the first surface in a fluid in which the cells are randomly dispersed over the first surface. In some embodiments, after the cells are delivered to the surface of the channel, the cells are randomly disposed on the surface. In some embodiments, the step of incubating under assay conditions may include only one step of an assay that includes more than one step, such as detecting a signal (or generating nucleic acid ready for sequencing in the case of a substance signal), or such a step of incubating under assay conditions may include multiple steps of a multi-step assay. In some embodiments, the cells may be treated or subjected to an assay step prior to loading into the channel of the fluidics device, such that the step of incubating may include only one step of a multi-step assay, such as signal generation and / or signal collection. In other embodiments, the incubating step may include performing an assay step or part of an assay, such as cDNA synthesis, second strand synthesis, capture of an assay component or cellular component, etc. In some embodiments, the assay conditions may include a series of steps, each with different conditions (e.g., temperature, pH, presence or absence of a particular reagent (e.g., primers or enzymes (e.g., ligases, polymerases, transposases, etc.)). For example, such steps may include loading primers such that they diffuse across the chamber walls and anneal to target sites in a template strand in the sample in the chamber, extending the annealed primers, heating the channel such that the extended primers melt from their template strands, capturing the melted extended primers with a capture agent attached to the first surface, etc.

[0040] In some embodiments, the cells are adhesive to the first surface such that they settle and, upon contact, remain on the surface even in the presence of fluid movement. In other embodiments, the cells are non-adhesive to the first surface such that they may settle to the first surface but may migrate or return to suspension in the presence of fluid movement. Those skilled in the art will recognize that the first surface may be treated (e.g., by attachment of a suitable capture element) to retain non-adherent cells either throughout the first surface (or the polymer matrix wall or the second surface) or over a selected portion thereof. In some embodiments, assay components that may be provided in or as part of the channel include, but are not limited to, capture elements, such as capture oligonucleotides, primers for surface amplification, antibodies, functional groups that can react with hydrogel components, beads, etc. In some embodiments, such assay components may be attached either exclusively to one of the first surface, the second surface, or the polymer matrix wall, or exclusively to a combination of such surfaces, or in combination with other reagents. In some embodiments, assay components that may be provided after synthesis of the gel chamber include, but are not limited to, lysis reagents, transcription reagents, reverse transcription reagents, antibodies, polymerase, primers, beads, etc. As used herein, "channel" refers to a container capable of holding a fluid (which may be stationary or flowing) and having at least one surface on which a cellular assay may be performed. In some embodiments, a channel may have a first surface and / or a second surface on which a chamber may be synthesized and / or to which a cellular component or assay component may be attached. Additionally, in some embodiments, a cellular component or assay component may be attached or captured by a capture element on the polymer matrix wall. As used herein, reference to a "first surface" (e.g., the first surface as a surface containing a capture element) may include the second surface, or the polymer matrix wall, if appropriate.As used herein, reference to a "surface" without reference to a "first" or "second" is intended to include a first surface or a second surface. In some embodiments, a channel may constrain the flow of fluid through the channel from an inlet to an outlet. In other embodiments, a channel may contain a non-flowing volume of fluid that may be removed, replaced, or added through an opening or inlet; i.e., in some embodiments, a channel of the invention may be a well or well-like structure, such as a depression. In some embodiments, hydrogel chambers are synthesized with the methods of the invention, and in other embodiments, degradable hydrogel chambers are synthesized with the methods of the invention. In some embodiments, particularly for CAR-T cells, the multiple cell characteristics may be selected from the following characteristics: cytotoxicity, proliferative potential or rate, activation state, cell identity, purity, gene expression profile or transcriptome, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, gene copy number, etc. Additional assays may include culture contamination assays, including, but not limited to, viral assays, bacterial assays, yeast assays, mold assays or mycoplasma assays, endotoxin assays, and cell morphology assays.

[0041] Another exemplary application of the present invention includes screening modified cell populations based on CRISPR / Cas9. For example, Shifrut et al. (Cell, 175(7):1958-1971(2018)) screened a population of T cells transduced by lentivirus producing a library of single guide RNAs targeting known T cell genes. The goal was to identify genes that, when knocked out, enhance T cell responses, as measured by growth rate. The present invention is perfectly suited to provide single cell growth rates at large scales as required by such studies.

[0042] An exemplary system for carrying out the above method is illustrated in FIG. 5A. The flow cell (500) is a component of a fluidics device that provides channels for carrying out various assays and liquid handling components under programmable control for delivering samples and reagents to the channels. In this illustration, four channels (502, 504, 506, and 508) are shown, with a close-up (512) of a segment (510) of channel 2 (504) shown below. In the cutaway view of the flow cell (500) in FIG. 5A, the inlets, outlets, and other features of the channels are not shown. At a first surface (514) of channel 2 (504), a plurality of cells (e.g., (518)) are each enclosed by a hydrogel chamber (e.g., (516)). In some embodiments, the porosity of the polymer matrix walls of the hydrogel chambers is selected to be impermeable to cells, but permeable to assay reagents. Thus, reagents may be introduced into and removed from the interior of the hydrogel chamber by flowing the reagents through the channel (520), but the cells are retained within the hydrogel chamber. Shown below the enlargement (512) of the channel segment (510) is an exemplary optical system (521) for photosynthesizing the hydrogel chamber at the location of the cells in the channel, in some embodiments, and for collecting optical signals, including images. Those skilled in the art will recognize that optical systems having configurations different from those of Figures 5A and 5B may be used to perform these functions. In some embodiments, multiple DMD-objective lens subsystems for synthesizing hydrogel constructs may be used to increase the rate of synthesis by synthesizing multiple constructs simultaneously.

[0043] Returning to FIG. 5A, to photosynthesize the hydrogel chambers, a light source (522) generates a light beam (523) of appropriate wavelength (e.g., UV light) that passes through an appropriate photomask or beam shaping or beam steering (Galvo) system to shape the beam to synthesize the desired structure(s) in the channel. In some embodiments, a digital micromirror device (DMD) (524) is used, while in other embodiments, a physical photomask may be used. The chamber location, shape and polymer matrix wall thickness are determined, at least in part, from cell location information derived from images collected by a detector (532). The reflected light from the DMD (524) is shaped using conventional optics, for example, using collimating optics (528), and directed through an objective lens system (534) into a segment of channel 2 (510). The objective lens (534) and flow cell (500) move relative to one another in the xy directions (536) to photosynthesize the chambers to any position in any of the channels. In some embodiments, the flow cell (500) moves and the optical system (521) is stationary. In some embodiments, the objective lens (534) may also direct the light beam (527) from the light source (529) to the targets (e.g., cells, etc.) at the first surface (514) and collect optical signals (e.g., fluorescent signals, etc.) from the assay occurring at the first surface (514). Alternatively, optical signal collection may be performed by a separate objective lens as shown in FIG. 5B. Information collected by the detector (532) or its counterpart in the embodiment of FIG. 5B, particularly the positions of the cells in their respective channels, is used by the computer (538) and / or auxiliary controller to direct the DMD (524) and translation device, which control the relative positions of the objective lens (534) and the flow cell (500) to synthesize hydrogel chambers of appropriate shapes and sizes at appropriate locations.

[0044] FIG. 5B illustrates an alternative optical system in which the detection portion (550) of the optical system moves (572) independently of the movement (568) of the combination portion (552) of the optical system. The detection portion (550) of the optical system includes a detector (556), an objective lens (558), a light source (560), and interconnecting optical elements, such as a dichroic mirror (562). As in the embodiment of FIG. 5A, the detector (556) is operably associated with a computer (564) and the combination portion (552) of the optical system to provide cell position information to the combination portion (552). The computer (564) and computer (538) are also operably associated with a stage and / or motors that control the relative position of the objective lens of the optical system and the position of the flow cell. In this embodiment, the combination portion (552) of the optical system is located on the opposite side of the first surface (564) from the detection portion (550). As with the embodiment of FIG. 5A, the optical system includes conventional components: objective lens (574), mirror (576), collimation optics (580), DMD (582), and light source (578).

[0045] In some embodiments, the cells (e.g., (518) in FIG. 5A) are randomly positioned on the first surface (514). In alternative embodiments, the first surface (514) may include regularly spaced sites or features for capturing cells, such that cells are located substantially only at such sites or features on the first surface. For example, in some embodiments, such sites or features may be linear or hexagonal arrays of spots. In some embodiments, such sites or features may include capture elements, such as antibodies specific to cell surface proteins, for capturing or immobilizing cells at the sites or features. In some embodiments, the first surface (514) may include capture elements designed to immobilize specific cell types. For example, lymphocytes are non-adherent on most surfaces; however, such cells may be allowed to settle on the first surface by gravity or may be immobilized on the surface using capture elements that include antibodies specific for lymphocyte cell surface proteins (e.g., CD3, CD4, CD8, etc.) (e.g., Sekine et al., J. Immunol. Meth., 313(1-2):96-109 (2006); Zhu et al., Anal. Chim. Acta, 608:186-196 (2008)). For adherent cells, in some embodiments, patterning of fibronectin protein or polylysine on the surface may be used to capture such cells at defined locations. In some embodiments, such methods may be used to separate different cell subsets in different regions within the same channel.

[0046] Cell proliferation assay 2A-2F illustrate exemplary assays for determining the proliferation rate of cells, followed by identification of the cells based on either cell surface proteins, protein secretion profiles, etc. Cells may also be identified or characterized by their transcriptomes, as determined as described below, or by similar techniques. It is understood that the term "proliferation rate" may include a measure of lack of proliferation. For example, a chamber encapsulating one or more cells may be exposed to an agent (e.g., a drug candidate), after which such cells may be returned to normal growth conditions. In some cases, the agent may kill cells or slow cell growth, for example, compared to a control not exposed to the agent. Thus, for treated cells, a negative "proliferation rate" may occur because the final number of cells counted in the chamber may be less than the original number; or a signal monotonically related to cell number may drop in value.

[0047] FIG. 2A shows the steps of a proliferation assay performed by the system of the present invention. Cells and polymer precursors are loaded into the channel (200) and placed on a first surface (202) (e.g., cell (204)). The position of the cell is determined by the detector (206): in this case, the position of the cell is used by the control system to generate instructions for the spatial energy regulating element (208) to generate a light beam to synthesize (210) a hydrogel chamber in the channel (200) around a single cell, as exemplified by hydrogel chambers (212, 213, and 214). The close-up (215) illustrates that the solid-looking structures (212, 213, and 214) have an interior (211) and a wall (221) with a predetermined thickness (216). Similarly, the hydrogel chamber has a predetermined shape (e.g., a circular shape with a diameter (217)) and encloses a predetermined area. In the figures, for convenience, the chambers are illustrated as being isolated with no connections to adjacent chambers and as having cylindrical or ring-like shapes. However, spatial energy modulating elements may synthesize chambers of different shapes and sizes as may be useful for a particular application. In some embodiments of the proliferation assay, each hydrogel chamber synthesized has the same shape and area (e.g., 0.001 to 0.01 mm2). 2 or .001~1.0mm 2In some embodiments, each hydrogel chamber synthesized has the same shape and area for each different type of cell to be assayed: for example, cytotoxic T lymphocytes may be confined in a chamber with one area, while helper T lymphocytes may be confined in a chamber with another area. After a desired number of hydrogel chambers are synthesized, the cells are incubated under growth conditions for a period of time, after which the cells in each chamber (for example) are counted to provide a measure of proliferation potential for each cell (220). In some embodiments, after the chambers are synthesized to encapsulate selected cells, unselected cells may be removed by a washing step or other modification of reagents in the channels. In some embodiments, cells may be stained with a membrane dye or an intracellular dye to determine proliferation by dye dilution, such that an independent measure of cell proliferation may be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. The desired number of single cells encapsulated by the chambers depends on the desired statistical confidence in the measured value. If the subpopulation of interest is present as only a small percentage of the total population, a larger number of chambers is required. In some embodiments in which mammalian cells are assayed, the number of hydrogel chambers synthesized around a single cell may be greater than 100, or greater than 1000, or greater than 10,000, or the number may range from 100 to 100,000, or range from 1000 to 100,000. After the counts are recorded for each chamber, further assays may be performed on the clonal populations within the chambers to identify the cell type, for example, by evaluation of cell surface proteins, cell protein secretion, transcriptome, etc. This approach is particularly useful for evaluating populations of immune cells, especially engineered immune cell populations.In some embodiments, a cell proliferation assay may be performed by the steps of: (a) providing a fluidics device including (i) a first surface, a channel including a cell disposed on or adjacent to the first surface, and one or more polymer precursors, (ii) a spatial energy modulating element in optical communication with the first surface, and (iii) a detector for determining the location of the cell in the channel based on one or more optical signals from the cell; (b) synthesizing one or more chambers in the channel encapsulating one or more cells by projecting light into the channel by the spatial energy modulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, where the location of the synthesized chamber is determined by the location of the cell encapsulated by the chamber as determined by the detector; (c) incubating the cells under growth conditions; and (d) determining the proliferation rate of the cells. In some embodiments, determining the proliferation rate includes counting cells in each chamber.

[0048] FIG. 2B illustrates a subsequent assay for identifying cells by cell surface markers. If the cell surface markers are to be measured directly (without first measuring cell proliferation), it is noted that prior to introduction into the channel, the cells may be incubated with a labeled antibody, where the label may vary widely, including but not limited to fluorescent labels, oligonucleotide labels, colorimetric labels, enzyme labels, and the like. It is also understood that binding compounds other than antibodies or fragments thereof (e.g., aptamers, etc.) may be used in the present invention. After the proliferation rate is determined for each chamber, the channel (200) is loaded with a mixture of different labeled antibodies, each specific for a different cell surface protein. While such antibodies may be prepared for any cell surface protein, surface proteins of particular interest are those that characterize various immune cells and their binding specificities (e.g., CARs). In some embodiments, the antibody is specific for a surface protein, including, but not limited to, cluster of differentiation (CD) markers, such as CD3, CD4, CD5, CD6, CD7, CD8, CD11, CD19, CD20, CD21, CD22, CD23, CD24, CD25, and the like. In some embodiments, the label of the antibody is an oligonucleotide. In some embodiments, such an oligonucleotide may be attached to the antibody by a scissile bond that may be cleavable by conventional methods, such as by chemical cleavage by reducing, acidic, or basic conditions, or by photocleavage by exposure to light of an appropriate wavelength and intensity. In some embodiments, exposing the antibody-labeled cells to a lysis reagent is sufficient to release the antibody-oligonucleotide conjugate for capture by the capture element, regardless of whether the scissile linkage is present. The oligonucleotide label may include a barcode sequence that uniquely identifies the protein to which the antibody is specific. The oligonucleotide label may also include a sequence complementary to the oligonucleotide attached to the first surface as the capture element.In some embodiments, either such complementary sequence or the complementary sequence of the capture oligonucleotide may be blocked by hybridization of a mismatch oligonucleotide or a shorter oligonucleotide (e.g., less stable than the oligonucleotide label-capture oligonucleotide duplex) to block spurious capture of the antibody-oligonucleotide conjugate by the capture oligonucleotide at the first surface when loaded into the channel. Such blocking oligonucleotides are then removed in a washing step prior to releasing the oligonucleotide label of the antibody bound to the cell surface. Alternatively, the oligonucleotide label may be configured to be linked to the capture oligonucleotide via a splice oligonucleotide. The splice oligonucleotide will block spurious hybridization of the oligonucleotide label prior to release from the antibody-surface protein complex. After such release and introduction of a ligase, the oligonucleotide label will be captured by the free end of the splice oligonucleotide and the oligonucleotide label will be linked to the end of the capture oligonucleotide.

[0049] In the embodiment of Figures 2A-2B, after a period of incubation (226) to allow binding of the antibody to its target protein, the oligonucleotide label is released and captured by the complementary sequence of the capture element. For example, the oligonucleotide label may be attached by a disulfide linker that can be released by a reducing agent (e.g., Hermanson, cited above). In some embodiments, after incubation, unbound antibody may be removed, for example, by a washing step. In some embodiments, the channel (200) may be loaded with a reaction mixture (228) containing a polymerase, dNTPs, and other components necessary to extend the complementary oligonucleotide of the capture element with the captured antibody oligonucleotide as a template. In some embodiments, the polymer matrix wall of the chamber may be degraded or depolymerized (226), after which the channel (200) may be loaded with such extension reagents (228). As explained more fully below, the DNA copies may be sequenced in situ (230), or may be released (possibly after amplification) from the first surface, eluted, and sequenced externally. In the latter process, the complementary oligonucleotides of the capture elements contain spatial barcodes to identify the chamber from which the antibody barcode sequence originates. In either case, the number of each type of antibody oligonucleotide calculated from the sequence data provides a cell surface protein profile (e.g., the relative frequency of each cell surface protein) of the cells encapsulated by each of the hydrogel chambers. Moreover, this data can be correlated to the proliferation potential of such cells.

[0050] 2C-D illustrate one embodiment of a protein secretion assay using commercially available protein capture beads (e.g., Biolegend, San Diego, Calif.). One of skill in the art will recognize that other surfaces besides (or in addition to) the bead surface may be used for localized capture of secreted proteins (e.g., cytokines, etc.). The top and bottom panels of FIG. 2C are top views of a cross section of a channel (200) containing chambers (212, 213, and 214). (Thus, Figures 2C-2D illustrate an alternative assay of cells after counting (to the assay of Figure 2B), or in some embodiments, the assay of Figure 2B can be performed after the assay of Figures 2C-2D to determine a protein secretion profile, followed by a surface protein profile.) After cell counting, the channel (200) is loaded (232) with protein capture beads (e.g., 234), which are beads (which are typically impermeable to the polymer matrix walls of the chamber) having covalently attached antibodies specific for a predetermined protein that may be secreted by cells enclosed by the chamber.Such beads may be prepared for any secreted protein, however proteins of particular interest include interferons, such as interferon-gamma (IFN-gamma) and interferon-alpha (IFN-alpha), 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), The cytokines and immunoactive proteins include, but are not limited to, colony stimulating factors (CSFs), such as granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), tumor necrosis factors (TNFs), such as TNF-α and TNF-β, and effector molecules, such as granzyme B. After such beads are loaded, concentric chambers are synthesized around each of the cell-containing chambers (236), after which the beads outside the concentric chambers are removed (238). In some embodiments, the size of the larger concentric chamber is sufficient to encapsulate a large number of protein capture beads, such that there is a high probability (e.g., greater than 90 percent, or greater than 95 percent, or greater than 99 percent) that at least one bead is present for each different cytokine to be measured. One skilled in the art will recognize that such sizing depends on the concentration of beads loaded into the channel (200). After synthesis of the concentric chambers, the polymer matrix wall of the inner chamber is depolymerized (240), such that cells and beads can freely mix, such that secreted proteins can be captured by the beads, after which (as in a sandwich assay) a labeled antibody ("detection antibody") is loaded into the channel to bind to a different epitope of the captured protein.In some embodiments, the porosity of the inner chamber is selected to prevent or inhibit the passage of secreted proteins through its polymer matrix wall. In some embodiments, the porosity of both the inner chamber and the outer concentric chamber is selected to prevent or inhibit the passage of secreted proteins, and prior to loading the detection antibody, the polymer matrix wall of the outer chamber is depolymerized to remove the obstacle to the detection antibody binding to the captured protein. Alternatively, in some embodiments, the porosity of the polymer matrix wall of the inner chamber can be selected to allow the passage of secreted proteins into the annular region containing the beads. The relative amount of secreted proteins can be estimated by counting each bead type that produces a characteristic fluorescent signal, or by integrating each different fluorescent signal over all the beads adjacent to the cells of the chamber. This embodiment may be carried out by the following steps, either initially after cells are placed on a first surface, or after another assay that does not destroy the cells: (a) loading into the channel a second reaction mixture comprising a second polymer precursor and protein capture beads comprising a protein capture antibody that binds to a protein secreted by the cells; (b) synthesizing second chambers enclosing each of the one or more chambers by projecting light into the channel with the spatial energy regulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the second chamber, wherein the position of the synthesized second chamber is determined by the position of the chamber enclosed thereby as identified by the detector; and (c) depolymerizing the chambers, such that the protein capture beads in each second chamber mix with the cells enclosing the depolymerized chamber. In some embodiments, the (first) polymer precursor may include a precursor that forms gel linkages degradable by reducing conditions (e.g., Structure 2 in Table 2B), while the second polymer precursor does not include such a precursor.In some embodiments, the method further comprises incubating the cells with the protein capture beads and detecting proteins secreted by the cells by the amount of labeled protein detection antibody for each protein bound to the protein capture beads adjacent to the cells.

[0051] Those skilled in the art will recognize that the function of protein capture antibody may be performed by other affinity reagents (such as aptamers) that have specific binding ability. As used herein, the term "affinity reagent" refers to a compound that can specifically bind to a target molecule, such as a protein. Affinity reagents include, but are not limited to, antibodies, antibody fragments, aptamers, and similar compounds.

[0052] An alternative embodiment for measuring protein secretion profiles is illustrated in Figures 2E-2F. In this embodiment, a uniform distribution of protein capture beads is attached to a first surface (250) prior to loading the cells. As illustrated in close-up (244), the uniform distribution can include beads with antibodies specific for different proteins. After the cells are deposited on the surface (250), the cells are located by a detector (245) and hydrogel chambers are synthesized (246) by a spatial energy control element (247). The cells are incubated under growth conditions, after which the cells in each chamber are counted to determine the proliferation rate (253). As described above, the secreted proteins are detected by capturing the protein capture beads and then labeling with a detection antibody (255). In some embodiments, the porosity of the polymer matrix walls of the chambers is selected to prevent the passage of cells, but to allow the free passage of proteins. In other embodiments, such porosity is selected to prevent the passage of both cells and proteins. In the latter embodiment, an additional step of depolymerizing the polymer matrix wall is included. The relative amount of secreted protein is determined either by counting the different types of labeled beads adjacent to or near the cells of each chamber (257) or by integrating the different fluorescent signals from the beads adjacent to or near the cells of each chamber (256). In addition to the beads carrying the protein capture antibodies as mentioned above, other surfaces capable of carrying the secreted protein capture antibodies include, but are not limited to, the first surface, the polymer matrix wall of the chamber, or the second surface. These surfaces capable of attaching the secreted protein capture antibodies are referred to herein as "protein capture surfaces."

[0053] In some embodiments, the method for correlating a cell characteristic with a proliferation rate may include the steps of: (a) providing a fluidics device including (i) a channel including a first surface, a cell disposed on or adjacent to the first surface, and one or more polymer precursors, (ii) a spatial energy modulating element in optical communication with the first surface, and (iii) a detector for identifying a position of a cell in the channel based on one or more optical signals from the cell; (b) synthesizing one or more chambers in the channel encapsulating one or more cells by projecting light into the channel by the spatial energy modulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, where the position of the synthesized chamber is determined by the position of the cell encapsulated by the chamber as identified by the detector; (c) incubating the cells under proliferation conditions; and (d) counting cells in each chamber to determine their proliferation rate. In some embodiments, the first surface may include one or more capture elements for capturing one or more biological components of said cells (e.g., secreted proteins, messenger RNA, genomic DNA, etc.). In some embodiments, the synthesizing step may be followed by a washing step to remove unreacted polymer precursors. In some embodiments, after such a washing step (or as part of such a washing step), cell culture medium may be loaded into the channel.In some embodiments, the method further comprises the steps of: (i) incubating the cells with antibodies specific for surface proteins whose relative expression allows for identification of the cells, each such antibody having an oligonucleotide label comprising an antibody-specific barcode capable of being captured by the capture element; (ii) optionally loading a releasing reagent into the channel, such that the oligonucleotide labels of antibodies attached to the one or more cells are released and captured by the capture element (if the oligonucleotide labels are attached by a scissile linkage); (iii) optionally depolymerizing the polymer matrix wall of the chamber; (iv) loading the channel with a reagent that copies the captured oligonucleotide label to generate its complementary DNA; and (v) sequencing the complementary DNA to identify the captured oligonucleotide label. In some embodiments, the cDNA of the captured labels can be sequenced at or near their capture location by (a) amplifying the complementary DNA, (b) sequencing the amplified complementary DNA, and (c) determining the relative expression of the surface proteins for each of the cells in the chamber. Oligonucleotide labels can be attached to the antibody (or other binding moiety) by a cleavable linkage known in the art. Such oligonucleotide labels attached to the antibody by a cleavable linkage can be cleaved or released from the antibody by a cleavage reagent or agent, which can be chemical, physical or electrical in nature, or (equivalently) a release reagent or agent. Reagents for copying the captured oligonucleotide labels include DNA polymerase, dNTPs, and associated buffers and salt solutions to extend the captured oligonucleotide using the oligonucleotide label as a template. In some embodiments, the cDNA of the captured labels can be sequenced on an external sequencing instrument after (optional amplification and) elution from the channel.For the latter sequencing, the capture elements on the first surface contain spatial barcodes and the captured oligonucleotide-labeled cDNAs each contain a spatial barcode. After sequencing the cDNA-spatial barcode conjugates, the cDNAs may be assigned to chambers based on their spatial barcodes.

[0054] In other embodiments, cell proliferation can be correlated to protein secretion by: (a) providing a channel comprising a protein capture surface comprising protein capture antibodies that bind to proteins secreted by the cells; and (b) detecting proteins secreted by the cells by the amount of labeled protein detection antibodies for each protein bound to the protein capture surface adjacent to the cells. In some embodiments, the protein capture surface comprises protein capture beads.

[0055] In some embodiments, cell proliferation rate can be correlated with cell transcriptome after the process described below for converting mRNA from cells to cDNA and sequencing the resulting cDNA. Briefly, after the counting process, the following additional steps can be carried out: (a) loading the channel with a lysis reagent, which results in the release of the cell's mRNA and capture by the capture element; and (b) loading the channel with a reverse transcription reagent that copies the captured oligonucleotide label and generates its complementary DNA; and (c) sequencing the complementary DNA. It is understood that the sequencing process may include additional steps in certain embodiments, including but not limited to tagmentation, adding adapters, truncating cDNA to form the appropriate length for sequencing, etc. In some embodiments, additional steps can be carried out to depolymerize or decompose the polymer matrix wall of the chamber after mRNA capture. The reverse transcription reagents include conventional reagents for reverse transcription, i.e., reverse transcriptase (e.g., Moloney Murine Leukemia Virus (MMLV)), dNTPs, RNase inhibitors as needed, and buffers. The sequencing step may be performed at the site of the captured mRNA (in situ), or the cDNA may contain spatial barcodes, be eluted, and sequenced on a separate sequencing instrument ("ex situ" sequencing). For in situ sequencing, further steps may include (i) amplifying the complementary DNA, e.g., by a method such as bridge amplification, (ii) sequencing the amplified complementary DNA, e.g., by a technique of sequencing by synthesis, and (iii) determining the relative expression of mRNA for each cell in the chamber. For external sequencing, further steps may include (i) providing a capture element containing a spatial barcode, (ii) synthesizing cDNA containing the spatial barcode, and (iii) eluting and sequencing the cDNA, correlating each cDNA with a chamber location by its spatial barcode.

[0056] Cytotoxicity assay As with cell proliferation, measures of cytotoxicity against a target cell population can be correlated against other cellular characteristics, including, but not limited to, cell membrane protein expression, cellular protein secretion profile, transcriptome, and the like. The target cells may comprise a sample of a patient's tumor cells, or the target cells may be a cell line, such as those derived from a tumor cell line, such as the liver tumor cell line, SK-HEP-1 (Chava et al., J. Vis. Exp., 2020 Feb 22:(156):10.3791 / 60714), and the like. Steps of an exemplary cytotoxicity assay are illustrated in Figures 3A-3B. Target cells (303) are loaded (304) into the channel (300) to be positioned on a first surface (302), after which effector cells are loaded (306) and positioned on the first surface (302) in addition to the target cells. By "effector cells" is meant cells for which cytotoxicity is being measured. In some embodiments, the effector cells are cells that are manipulated for therapeutic purposes, such as for the treatment of cancer. The target cells and the effector cells are incubated (308) with a vital dye that generates an optical signal in response to a characteristic of live or dead cells that allows dead cells to be counted. Exemplary vital dyes for live cells include, but are not limited to, Hoechst33342. Exemplary vital dyes for dead cells include, but are not limited to, 7-AAD; see, for example, Schmid et al., Cytometry, 15:12-20 (1994); Bradford et al., poster entitled "Dead cell stains in flow cytometry: a comprehensive analysis", Molecular Probes (Eugene, OR). After such incubation, a measure of cytotoxicity can be determined by counting dead target cells (310) adjacent to or in the vicinity of each effector cell. In some embodiments, individual effector cells can be encapsulated by the chamber immediately after loading to ensure that the only dead target cells counted are those target cells killed by a single encapsulated effector cell.In other embodiments, effector cells may be encapsulated by the chamber at a later time, if the dead target cells are easily associated with the dead target cells, for example, by being in close proximity. The effector cells may then be identified by (for example) cell surface markers, protein secretion, transcriptome, etc. For identification by cell surface markers, the effector cells may be encapsulated by the hydrogel chamber (312) (if not already encapsulated) and antibodies with oligonucleotide labels may be loaded into the channel (314). In some embodiments, unencapsulated cells may be removed by a washing step or other modification of the reagents in the channel. After incubation for antibody binding to the target surface protein (316), the oligonucleotide labels are released and captured by the capture element. After such capture, the chamber is optionally depolymerized and an extension reagent is loaded to copy the captured oligonucleotide by extending the capture element oligonucleotide with the captured oligonucleotide as a template (318). The resulting cDNA is amplified and sequenced (320) as described above for the cell proliferation assay.

[0057] Similarly, cells from different channels encapsulated as shown in step (312) may be subjected to different assays in the different channels to determine cell characteristics other than cell surface markers, where the cells are being measured for cytotoxicity. Such different assays include, but are not limited to, protein secretion, vector copy number, transcriptome, etc.

[0058] In some embodiments, additional steps can be performed to reduce or eliminate false signals generated by target cell proteins or mRNA. For example, after incubation for killing and after the labeled antibody binds to cell surface proteins, the effector cells in the chamber can be encapsulated or encased in a gel that separates the effector cells from the target cells in the first chamber (or a second chamber can be synthesized). After counting, the target cells in the first chamber can then be lysed and removed by washing. After such removal, the gel encasing the effector cells can then be depolymerized to allow the antibody label or mRNA to be released and captured by the capture element of the first surface. In some embodiments, the gel encasing the effector cells can be degraded, for example, by a reducing agent, and has an average pore size that is small enough to prevent the passage of most mRNAs of interest (e.g., mRNAs of 300 nucleotides or more) until depolymerization.

[0059] In some embodiments, a method of correlating cell characteristics with cytotoxicity includes the steps of: (a) providing a fluidics device including (i) a channel comprising a first surface and a population of target cells disposed on the surface, (ii) a spatial energy modulating element in optical communication with the first surface, and (iii) a detector that identifies a location of a cell in the channel based on one or more optical signals from the cell; (b) loading each channel with an effector cell and one or more polymer precursors such that the effector cell is disposed on or adjacent to the first surface; (d) loading one or more chambers in the channel each encapsulating an effector cell. The method may include: (e) synthesizing a bar by projecting light into the channel by the spatial energy modulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, where the position of the synthesized chamber is determined by the position of the effector cell encapsulated by the chamber identified by the detector; (e) incubating the target cell and the effector cell with a vital dye that identifies dead cells; and (f) counting dead cells in each chamber to determine a measure of cytotoxicity of the effector cell encapsulated by the chamber. In some embodiments, the vital dye generates an optical signal in dead cells, but not in live cells. In some embodiments, the synthesizing step may be followed by a washing step to remove unreacted polymer precursors. In some embodiments, after such washing step (or as part of such washing step), cell culture medium may be loaded into the channel.

[0060] In some embodiments, after the cytotoxicity of effector cells is measured, for example, by counting adjacent dead cells or dead cells in the chamber, the effector cells can be identified by other assays measuring properties such as surface proteins, protein secretion, etc. An assay for identifying effector cells by their cell surface proteins can include the following steps: (i) incubating the cells with antibodies specific for surface proteins whose relative expression allows the cells to be identified, each such antibody having an oligonucleotide label comprising an antibody-specific barcode that can be captured by the capture element; (ii) loading a release reagent into the channel, such that the oligonucleotide label of the antibody attached to the one or more cells is released and captured by the capture element; (iii) optionally depolymerizing the polymer matrix wall of the chamber; (iv) loading the channel with a reagent that copies the captured oligonucleotide label to generate its complementary DNA; and (v) sequencing the complementary DNA to identify the captured oligonucleotide label, thereby determining the cell surface protein profile of each effector cell. As noted above, in some embodiments, the following steps may be performed prior to loading of the release reagent: (a) encapsulating the effector cells in each chamber in a degradable encapsulating gel that prevents leakage of bound oligonucleotide-labeled antibodies or mRNA, (b) lysing the target cells by loading a lysis reagent into the channel, and (c) washing to remove the oligonucleotide-labeled antibodies and mRNA bound to the target cells.

[0061] In some embodiments, such cDNA may be sequenced in situ by: (a) amplifying the complementary DNA, (b) sequencing the amplified complementary DNA, and (c) determining the relative expression of the surface proteins for the cells in each of the chambers. In alternative embodiments, such cDNA may be sequenced exogenously by providing a capture element with a spatial barcode such that cDNA synthesized from captured oligonucleotide labels contains a spatial barcode, amplifying the cDNA, releasing (or dehybridizing) and eluting the amplified cDNA (or their complements), and sequencing the cDNA, where the spatial barcode allows identification of the chamber from which the cDNA originated.

[0062] The cytotoxicity of cells can be correlated to protein secretion profile using additional steps as described for cell growth.In one embodiment, such steps may include: (i) loading the channel with a second reaction mixture that includes a second polymer precursor and a protein capture bead that includes a protein capture antibody that binds to the protein secreted by the cell; (ii) synthesizing a second chamber that encloses each of the one or more chambers by projecting light into the channel with the spatial energy control element, whereby the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the second chamber (wherein the position of the synthesized second chamber is determined by the position of the (first) chamber that is enclosed by the chamber identified by the detector); and (iii) depolymerizing the chamber, whereby the protein capture bead in each second chamber mixes with the cell that is enclosed by the depolymerized chamber therein. The method may further include incubating the cells with the protein capture beads and detecting proteins secreted by the cells by the amount of labeled protein detection antibody for each protein bound to the protein capture beads adjacent to the cells.

[0063] In an alternative embodiment, the channel further comprises protein capture beads disposed in the channel comprising protein capture antibodies that bind to proteins secreted by the cell, and the method further comprises detecting the proteins secreted by the cell by the amount of labeled protein detection antibodies for each protein bound to the protein capture beads adjacent to the cell.

[0064] Vector copy number assay Vector copy number in therapeutically engineered cells is important due to the increased risk of carcinogenesis if the copy number is too high, e.g., greater than about 5 copies per cell (e.g., Chen et al., Cytotherapy, 22(5), Supplement, S142 (2020); Paugh et al., Scientific Reports, 11:389 (2021)). In accordance with the present invention, vector copy number can be measured by single-cell quantitative PCR in the chamber or by counting unique molecular identifiers (UMIs) or similar measures that are amplified and sequenced from single cells in the chamber. For either assay, cells can be prepared as illustrated in FIG. 4A. That is, engineered or transduced cells are loaded into the channel (400) such that the cells (e.g., 401) are positioned on a first surface (402), after which a detector (404) records the location of the cells on the first surface (402) and a spatial energy regulating element (406) synthesizes a chamber (411) that separately encapsulates the single cell (408). The porosity of the polymer matrix wall of the chamber can be selected to allow for the transfer of proteins, lysis reagents, mRNA, etc., but retain genomic DNA, using polymer precursors and conditions such as those described in Spencer et al., ISME Journal, 10:427436 (2016); and Tamminen et al., Frontiers Microbiology: Methods, 6:195 (2015). Cells can be lysed as described in Tamminen et al. (proteinase K followed by lysozyme) or by Zhang et al., Proc. Natl. Acad. Sci., 89:5847-5851 (1992) (alkaline lysis: 200 mM KOH / 50 mM dithiothreitol followed by neutralization with 900 mM Tris-HCL (pH 8.3) / 300 mM KCl / 200 mM HCl) or similar methods (413).Following such treatment, the genomic DNA retained in the chamber may be further analyzed by qPCR (420), such as the qPCR described by Vaninsberghe et al., PlosOne, 13(1):e0191601 (2018), or, if the vector contains a UMI, by sequencing (421) as outlined in Figure 4B (e.g., Porter et al., Genome Biology, 15:R75 (2014)).

[0065] For qPCR (420), in some embodiments, the channel (400) is loaded with a PCR reaction mixture that includes polymerase, primers, taqman probes, buffers, and salts. In some embodiments, the flow cell is moved to a temperature cycle station to perform the PCR. As the PCR proceeds in the various chambers, a detector (404) records the accumulation of signal from the taqman probes in each chamber, which can be converted to copy number. In some embodiments, the channel containing the chamber in which the qPCR reaction is occurring can be loaded with a blocking agent to prevent diffusion of labeled taqman fragments out of the chamber. Such a blocking agent can include polymer precursors that can be polymerized in the space surrounding the chamber.

[0066] For sequencing (412), the retained genomic DNA may be pre-amplified, if desired, by whole genome amplification techniques, e.g., as described by Zhang et al. (cited above), after which the desired sequence, e.g., a sequence containing a viral UMI, is specifically amplified (e.g., by PCR or linear amplification techniques) and captured by the capture element (412). In some embodiments, the UMI is amplified along with a "handle" segment (an oligonucleotide segment complementary to the capture oligonucleotide) to facilitate capture by the capture element on the first surface. As described above, the chamber is depolymerized, extension reagents are loaded, and cDNA containing copies of the UMI may be synthesized (416). After amplification, the cDNA may be sequenced either in situ or ex vivo, and the number of UMIs with different sequences is determined to give the viral copy number for the cells in each chamber.

[0067] The method may be carried out by first encapsulating the engineered cells in hydrogel chambers according to the present invention, i.e., by the steps of: (a) providing a fluidics device comprising: (i) one or more channels, each having a first surface, (ii) a spatial energy modulating element in optical communication with each of the first surfaces, and (iii) a detector for identifying the location of a cell in each of the channels based on one or more optical signals from the cell; (b) loading each of the channels with a cell and one or more polymer precursors, such that the cell is positioned at or adjacent to the first surface; (c) isolating one or more chambers in each of the channels by the spatial energy modulating element, each chamber encapsulating a cell. (d) synthesizing one or more cells by projecting light onto the one or more polymer precursors, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, wherein the location of the synthesized chamber is determined in each channel by the location of the cell encapsulated by that chamber as identified by the detector; (d) loading assay reagents (e.g., lysis reagents followed by amplification reagents followed by cDNA synthesis reagents, etc.) into one or more channels; and (e) incubating the cells in each channel under assay conditions, including PCR conditions, rolling circle amplification conditions and / or sequencing conditions, to generate a signal from the chamber in each channel indicative of sequence copy number, e.g., vector copy number (VCN), etc.To count the number of copies of a particular (or predetermined) nucleotide sequence (such as a UMI), in some embodiments, the following steps may be performed: (i) lysing the cells to release genomic DNA; (ii) amplifying the one or more nucleotide sequences; (iii) capturing the one or more amplified nucleotide sequences with the capture element; (iv) loading the channel with a reagent that copies the captured one or more nucleotide sequences to generate complementary DNA; and (v) sequencing the complementary DNA to identify the number of copies of the one or more nucleotide sequences. Particularly noteworthy embodiments include a predetermined nucleotide sequence that is a barcode, particularly a barcode that is a UMI or a barcode that contains a UMI. The amplified nucleotide sequence typically includes one or two primer binding sites, a barcode, and a sequence complementary to the capture oligonucleotide of the capture element. The amplified nucleotide may include an additional segment to increase the molecular weight of the amplification product to prevent or inhibit passage through the polymer matrix wall of the hydrogel chamber. From the sequencing data, a number of different UMIs can be identified that give the viral copy number for the cell.

[0068] To count copy numbers by quantitative PCR or other amplification methods, in some embodiments, the following steps may be performed: (i) lyse the cells to release genomic DNA; (ii) load the channel with an amplification reagent that generates a signal proportional to the copy number of the one or more nucleotide sequences of the genomic DNA; (iii) amplify the one or more nucleotide sequences to generate a signal that is monotonically related to the copy number of the one or more nucleotide sequences. In some embodiments, the amplification reagent may be a quantitative PCR reagent, a bridge PCR reagent, or a rolling circle amplification reagent. In some embodiments, the quantitative PCR reagent includes an amplification buffer, a polymerase with 5'→3' exonuclease activity, a primer, dNTPs, and a taqman probe.

[0069] In further embodiments, copy number can be determined by copying selected genomic sequences, capturing them with a capture element, synthesizing cDNA from the selected genomic sequences, then forming clusters of cDNA either by bridge PCR or DNA nanoballs by rolling circle amplification, after which the clusters or DNA nanoballs can be counted for a measure of copy number. When quantitative PCR is used, the signal related to the copy number can be an optical signal or cycle number. When bridge PCR is used, the first surface can include bridge PCR primers, and the signal related to the copy number can be the number of clusters formed. When rolling circle amplification is used, the signal related to the copy number can be the number of DNA nanoballs formed. Clusters and DNA nanoballs can be detected by a wide variety of techniques, including but not limited to the use of fluorescently labeled dNTPs, double-stranded DNA dyes, and the like.

[0070] Integration site analysis The site(s) of integration of the viral vector in the genome of the therapeutic cell is of great interest due to possible abolishment or alteration of expression of one or more important genes, sometimes manifested as genotoxicity or insertional mutagenesis; see, e.g., Biasco et al., Molecular Therapy: Methods & Clinical Development, 8:21-30 (2018); Cornetta et al., Molecular Therapy: Methods & Clinical Development, 28:28-39 (2023); and Desfarges et al., Viruses, 2:111-130 (2010). The insertion site can be detected in a population of cells according to the present invention, as illustrated in Figures 4C-4F. The general concept of the measurement approach is to anneal a primer to a known sequence of the vector to be integrated into the genome, extend the primer into the host genomic DNA, and then identify the host gDNA and the integration site. Individual cells are encapsulated in hydrogel chambers as depicted in Figure 4A, where the porosity of the hydrogel is selected such that the walls of the chamber prevent the passage of genomic DNA but allow the passage of reagents (e.g., deoxynucleoside triphosphates (dNTPs), DNA polymerase, primers, etc.) (420), as disclosed, for example, by Spencer et al., ISME J., 10:427-436 (2010). The cells are lysed (422) to release genomic DNA (gDNA) (424), after which the released gDNA is amplified, denatured, and combined with vector-specific primers (e.g., 425). Such lysis can be carried out by various methods known in the art by loading a lysis reagent into the channel; e.g., Spencer (cited above); Cui et al., Proc. Natl. Acad. Sci., 86:9389-9393 (1989); Deleye et al., Scientific Reports, 5:11711 (2015), etc.After lysis, whole genome amplification (WGA) can be performed using techniques known in the art, such as primer extension pre-amplification PCR (PEP-PCR), degenerate oligonucleotide primer PCR (DOP-PCR), multiple displacement amplification (MDA), multiple annealing-loop formation-based amplification cycles (MALBAC), and the like, as disclosed in the following references: Volozonoka et al., Int. J. Mol. Sci., 23:4819 (2022); Hou et al., GigaScience, 4:37 (2015); Yu et al., Anal. Chem., 86:9386-9390 (2014), and the like. FIG. 4C shows the integrated vector (426) as a linear segment of DNA flanked by long terminal repeat (LTR) sequences. In some embodiments, other vector regions can be used as primer binding sites. The LTR primer (425) has a 5' oligonucleotide tail (427) that has a sequence complementary to a capture oligonucleotide (430) on a first surface (428) within the chamber. The LTR primer is extended by a DNA polymerase in the presence of dNTPs (e.g., 431), after which the chamber is washed (429) with stringency (e.g., salt, heat, etc.) high enough to destabilize and remove the unextended LTR primer, but not so high as to remove the extended LTR primer. The extended LTR primer is then melted from the gDNA and captured (436) by the capture oligonucleotide (430) by hybridizing to the 5' tail (427) of the extended LTR primer. The capture oligonucleotide (430) is attached to the surface (428) by its 3' end, and its 5' end has a 5'-phosphate group attached. The capture oligonucleotide may have additional features depending on the embodiment. For example, if the sequence of the extended region of the extended LTR primer is to be determined in situ, the capture oligonucleotide may include a primer for bridge amplification, whereas if the sequence of the extended region is to be determined off-instrument, the capture oligonucleotide may include a spatial barcode to identify sequences associated with the same cell.In Figure 4D, surface (428) is illustrated with so-called P7' sequence (432) and P5 sequence (434) for bridge PCR; thus, capture oligonucleotide (430) contains the P7 sequence to initiate bridge PCR after the LTR primer extension has been copied and removed. The actual sequences of the P5 and P7' regions are a matter of design choice for those skilled in the art.

[0071] After capture of the extended LTR primer (436), a random sequence hexanucleotide primer (441) is annealed to the extension region (438) and itself extended (440), after which the synthesized strand is ligated to the 5' end of the capture oligonucleotide using protocols described, for example, by Schmidt et al., Nature Methods, 4(12):1051-1057 (2007); Kalle et al., U.S. Patent No. 6,514,706, which are incorporated herein by reference. Alternatively, the 3' end of the extension region (438) can be extended by terminal deoxynucleotidyl transferase (TdT) to generate a homopolymeric tail to which a complementary primer can be annealed and extended to the 5' end of the capture oligonucleotide (430). The copied sequence is then ligated to a capture oligonucleotide (430), after which the resulting construct is treated with a restriction endonuclease to provide a known sequence end distal to the surface (428). The restriction endonuclease is selected to maximize the probability of retaining a sufficient extension region (e.g., about 18 nucleotides for humans) to uniquely identify its location in the genome of the engineered cell. In some embodiments, a restriction endonuclease is selected that has a 4-nucleotide recognition site and leaves a 4-nucleotide overhang after cleavage. After such cleavage, a double-stranded adapter is ligated to the end of the double-stranded fragment attached to the surface (428). As noted in FIG. 4F (446), the sequence of the adapted strand may be determined in situ by performing surface amplification followed by a sequencing reaction, or may be determined away from the surface (428) by amplifying by PCR and eluting the amplicon. In the former case, the capture oligonucleotide (430) contains a P7 sequence at its end proximal to the surface (428). In the latter case, the capture oligonucleotide (430) contains a spatial barcode at its end proximal to the surface (428).

[0072] In some embodiments, the method for determining vector integration sites for a population of cells can be performed by: (a) synthesizing one or more hydrogel chambers that encapsulate each of one or more cells arranged on the surface of a channel; (b) lysing the cells, whereby the genomic DNA of each cell is released into the hydrogel chamber; (c) amplifying the genomic DNA of each cell; (d) annealing a vector-specific primer to the amplified genomic DNA; (e) extending the vector-specific primer, whereby an extension product is formed that includes a copy of a segment of the genomic DNA; and (f) identifying the site of each vector integrated into the genomic DNA of each of the cells from the segment. In some embodiments, the vector-specific primer comprises a primer that is complementary to the vector sequence, but not to the genomic sequence of the cell. In some embodiments, whenever the vector is a retrovirus, the vector-specific primer can be complementary to the sequence of a long terminal repeat (LTR) element. Such vector-specific primers are sometimes referred to herein as "LTR primers." Those skilled in the art will recognize that the extent or length of the extension of the vector-specific primer into the flanking region of genomic DNA must be large enough to uniquely identify the location of the integrated vector in the cellular genome. For the human genome, in some embodiments, the length of such extension comprises at least 18 nucleotides of cellular genomic DNA.

[0073] Genomic copy number variation A key concern for stem cell therapy is the genetic stability of stem cells, since stem cells can exhibit aneuploidy and genome copy number variability, which are common characteristics of tumorigenic cells (e.g., Goldring et al., Cell Stem Cell, 8:618-628 (2011)). Single-cell genome copy number variation can be detected in a population of cells according to the present invention, as illustrated in Figures 4G-4H. In some embodiments, the methods described herein include the steps described in Example 3 of Khurana et al., US Patent Application Publication No. 2022 / 0219170, which is incorporated herein by reference. As described above, individual cells can be encapsulated in hydrogel chambers (450) as described in FIG. 4A, where the porosity of the hydrogel is selected such that the walls of the chamber prevent the passage of genomic DNA but allow the passage of reagents (e.g., deoxynucleoside triphosphates (dNTPs), DNA polymerase, primers, WGA reagents, tagmentation reagents, etc.) (e.g., gel porosity as disclosed by Spencer et al., ISME J., 10:427-436 (2010)). As shown in FIG. 4G, after the cells are encapsulated in the hydrogel chambers, the cells are lysed to release genomic DNA, after which WGA reagents are loaded to increase the amount of gDNA in the chambers, such that after fragmentation, the sequenced fragments will provide sufficient coverage for copy number variation (CNV) to be determined with acceptable resolution. In some embodiments, a sequencing depth of 0.3x results in a resolution of 3 megabases (Mb) for CNV determination (e.g., Deleye et al., Scientific Reports, 7:3422 (2017)). For 0.3x coverage in human cells, approximately 1 billion nucleotide sequence reads are required. One of skill in the art will recognize that the degree of amplification from WGA, the surface area enclosed by the chamber, and the capture oligonucleotide density can be adjusted to achieve the desired CNV resolution. In some embodiments, for CNV measurement, the surface area enclosed by the chamber is at least 103 μm 2 , or at least 10 4 μm 2 , or at least 10 5 μm 2 , or at least 10 6 μm 2 and the density of the capture oligonucleotides is 1 cm 2 At least 1×10 per 12 The capture oligonucleotides are 100 times larger than the number of capture oligonucleotides. In some embodiments, WGA increases the amount of genomic DNA in each chamber by at least 100 times. From such genome sequence data, genome copy number variation can be estimated by known methods (e.g., Mallory et al., Genome Biology, 21:208 (2020); Wang et al., Briefings in Bioinformatics, 19 (5); 731-736 (2018) etc.). In some embodiments, the resolution of CNV measurement is 3 megabases (Mb) or more, or 2 Mb or more, or 1 Mb or more (wherein the larger resolution corresponds to a smaller unit of genome length, e.g., a smaller Mb value).

[0074] Returning to FIG. 4G, the amplified gDNA (454) is treated with a tagmentation reagent, such as Tn5 transposase (456), which generates gDNA fragments (466) having upstream adapters (460) and downstream adapters (464) of predetermined sequence, which may include amplification primer binding sites, sequencing primer binding sites, etc.

[0075] The adaptor-tagged tagmentation fragments are then denatured so that the strands can be captured and extended (467) by a capture oligonucleotide (469). The capture oligonucleotide (469) comprises a capture moiety ("CM"), a spatial barcode ("sBC"), and an amplification primer ("Primer 1"). The depicted capture oligonucleotide (469) is on a small region of the surface (428) enclosed by the chamber. Of course, the entire surface (428) may have such capture oligonucleotides attached, whether enclosed by a chamber or not. A second amplification primer ("Primer 2") is provided by the captured tagmentation fragment. These sequences may be amplified by conventional PCR, and the amplicons may be eluted from the channel for sequencing (470). One of skill in the art will recognize that such elution and / or amplification may be preceded by disassembly of the hydrogel of the chamber.

[0076] In some embodiments, the method for genome copy number determination for a population of cells may be performed by (a) synthesizing one or more hydrogel chambers encapsulating each of one or more cells disposed on a surface of a channel; (b) lysing the cells, such that the genomic DNA of each cell is released into the hydrogel chamber; (c) amplifying the genomic DNA of each cell; (d) sequencing fragments of the amplified genomic DNA; and (e) determining genome copy number variation for each of the cells from the sequences of the genomic DNA fragments. In some embodiments, the sequencing step includes fragmenting the genomic DNA and attaching adapters to the genomic DNA fragments by tagmentation. In some embodiments, such adapters include strands that are complementary to the oligonucleotides of the capture elements. In some embodiments, the sequencing step includes obtaining 0.25× or more sequence coverage of the cellular genomic DNA. In some embodiments, the sequencing step includes obtaining about 0.25× to about 100× sequence coverage of the cellular genomic DNA. In some embodiments, the sequencing step comprises obtaining a sequence coverage of the cellular genomic DNA of about 0.25× to about 0.5×, about 0.25× to about 1×, about 0.25× to about 10×, about 0.25× to about 30×, about 0.25× to about 100×, about 0.5× to about 1×, about 0.5× to about 1×, about 0.5× to about 30×, about 0.5× to about 100×, about 1× to about 10×, about 1× to about 30×, about 1× to about 100×, about 10× to about 30×, about 10× to about 100×, or about 30× to about 100×. In some embodiments, the sequencing step comprises obtaining a sequence coverage of the cellular genomic DNA of about 0.25×, about 0.5×, about 1×, about 10×, about 30×, or about 100×. In some embodiments, the sequencing step comprises obtaining at least about 0.25×, about 0.5×, about 1×, about 10×, or about 30× sequence coverage of the cellular genomic DNA.In some embodiments, the sequencing step comprises obtaining at most about 0.5×, about 1×, about 10×, about 30×, or about 100× sequence coverage of the cellular genomic DNA.

[0077] In some embodiments, the genome copy number variation is determined to a resolution of 3 megabases or greater. In some embodiments, the genome copy number variation is determined to a resolution of about 1 megabase to about 6 megabases. In some embodiments, the genome copy number variation is determined to a resolution of about 1 megabase to about 2 megabases, about 1 megabase to about 3 megabases, about 1 megabase to about 4 megabases, about 1 megabase to about 5 megabases, about 1 megabase to about 6 megabases, about 2 megabases to about 3 megabases, about 2 megabases to about 4 megabases, about 2 megabases to about 5 megabases, about 2 megabases to about 6 megabases, about 3 megabases to about 4 megabases, about 3 megabases to about 5 megabases, about 3 megabases to about 6 megabases, about 4 megabases to about 5 megabases, about 4 megabases to about 6 megabases, or about 5 megabases to about 6 megabases. In some embodiments, the genome copy number variation is determined to a resolution of about 1 megabase, about 2 megabases, about 3 megabases, about 4 megabases, about 5 megabases, or about 6 megabases. In some embodiments, the genome copy number variation is determined to a resolution of at least about 1 megabase, about 2 megabases, about 3 megabases, about 4 megabases, or about 5 megabases. In some embodiments, the genome copy number variation is determined to a resolution of at most about 2 megabases, about 3 megabases, about 4 megabases, about 5 megabases, or about 6 megabases.

[0078] Barcodes, genome fragments and transcriptome sequencing Oligonucleotide labels, barcodes, genome fragments, messenger RNA and similar polynucleotide targets can be sequenced by the methods and systems of the present invention. In some embodiments, capture elements for this purpose comprise oligonucleotides attached to a surface in the channel, where such oligonucleotides comprise a sequence segment that is complementary to a sequence segment of the nucleic acid to be captured, which may be a polyA segment of an mRNA, or a "handle" sequence region of any sequence adjacent to the barcode or oligonucleotide label. The channel is provided with such capture elements when a sequencing operation is to be performed. Such capture oligonucleotides can be attached to the first surface by many chemistries known in the art (e.g., Integrated DNA Technologies' brochure entitled "Strategies for attaching oligonucleotides to solid supports" (2014)). The sequencing step may be performed on the surface of the channel ("in situ" sequencing), or the template may be optionally amplified, released and eluted from the channel, and sequenced on an external sequencing instrument ("external" sequencing). In the latter approach, the capture element may contain spatial barcodes that provide channel position information and allow externally determined sequences to be associated with individual chambers. In some embodiments, one or more spatial barcodes are present at a sufficiently high density that each chamber covers an area of ​​the first surface that is usually uniquely associated with a single spatial barcode. In some embodiments, preparation of the polynucleotide for sequencing operation occurs after the target template (e.g., oligonucleotide label, mRNA, genome fragment) is released and captured by a complementary sequence in the capture element. The release step depends on the nature of the target template. For example, an oligonucleotide label attached to an antibody by a disulfide linkage can be released by a reducing agent (which may be the same as the lysis reagent).mRNA can be released by treating cells with conventional lysis agents. Releasing genomic fragments may require lysis and pre-amplification steps. Lysis conditions may vary widely and may be based on the action of heat, detergents, proteases, alkalinity, or a combination of such factors. The following references provide guidance for the selection of lysis reagents or lysis buffers for single cell lysis conditions for mRNA and / or genomic DNA: Thronhill et al., Prenatal Diagnosis, 21:490-497 (2001); Kim et al., Fertility and Sterility, 92:814-818 (2009); Spencer et al., ISME Journal, 10:427-436 (2016); Tamminen et al., Frontiers Microbiol. Methods, 6: Paper 195 (2015), etc. Exemplary lysis conditions include: 1) cells in HO at 96° C. for 15 min, followed by 10° C. for 15 min; 2) 200 mM KOH, 50 mM dithiothreitol, heated to 65° C. for 10 min; 3) 4 μL of protease-based lysis buffer, 1 μL of 17 μM SDS combined with 3 μL of 125 μg / mL proteinase K, followed by incubation at 37° C. for 60 min. for 10 min, then at 95 °C for 15 min (to inactivate proteinase K); 4) 10 µL detergent-based lysis buffer: 2 µL HO, 2 µL 10 mM EDTA, 2 µL 250 mM dithiothreitol, 2 µL 0.5% N-lauryl sarcosine salt solution; 5) 200 mM Tris (pH 7.5), 20 mM EDTA, 2% sarcoyl, 6% Ficoll.

[0079] 7A-7B illustrate exemplary capture and cDNA synthesis methods for performing a sequencing operation. FIG. 7A illustrates one process for capturing a target template and preparing cDNA for external sequencing. Those skilled in the art will recognize that the details of the following examples of target template capture and cDNA synthesis may vary widely depending on the sequencing system used. In some embodiments, the preparation of cDNA includes a tagmentation step. Guidance for specific embodiments can be found in Picelli et al., Genome Research, 24:2033-2040 (2014); Bose et al., Genome Biology, 16:120 (2015); Hashimshony et al., Genome Biology, 17:77 (2016); Yuan et al., Scientific Reports, 6:33883 (2016), and the like. Various oligonucleotides with the following components are attached by their 5' ends to the surface (701): primer binding site P7 (for Illumina sequencers) (702); optional primer binding site R1 (for Illumina paired-end sequencing); barcode oligonucleotide (706) (which may be or include a spatial barcode); an optional unique molecular identifier (708); and capture oligonucleotide (710) (which may be a poly-T segment whenever mRNA is to be captured). The target template (712) is captured by hybridization of a poly-A segment or sequence handle (714) to the capture oligonucleotide (710). After capture, the capture oligonucleotide (710) and poly-A segment (714) are extended by a polymerase (e.g., reverse transcriptase of Moloney Murine Leukemia Virus (MMLV)) that leaves a single-stranded poly-C tail (716). In some embodiments, as shown at (730), a template switching oligonucleotide (718) is hybridized thereto to further extend the poly-C tail (e.g., Zhu et al., Biotechniques, 30:892-897 (2001)).The unattached strand is melted and the attached strand is amplified, for example by PCR, and eluted for external sequencing (732). For in situ sequencing, the surface (753) contains an attached P5 primer (Illumina) and a capture oligonucleotide (750), which may have the same structure as for external sequencing: primer binding site P7 (for Illumina sequencer) (752); optional primer binding site R1 (for Illumina paired-end sequencing); barcode oligonucleotide (756) (which may be or include a spatial barcode); an optional unique molecular identifier (758); and a capture oligonucleotide (760) (which may be a polyT segment whenever mRNA is to be captured). The target template (762) is captured by hybridization of a polyA segment or sequence handle (764) to the capture oligonucleotide (710). After capture, the capture oligonucleotide (710) and polyA segment (764) are extended by a polymerase leaving a single-stranded polyC tail (766). In some embodiments, a template switching oligonucleotide (768) is hybridized thereto, as shown at (780), and the polyC tail is further extended, after which a double-stranded segment (781) is ligated thereto. Segment (781) comprises a complementary strand R2' (765) to primer binding site R2 and a complementary strand P5' to primer binding site P5. The unattached strand of the resulting polynucleotide (785) is melted, and the attached strand of (785) is bridge amplified. In some embodiments, the DNA of the resulting cluster may be sequenced using a sequencing-by-synthesis technique that generates a sequence of fluorescent signals for nucleotide identification. It is understood that there are many alternatives to the above process steps that may be substituted or added by other process steps while still being within the scope of the invention.

[0080] In embodiments in which spatial barcodes are used on a surface, a wide variety of methods may be used to generate spatial barcodes, including but not limited to those described in the following references, which are incorporated by reference: Horgan et al., International Patent Application Publication No. WO2022 / 013094; Fan et al., U.S. Patent Application Publication No. 2019 / 0360121; Chen et al., bioRxiv (https: / / doi.org / 10.1101 / 2021.01.17.427004); Cho et al., bioRxiv (https: / / doi.org / 10.1101 / 2021.01.25.427807); Quan et al., Nature Biotechnology, 29(5):449-453 (2011); Singh-Gasson et al., Nature Biotechnology, 17:974-(1999), and the like.

[0081] In some embodiments, a hydrogel chamber can be synthesized for a single cell on a first surface by the following steps: (a) providing a fluidics device including (i) a channel including a first surface and a biological sample including biological cells disposed on or adjacent to the first surface, (ii) a spatial energy modulating element in optical communication with the first surface, and (iii) a detector for identifying the location of the one or more biological components in the channel based on one or more optical signals from the cells; and (b) synthesizing one or more chambers in the channel encapsulating each of the biological cells by projecting light into the channel by the spatial energy modulating element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, where the location of each of the synthesized chambers is determined by the location of the biological cell encapsulated by the chamber identified by the detector. In some embodiments, the first surface includes a capture element, such as a capture oligonucleotide covalently attached to the first surface. Exemplary capture oligonucleotides are described in Figures 7A-7B. Transcriptome sequencing may be performed with the following additional steps: (i) loading a lysis reagent into the channel that ruptures cell membranes, resulting in the release of messenger RNA (mRNA) that is captured by the capture element; (ii) loading the channel with a transcription reagent that copies the captured mRNA and produces its complementary DNA; (iii) sequencing the complementary DNA to identify the captured mRNA. In some embodiments, a wash step may be included, for example, to remove the lysis reagent prior to loading the transcription reagent, or to remove the transcription reagent prior to loading the amplification reagent, or to remove the amplification reagent prior to loading the sequencing reagent, or otherwise. In some embodiments, the reverse transcription reagent comprises a reverse transcriptase enzyme.In some embodiments, such a reverse transcription reagent is MMLV reverse transcriptase. As mentioned above, cDNA sequencing can be performed "in situ" or external to the system. In situ sequencing can be performed by the following additional steps: (a) amplifying the complementary DNA; (b) sequencing the amplified complementary DNA, for example, using a sequencing-by-synthesis technique. After sequencing, the relative expression levels of mRNA can be determined, thereby providing a transcriptome. In some embodiments, external, i.e., off-channel, sequencing requires that the capture element contains a spatial barcode as described in the above example. The cDNA can be optionally amplified, after which it is eluted and sequenced by an external sequencing instrument.

[0082] Hydrogel Chamber Function. A wide variety of photosynthesizable gels may be used in conjunction with the present invention. In some embodiments, hydrogels are used with the present invention, particularly due to their compatibility with living cells, as well as the versatility to formulate gels with desired properties, including but not limited to porosity (which in large part determines what is contained by the gel (or polymer matrix) walls and what is allowed to pass through the gel (or polymer matrix) walls), degradability, mechanical strength, ease and speed of synthesis, and the like.

[0083] Porosity. In some embodiments, the porosity of the hydrogel is selected to allow the passage of selected reagents while simultaneously preventing the passage of other reagents or objects (such as cells). In some embodiments, the porosity of the hydrogel is selected to prevent the passage of biological cells but allow the passage of reagents including proteins (such as polymerases). In some embodiments, such reagents that are permeable to the polymer matrix walls include lysozyme, proteinase K, random hexamers, polymerases, transposases, ligases, deoxynucleotide triphosphates, buffers, cell culture media, or divalent cations. In some embodiments, at least one polymer matrix includes pores that are sized to allow diffusion of reagents through the at least one polymer matrix, but are too small to allow DNA or RNA for analysis (having a size greater than 100 nucleotides or base pairs, or greater than 300 nucleotides or base pairs) to cross the pores. In some embodiments, crosslinking the polymer chains of the hydrogel construct forms a hydrogel matrix with pores (i.e., a porous hydrogel matrix). In some versions, the size of the pores in the hydrogel structure may be adjusted or tailored and may be formulated to encapsulate sufficiently large genetic material, such as cells or nucleic acids (e.g., greater than about 300 base pairs), but allow smaller materials, such as reagents, or smaller sized nucleic acids (e.g., smaller than about 50 base pairs), such as primers, to pass through the pores, thereby entering or exiting the hydrogel structure. In some embodiments, the hydrogel can have any pore size that allows diffusion of the above-listed reagents through the structure while having a diameter sufficient to retain nucleic acid molecules greater than 500 nucleotides or 500 base pairs in length. In some embodiments, the hydrogel structure can swell when the hydrogel is hydrated.The size of the pores can then be varied depending on the water content in the hydrogel of the hydrogel construct. In some embodiments, the pores have a diameter of about 10 nm to about 100 nm. In some embodiments, the pore size of the hydrogel construct is adjusted by routine experimentation by varying the ratio of the concentration of polymer precursor to the concentration of crosslinker, varying pH, salt concentration, temperature, light intensity, etc. In some embodiments, the average diameter of the pores in the polymer matrix wall prevents the passage of molecules having a molecular weight of 25 kilodaltons (kDa) or more, or molecules having a molecular weight of 50 kDa or more, or molecules having a molecular weight of 75 kDa or more, or molecules having a molecular weight of 100 kDa or more, or molecules having a molecular weight of 150 kDa or more.

[0084] In some embodiments, the retained DNA or RNA has a length that is sequenceable using conventional techniques of sequencing by synthesis. For example, such DNA or RNA comprises at least 50 nucleotides, or in some embodiments at least 100 nucleotides. In some embodiments, the pores may have an average diameter of 5 nm to 100 nm. In some embodiments, the pores may have an average diameter of 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have an average diameter greater than 100 nm. In some embodiments, the pores may have an average diameter less than 5 nm. The reagents may include an enzyme, or a primer having a size of less than 50 base pairs (bp). The primer may include single stranded DNA (ssDNA). In some embodiments, the primer may have a size of 5 bp to 50 bp. In some embodiments, the primers may have a size of 5 bp to 10 bp, 10 bp to 20 bp, 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, the primers may have a size of more than 50 bp. In some embodiments, the primers may have a size of less than 5 bp. In some embodiments, the pores may have a diameter of 5 nm to 100 nm. In some embodiments, the pores may have a diameter of 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores may have an average diameter smaller than 5 nm. The polymer matrix may have a pore size of about 5 nanometers (nm) to about 100 nm.The polymer matrix may be about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 110 nm, about 10 nm to about 20 nm, about 10 nm to about 30 nm, about 10 nm to about 40 nm, about 10 nm to about 50 nm, about 10 nm to about 60 nm, about 10 nm to about 70 nm, about 10nm to about 80nm, about 10nm to about 90nm, about 10nm to about 100nm, about 10nm to about 110nm, about 20nm to about 30nm, about 20nm to about 40nm, about 20nm to about 50nm, about 20nm to about 60nm, about 20nm to about 70nm, approximately 20nm to approximately 80nm, approximately 20nm to approximately 90nm, approximately 20nm to approximately 100nm, approximately 20nm to approximately 110nm, approximately 30nm to approximately 40nm, approximately 30nm to approximately 50nm, approximately 30nm to approximately 60nm, approximately 30nm to approximately 70nm, approximately 30nm to about 80nm, about 30nm to about 90nm, about 30nm to about 100nm, about 30nm to about 110nm, about 40nm to about 50nm, about 40nm to about 60nm, about 40nm to about 70nm, about 40nm to about 80nm, about 40nm to about 90nm, about 40nm to about 100nm, about 40nm to about 110nm, about 50nm to about 60nm, about 50nm to about 70nm, about 50nm to about 80nm, about 50nm to about 90nm, about 50nm to about 100nm, about 50nm to about 110nm , about 60 nm to about 70 nm, about 60 nm to about 80 nm, about 60 nm to about 90 nm, about 60 nm to about 100 nm, about 60 nm to about 110 nm, about 70 nm to about 80 nm, about 70 nm to about 90 nm, about 70 nm to about 100 nm, about 70 nm to about 110 nm, about 80 nm to about 90 nm, about 80 nm to about 100 nm, about 80 nm to about 110 nm, about 90 nm to about 100 nm, about 90 nm to about 110 nm, or about 100 nm to about 110 nm. The polymer matrix may have a pore size of about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, or about 110 nm.The polymer matrix may have a pore size of at least about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm or less. The polymer matrix may have a pore size of at most about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm or more.

[0085] Porosity control. The pore size in the polymer matrix can be controlled using chemical reagents or by applying heat, electric field, light or another suitable stimulus. In other words, the polymer matrix can include a tunable property (e.g., pore size). In some cases, the polymer matrix can include a thermoresponsive or temperature-responsive polymer. Thermoresponsive polymers (e.g., poly(N-isopropylacrylamide) (NIPAAM)) can phase separate from the solution when heated or cooled (e.g., polymers that exhibit a lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix can include a polymer that can collapse at high temperatures, for example, to control the pore size of a hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that can be used to form hydrogel / polymer matrices with tunable properties can include poly(N-vinylcaprolactam), poly(N-ethyloxazoline), poly(methyl vinyl ether), poly(acrylic acid-co-acrylamide), or combinations thereof. A change in temperature can expand or contract the average pore size in the polymer matrix, allowing selected molecules (such as nucleic acid molecules, proteins, or any biomolecules or molecules smaller than the adjusted pore size) to be released from the hydrogel chamber.

[0086] Size and shape of the hydrogel chamber. In some embodiments, the polymer matrix wall of the chamber inhibits passage of a given component, such as mammalian cells, genomic DNA, larger polynucleotides (e.g., mRNAs larger than 200 ribonucleotides, or larger than 300 ribonucleotides, or larger than 500 ribonucleotides). In some embodiments, the polymer matrix wall extends from a first surface to a second surface (parallel to the first surface) to form a chamber within a channel. In some embodiments, the chamber has a polymer matrix wall and an interior. In some embodiments, the interior of the chamber is sized to encapsulate cells. For example, such a chamber may include a cylindrical or polygonal shell that includes an inner space, i.e., an interior, and a polymer matrix wall. In some embodiments, such a chamber has a ring-like cross section. As used herein, the term "ring-like cross section" refers to a cross section that is topologically equivalent to a ring. In some embodiments, the interior space of the chamber, i.e., the interior, has an inner diameter of 1 μm to 500 μm and a volume in the range of 1 picoliter to 200 nanoliters, or 100 picoliters to 100 nanoliters, or 100 picoliters to 10 nanoliters. In some embodiments, the polymer matrix wall has a thickness of at least 1 μm (micrometer). In some embodiments, the height of the chamber with a ring-like cross section has a value in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm. In some embodiments, the polymer matrix wall with a ring-like cross section has an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, the aspect ratio and the polymer matrix wall thickness are selected to maximize the chamber stability against forces, e.g., flow of reagents through the channel, washing, etc. In some embodiments, at least one polymer matrix wall is a hydrogel wall. In some embodiments, at least one polymer matrix wall is degradable.In some embodiments, the degradation of at least one polymer matrix is ​​"on demand". In some embodiments, the chambers in the channel are non-contiguous. In some embodiments, the chambers in the channel may be continuous with adjacent chambers. In some embodiments, the chambers may share polymer matrix walls with each other. In some embodiments, the chambers may be synthesized with slits or other orifices that are large enough to allow the passage of certain components (e.g., beads) but small enough to prevent the passage of other components (e.g., cells).

[0087] Hydrogel Compositions. In some embodiments, the channels of the fluidics devices of the systems of the invention comprise one or more polymer precursors for forming the chambers. In some embodiments, the one or more polymer precursors comprise a hydrogel precursor. Such precursors include polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide, N,N'-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, sulfated alginate, etc. The hydrogel may be selected from a wide variety of compounds including, but not limited to, glyceryl sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethylene glycol diallyl ether, ethylene glycol diacrylate, polymethylene glycol diacrylate, polyethylene glycol diacrylate, trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEG thiol / PEG-acrylate, acrylamide / N,N'-bis(acryloyl)cystamine (BACy), or PEG / PPO. In some embodiments, the precursors and crosslinkers described below may be used to form chambers with degradable polymer matrix (hydrogel) walls.A polymer precursor can be formed by using any of the hydrogel precursors and crosslinkers (columns 1 and 3, respectively) in Table 2A. The resulting polymer matrix can be degraded by the indicated degradation agents in Table 2A (column 4). [Table 2A] [Table 2B]

[0088] Hydrogel Degradation. In some embodiments, the hydrogel chambers of the present invention are degradable or depolymerizable either generally in the channel or "on demand" in the channel. Hydrogel chambers that are generally degradable are degraded by treatment with a degrading agent, or equivalently, a depolymerizing agent that is exposed to all chambers in the channel. Exemplary depolymerizing agents include, but are not limited to, heat, light, and / or chemical depolymerizing agents (which are sometimes referred to as cleaving or decomposing agents). In some embodiments, on-demand degradation can be performed using polymer precursors that allow for photocrosslinking and photodecomposition, for example, using different wavelengths for crosslinking and decomposition. For example, Eosin Y can be used for radical polymerization in a defined area using a wavelength of 500 nm, after which irradiation at 380 nm can be used to cleave the crosslinker. In other embodiments, a photocaged hydrogel cleaving agent can be included in the formation of the polymer matrix wall. For example, an acid-labile crosslinker (such as an ester) can be used to produce a hydrogel, and then UV light can be used to generate localized acidic conditions that in turn degrade the hydrogel. In some embodiments, the at least one polymer matrix is ​​degradable by at least one of the following: (i) contacting the at least one polymer matrix with a cleavage reagent; (ii) heating the at least one polymer matrix to at least 90° C.; or (iii) exposing the at least one polymer matrix to light of a wavelength that cleaves a photocleavable crosslinker that crosslinks the polymers of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleavage reagent degrades the hydrogel. In some embodiments, the cleavage reagent comprises a reducing agent, an oxidizing agent, an enzyme, a pH-based cleavage reagent, or a combination thereof.In some embodiments, the cleavage reagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydrogel may be functionalized by coupling a functional group to the polymer matrix or hydrogel. Some non-limiting examples of functional groups may include capture reagents (e.g., pyridinecarboxaldehyde (PCA)); acrylamide; agarose; biotin; streptavidin; strept-tag II; linkers; functional groups including aldehydes, phosphates, silicates, esters, acids, amides, aldehyde dithiolanes, PEG, thiols, alkenes, alkynes, azides, or combinations thereof. In some cases, the functionalized polymer matrix may be used to capture biomolecules inside the polymer matrix compartments formed adjacent to (e.g., around or on) the biological component. The biomolecules may be produced by the biological component (e.g., cell-derived secretome). The functionalized surface of the polymer matrix inside the compartment can be used to capture reagents or molecules from the outside of the compartment. The functionalized surface may increase the surface area covered by the reagent, molecular sensor, or any molecule of interest (e.g., an antibody).

[0089] Partial Degradation. In some embodiments, an existing polymer matrix wall can be partially degraded, for example, to change porosity. In some embodiments, polymer precursors can include degradable beads that form part of the polymer matrix wall when synthesized and can be embedded in the polymer matrix wall and then subsequently degraded, either on demand or generally, thereby causing an increase in porosity.

[0090] Photosynthesis. In some embodiments, the generation of the polymer matrix in the fluidics device comprises exposing the one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light between 350 nm and 800 nm. In some embodiments, the light generating device generates light between 350 nm and 600 nm. In some embodiments, the light generating device generates light between 350 nm and 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymer matrix in the fluidics device is performed using a spatial light modulator (SLM) (i.e., a spatial energy modulating element capable of spatially generating a desired light intensity pattern). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam directed using a galvanometer. In some embodiments, the SLM is liquid crystal based.

[0091] Systems and Equipment In some embodiments, the system of the invention includes: (a) a channel including a first surface, a plurality of cells disposed on the first surface, and one or more polymer precursors; (b) a spatial energy modulation element in optical communication with the first surface; (c) a detector in optical communication with the first surface and operably associated with the spatial energy modulation element, the detector detecting each of the plurality of cells and determining its location at the first surface; and (d) a plurality of gel chambers, each gel chamber encapsulating a single cell of the plurality of cells, the gel chambers being synthesized by projecting light into the channel by the spatial energy modulation element, such that the projected light causes crosslinking of the one or more polymer precursors to form a polymer matrix wall of the chamber, wherein the location of the synthesized chamber is determined by the location of the cell encapsulated by the chamber identified by the detector. It is understood that the term "detector" as used herein may include, but is not limited to, a microscopic component that collects and optionally magnifies an image of a portion of the channel, as well as an image analysis component that includes software for identifying cells, cell features, chambers, and other objects, and storing such information and associated location information. The computer component uses such information generated by the detector, along with user input, to generate commands for other components (e.g., spatial energy modulation components) to perform various functions, including, but not limited to, synthesizing chambers, disassembling chambers "on demand," photolyzing cells, and the like. Exemplary configurations of such embodiments are illustrated in Figures 5A-5B described above. In some embodiments, the channel of the fluidics device further includes a second surface, where the first surface and the second surface are disposed opposite each other across the channel, and the polymer matrix walls of the chambers extend from the first surface to the second surface to form chambers, each having an interior.In some embodiments, each chamber in the channel encloses a single cell. In some embodiments, both the first and second walls are made of optically transparent materials, such as glass, plastic, etc., and are arranged such that the first and second surfaces are substantially parallel to each other. The vertical distance between the first and second surfaces may be in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm. In some embodiments, the vertical distance between the first and second surfaces may be in the range of 2 times the average size of the cells to be analyzed to 5 times the average size of the cells to be analyzed.

[0092] In other embodiments, the first surface may include a capture element for capturing cells at a predetermined location. For example, the capture element may include (but is not limited to) a capture antibody specific to all or a subpopulation of cells. The capture element may also include non-specific capture materials, such as, but not limited to, polylysine, fibronectin, treated plastic (e.g., Maxysorb™ plastic, ThermoFisher), and the like. In some embodiments, such cell capture moieties (e.g., antibodies) may be restricted to spots or reaction sites arranged in a regular pattern on the first surface; thus, cells captured at such reaction sites may be arranged on the first surface in a regular pattern that may be more efficient than random placement for chamber synthesis and / or optical signal detection. Guidance for providing cell capture antibodies on surfaces can be found in the following references: Zhu et al., Analytica Chemica Acta, 608:186-196 (2008); Sekine et al., J. Immunol. Methods, 313(1-2):96-109 (2006), etc. In some embodiments, such reaction sites or spots have a diameter in the range of 5-500 μm, or in the range of 10-1000 μm. In some embodiments, such spots or reaction sites are arranged in a linear array, or in a hexagonal array. In some embodiments, such an array of such spots or reaction sites has a diameter in the range of 10-2500 sites / mm. 2 , or 10-1000 sites / mm 2 , or 10-500 sites / mm 2 , or 10-100 sites / mm 2 It has a density in the range of

[0093] In some embodiments, the cells may be randomly disposed on the first surface. In some embodiments, the cells are randomly disposed on the first surface with a Poisson distribution. In some embodiments, in such a Poisson distribution, the cells have a nearest neighbor distance equal to or greater than 10 μm, equal to or greater than 20 μm, equal to or greater than 30 μm, equal to or greater than 40 μm, equal to or greater than 50 μm, or equal to or greater than 100 μm. In some embodiments, each subset of such Poisson distributed cells is enclosed by a ring-like shaped chamber having a diameter in the range of 10-500 μm. In some embodiments, the cells are distributed at a density of 10-2500 cells / mm. 2 , or 10-1000 cells / mm 2 , or 10-500 cells / mm 2 , or 10-100 cells / mm 2 The particles are randomly placed on the first surface with a Poisson distribution having a density in the range

[0094] In some embodiments, multiple channels may be arranged together in a flow channel as illustrated in Figures 6A-6B. In some embodiments, the multiple channels may range from 2 to 12, or from 2 to 8, or from 2 to 6, or from 2 to 4. An exemplary flow cell (600) is shown in cross-section and top view. The flow cell (600) has a bottom, or first wall (606) with a first surface (605); a top, or second wall (602) with a second surface (601); and a spacer (604) closely sandwiched between them that forms longitudinal holes for channels 1 to 6, one of which is shown by (608) in cross-section and (612) in top view. In some embodiments, the spacer (604) may have a thickness in the range of 10 μm to 500 μm, or in the range of 50 μm to 250 μm, which determines the internal height of the channel. The upper wall (602) includes an inlet (614) and an outlet (616) for loading and removing reagents and cells into or from channels 1-6, either separately or in concert. In some embodiments, at least one of the walls (602) and (606) is made from an optically transparent material, such as glass, plastic, or the like. The flow cell (600) may be operably associated with a fluidics device that delivers reagents and cells to any of channels 1-6 under programmatic control. Guidance for specific designs, including fluid handling and valving for such fluidics systems may be found in U.S. Pat. Nos. 8,921,073, 8,173,080, 8,900,828, and the like, which are incorporated herein by reference. FIG. 6B illustrates a channel of a flow cell (600) with a random distribution (not to scale) of hydrogel chambers having a ring-like cross-section (such as (620)) on a first surface thereof.

[0095] As noted above, either the first surface, the second surface, or the polymer matrix wall of the chamber may include capture elements and other functional groups for performing various operations, including, but not limited to, capturing cells, capturing components of cells (e.g., mRNA, secreted proteins, intracellular proteins or genomic sequences, etc.), capturing components of analytical reagents (e.g., oligonucleotide labels from antibodies, etc.), etc. Derivatization of surfaces for such purposes is well known to those of skill in the art, as evidenced by the following exemplary references: Zhu et al. (cited above); Sekine et al. (cited above); Integrated DNA Technologies Company Brochure (cited above); Hermanson (cited above), etc.

[0096] As noted above, in some embodiments, the fluidics device of the method includes or is operably associated with a detector that may share the optical path of the spatial energy regulation element, or in embodiments having only a first wall and a first surface (e.g., a well, etc.), may be located adjacent to the second wall or on the opposite side of the first wall from the spatial energy regulation element. The detector is located so as to be able to detect optical signals from cells in the channel, for example, distributed over the first surface in the chamber, or adjacent to cells in the channel. In some embodiments, the first wall and the second wall each include an optically transparent material, for example, so that the spatial energy regulation element can project optical energy into the interior of the channel, and the detector can detect optical signals, such as fluorescent emissions or reflected light from biological components. In some embodiments, the projected energy from the spatial energy regulation element is optical energy from a light beam. In some embodiments, the light beam projected by the spatial energy regulation element may have a complex cross-section that allows for simultaneous synthesis of multiple chambers (in various embodiments). Optically transparent materials include, but are not limited to, materials such as glass, quartz, plastic, etc. In some embodiments, the step of synthesizing the chamber includes positioning the chamber such that the chamber encapsulates one or more biological components based on an optical signal detected by the detector. That is, in some embodiments, the detector is operably associated with the spatial energy adjustment element to selectively project one or more light beams to a location where the detected optical signal indicates the presence of a cell of interest. In such an embodiment, the detector and the spatial energy adjustment element are operably associated such that the spatial energy adjustment element is configured to generate an energy beam having a predetermined beam characteristic. For example, one such characteristic can be a beam cross-section that results in the biological component of interest being encapsulated by the ring-like shaped chamber.In such operational contexts, the optical signals detected by the detector may include, but are not limited to, the morphology of the biological component, e.g., cell morphology; cell motility; the interaction of one cell type with another, e.g., binding of one cell type to another; the presence, absence or amount of a label on a cell, and the like.

[0097] The spatial energy modulating element that uses light energy for polymerization may include a physical or virtual photomask, such as a digital micromirror device (DMD). The following references, which are incorporated herein by reference, provide guidance in selecting and operating a DMD for photopolymerizing gels: Chung et al., U.S. Pat. No. 10,464,307; Hribar et al., U.S. Pat. No. 10,351,819; Das et al., U.S. Pat. No. 9,561,622; Huang et al., Biomicrofluidics, 5:034109 (2011), and the like.

[0098] While the present invention has been described with reference to certain specific exemplary illustrative embodiments, those skilled in the art will recognize that many modifications may be made thereto without departing from the spirit and scope of the invention. The present invention is applicable to a variety of sensor implementations and other subject matter in addition to those discussed above.

[0099] definition Unless otherwise specifically defined herein, the terms and symbols of nucleic acid chemistry, biochemistry, genetics and molecular biology used herein follow the terms and symbols of standard treatises and textbooks in the field, e.g., Kornberg and Baker, DNA Replication, 2nd Edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd Edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd Edition (Wiley-Liss, New York, 1999); Abbas et al., Cellular and Molecular Immuology, 6th Edition (Saunders, 2007).

[0100] "Chimeric antigen receptor T cells" (CAR-T cells) refer to T cells engineered to express an antigen-specific receptor formed by fusing an antigen-binding component with a transmembrane component and a signaling component, and capable of inducing a cytotoxic T cell response whenever the antigen-specific component binds to its target. Exemplary references teaching the manufacture and application of CAR-T cells include the following references, which are incorporated herein by reference: U.S. Patent No. 8,822,647; and U.S. Patent Publication Nos. 2014 / 0134142; 2014 / 0314795; 2012 / 0148552; 2013 / 0288368, etc.

[0101] "Cleaving linkage" or "cleavable nucleotide" means that any of a wide variety of cleavable linkages, or more specifically, cleavable nucleotides, can be used with the embodiments of the present invention. As used herein, the term "cleavable site" refers to a nucleotide linkage or backbone linkage of a single-stranded nucleic acid sequence that can be cut or cut under a given condition, thereby dividing the single-stranded nucleic acid sequence into two parts. In some embodiments, the step of cleaving the cleavable nucleotide or cleavable linkage leaves a free 3'-hydroxyl on the cleaved strand, thereby allowing the cleaved strand to be extended, for example, by a polymerase. The cleaving step can be performed chemically, thermally, enzymatically, or by light-based cleavage. Sometimes, the term "releasing" can be used in reference to cleaving an oligonucleotide label, for example, by a releasing agent or releasing agent, which can be one or more of those listed above. In some embodiments, the cleavable nucleotide may be a nucleotide analog, such as deoxyuridine or 8-oxo-deoxyguanosine, which is recognized by a specific glycosylase (e.g., uracil deoxyglycosylase followed by endonuclease VIII, and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage by glycosylases and / or endonucleases may require a double-stranded DNA substrate. Methods for synthesizing and cleaving nucleic acids containing chemically cleavable, thermally cleavable, and photolabile groups are described, for example, in U.S. Pat. No. 5,700,642, which is incorporated herein by reference.Further cleavable linkages are disclosed in the following references: Pon, R., Methods Mol. Biol. 20: 465-496 (1993); Verma et al., Ann. Rev. Biochem. 67: 99-134 (1998); U.S. Patent Nos. 5,739,386, 5,700,642 and 5,830,655; U.S. Patent Application Publication Nos. 2003 / 0186226 and 2004 / 0106728; Urdea et al., U.S. Patent No. 5,367,066, which are incorporated herein by reference. The synthesis and cleavage conditions of chemically cleavable oligonucleotides are described in U.S. Patent Nos. 5,700,642 and 5,830,655. Phosphorothioate internucleotide linkages can be selectively cleaved under mild oxidizing conditions. Selective cleavage of phosphoramidate bonds may be achieved under mild acid conditions (e.g., 80% acetic acid, etc.). Selective cleavage of ribose may be achieved by treatment with dilute ammonium hydroxide. In another embodiment, the cleavable linking moiety may be an amino linker. The resulting oligonucleotides attached to the linker via phosphoramidite linkages may be cleaved with 80% acetic acid resulting in 3'-phosphorylated oligonucleotides that may be removed by phosphatase (if desired). In some embodiments, the cleavable linking moiety may be a photocleavable linker, such as an ortho-nitrobenzyl photocleavable linker. The synthesis and cleavage conditions of photolabile oligonucleotides on solid support are described, for example, in Venkatesan et al., J.Org.Chem.61:525-529(1996);Kahl et al., J.Org.Chem.64:507-510(1999);Kahl et al., J.Org.Chem.63:4870-4871(1998);Greenberg et al., J.Org.Chem.59:746-753(1994);Holmes et al., J.Org.Chem.62:2370-2380(1997);and U.S. Patent No. 5,739,386.Ortho-nitrobenzyl-based linkers, such as hydroxymethyl linkers, hydroxyethyl linkers, and Fmoc-aminoethyl carboxylic acid linkers, may also be commercially available.In some embodiments, ribonucleotides may be used as cleavable nucleotides, in which case the cleavage step may be carried out using a ribonuclease, such as RNase H. In other embodiments, the cleavage step may be carried out by treatment with a nickase.

[0102] "Hydrogel" means a gel comprising a crosslinked hydrophilic polymer network that has the ability to absorb and retain large amounts of water (e.g., 60-90 percent water, or 70-80 percent water) without dissolution due to the establishment of physical or chemical bonds between the polymer chains, which may be covalent, ionic, or hydrogen bonds. Hydrogels exhibit high permeability to oxygen and nutrients, making them attractive materials for cell encapsulation and cell culture applications. Hydrogels may comprise natural or synthetic polymers and may be reversible (i.e., degradable or depolymerizable) or irreversible. Exemplary synthetic hydrogel polymers include polyethylene glycol (PEG), poly(2-hydroxyethyl methacrylate), and poly(vinyl alcohol). Exemplary natural hydrogel polymers include alginate, hyaluronic acid, and collagen. The following references describe various hydrogels and their biomedical uses: Drury et al., Biomaterials, 24:4337-4351 (2003); Garagorri et al., Acta Biomatter, 4(5):1139-1147 (2008); Caliari et al., Nature Methods, 13(5):405-414 (2016); Bowman et al., U.S. Pat. No. 9,631,092; Koh et al., Langmuir, 18(7):2459-2462 (2002).

[0103] "On demand" means that the operation can be directed to individual, separate selected locations (e.g., spatial locations of polymer precursor solutions; or selected polymer matrix chambers). Such selection can be based on manual observation of optical signals or data collected by the detector, or such selection can be based on a computer algorithm operating on optical signals or data collected by the detector. Manual observation of optical signals or data collected by the detector can include either real-time detection, or detection at a period of time prior to adjusting the unit of energy for polymerizing the polymer precursors or decomposing the chambers. For example, a subset of chambers (all formed with photodegradable polymer matrix walls) can be preselected to release or remove their contents based on position information and optical signal values ​​from an analytical assay performed in the chambers. The preselected chambers can be photolyzed by selectively projecting (e.g., by a spatial energy adjustment element) a light beam of appropriate wavelength characteristics to decompose the polymer matrix walls of the preselected chambers. In another example, a plurality of chambers may be observed in real time (e.g., by fluorescence microscopy) for detection of an analyte of interest, and one or more of the plurality of chambers are selected for degradation in real time upon detection of the analyte of interest.

[0104] A "physical photomask" generally refers to a physical structure having a plurality of openings or holes through which light can be projected. A physical photomask can be used to produce a hydrogel matrix as described herein by causing a polymer precursor solution to polymerize and form a three-dimensional structure corresponding to the pattern in the photomask. A physical photomask can be patterned with a specific layout or geometric pattern. A physical photomask can be attached to the top surface of a flow cell.

[0105] "Polymerase chain reaction" or "PCR" refers to a reaction for in vitro amplification of a specific DNA sequence by simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicas of a target nucleic acid flanked by primer binding sites, such a reaction includes one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. The specific temperature, the duration at each step, and the rate of change between steps depend on many factors well known to those skilled in the art, such as those exemplified by the following references: McPherson et al. (eds.), PCR: A Practical Approach, and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively). For example, in conventional PCR using Taq DNA polymerase, double-stranded target nucleic acid may be denatured at a temperature above 90°C, primers may be annealed at a temperature ranging from 50-75°C, and primers may be extended at a temperature ranging from 72-78°C. The term "PCR" encompasses various derivatives of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, and the like. Reaction volumes range from hundreds of nanoliters, e.g., 200 nL, to hundreds of μL, e.g., 200 μL. "Reverse transcription PCR" or "RT-PCR" refers to PCR preceded by a reverse transcription reaction that converts target RNA into complementary single-stranded DNA that is subsequently amplified (e.g., Tecott et al., U.S. Pat. No. 5,168,038, which is incorporated herein by reference). "Real-time PCR" or "quantitative PCR" refers to PCR in which the amount of reaction product, i.e., the amount of amplicon, is monitored as the reaction proceeds.There are many forms of real-time PCR that differ primarily in the detection chemistry used to monitor the reaction products: for example, Gelfand et al., U.S. Pat. No. 5,210,015 ("taqman"); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons); these patents are incorporated herein by reference. Various detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002), also incorporated herein by reference.

[0106] "Polymer matrix" generally refers to a phase material (e.g., a continuous phase material) that includes at least one polymer. In some embodiments, a polymer matrix refers to at least one polymer and the interstitial space not occupied by said 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 that are intercalated in the interstitial space not occupied by the polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term "polymer matrix" may encompass dried hydrogels, wetted hydrogels, and hydrogels containing glass fibers. A polymer matrix may include polymer precursors, which generally refer to one or more molecules that can induce or initiate a polymer reaction upon activation. The polymer precursors may be activated by electrochemical energy, photochemical energy, photons, magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (which are polymerized to provide a polymer matrix) and crosslinking compounds, which may include photoinitiators, polymer matrices, and other compounds that are necessary or useful to generate polymer matrices that are, inter alia, hydrogels.

[0107] "Transfection" and / or "transformation" and / or "transduction" are used interchangeably herein and refer to the transfer of exogenous genetic material into a target mammalian cell. Such transfer may result in the temporary or transient expression of a transgene or the temporary or transient transcription of RNA, for example, due to exhaustion of genetic material, loss or degradation of genetic material, lack of replication of genetic material, etc. In some embodiments, "transfection" refers to "stable transfection," as the latter term is commonly used (e.g., Kim et al., Anal. Bioanal. Chem., 379:3173-3178 (2010)). Exogenous genetic material may include plasmids, viral vectors, transgenes, transposons, etc. "Stable," as used herein, means that the exogenous genetic material persists through multiple cell divisions or for the life of the cellular host. The exogenous genetic material may be integrated into the genome of the target mammalian cell or may constitute episomal DNA, e.g., plasmids.

Claims

1. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device; and (e) A step of measuring the cytotoxicity of the first cell to the second cell. Methods that include...

2. The method according to claim 1, wherein a plurality of second cells are introduced into the fluid engineering device in (b).

3. The method according to claim 2, wherein the step of measuring cell damage includes counting dead cells, living cells, or combinations thereof from among the plurality of second cells.

4. The method according to claim 3, wherein the counting includes staining the dead cells with a biological dye.

5. The method according to claim 1, wherein the first cell and the second cell are selectively encapsulated in a chamber including a polymer matrix wall.

6. The method according to claim 5, wherein the polymer matrix wall extends from a first surface of the fluid engineering device to a second surface opposite the first surface, thereby forming the interior of the chamber, the interior of the chamber comprising the first cell and the second cell.

7. The method according to claim 1, further comprising the step of detecting the first cell, the second cell, or a protein secreted by the first cell and the second cell.

8. The method according to claim 7, wherein the detection step includes binding the protein to a protein affinity reagent coupled to the protein capture surface.

9. The method according to claim 8, wherein the protein-capturing surface includes beads.

10. The method according to claim 8, wherein the first cell and the second cell are selectively encapsulated in a chamber having a polymer matrix wall, and the protein capture surface is located within the chamber.

11. The method according to claim 7, wherein the detection step further comprises binding a protein detection antibody to the protein and detecting the protein detection antibody.

12. The method according to claim 1, wherein the introduction of the first cells in (a), the introduction of the second cells in (b), and the introduction of the polymer precursor in (c) are carried out simultaneously.

13. The method according to claim 7, wherein the protein is secreted by the first cell and the protein is a cytokine or an immunoactive protein.

14. The method according to claim 13, wherein the protein secreted by the first cell is interferon-γ (IFN-g), interferon-α (IFN-a), interleukin, colony-stimulating factor (CSF), tumor necrosis factor (TNF), or an effector molecule.

15. A step of introducing a plurality of protein capture surfaces, each coupled to a plurality of protein affinity reagents, into the fluid engineering device; A step of co-encapsulating at least one of the plurality of protein-capturing surfaces in the chamber, wherein the plurality of protein affinity reagents are configured to bind to the plurality of proteins secreted by the first cell; and A step of measuring the binding of the plurality of proteins secreted by the first cell to the plurality of protein affinity reagents, thereby detecting the plurality of proteins secreted by the first cell. The method according to claim 5, further comprising:

16. The method according to claim 15, wherein each of the plurality of protein capture surfaces contains a single type of protein affinity reagent from among the plurality of protein affinity reagents.

17. The method according to claim 1, further comprising the step of detecting the first cell, the second cell, or a surface protein expressed by the first cell and the second cell.

18. The method according to claim 17, wherein the detection step includes binding the antibody to the surface protein and detecting the antibody.

19. The method according to claim 1, further comprising the step of measuring the proliferative capacity, proliferation rate, activation state, cell identity, purity, gene expression profile, transcriptome, epigenetic profile, sequence copy number, incorporated virus copy number, plasmid copy number, gene copy number, or any combination thereof of the first cells.

20. The method according to claim 1, wherein the step of measuring cell damage includes incubating the first cells together with a dye configured to generate an optical signal in response to the characteristics of dead cells.

21. The method according to claim 1, further comprising the step of detecting the activation of the first cell.

22. The method according to claim 1, further comprising the step of identifying the first cell.

23. The method according to claim 22, wherein the identifying step includes detecting a surface protein expressed by the first cell, detecting a protein secreted by the first cell, detecting an mRNA transcript expressed by the first cell, or any combination thereof.

24. The method of claim 5, further comprising the step of removing additional cells not enclosed by the chamber from the fluid engineering device.

25. The method according to claim 5, wherein the polymer matrix wall comprises a hydrogel.

26. The method according to claim 5, further comprising the step of disassembling the chamber at least partially.

27. ​​The method according to claim 1, further comprising the step of determining the location of at least the first cell in the fluid engineering device before (d).

28. The method according to claim 27, wherein the step of determining the location is carried out using a detector.

29. The method according to claim 1, wherein the virtual photomask is generated from a spatial light modulator (SLM).

30. The method according to claim 1, wherein the first cell comprises an effector cell and the second cell comprises a target cell.

31. The method according to claim 30, wherein the effector cell is an immune cell.

32. The method according to claim 31, wherein the immune cells are cytotoxic T lymphocytes, regulatory T cells, CD4+ T cells, CD8+ T cells, natural killer cells, antigen-presenting cells, or dendritic cells.

33. The method according to claim 30, wherein the target cells include cancer cells.

34. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device; and (e) A step of detecting the first cell, the second cell, or a protein secreted by the first cell and the second cell. Includes, The detection step includes binding the protein to a protein affinity reagent coupled to the protein capture surface, wherein in (d), the first cell and the second cell are selectively enclosed in a chamber including a polymer matrix wall, and the protein capture surface is located within the chamber. method.

35. The method according to claim 34, wherein the protein-capturing surface includes beads.

36. The method according to claim 34, wherein the protein is secreted by the first cell and the protein is a cytokine or an immunoactive protein.

37. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device; and (e) Step of detecting the protein secreted by the first cell. Includes, The protein secreted by the first cell is interferon-γ (IFN-g), interferon-α (IFN-a), interleukin, colony-stimulating factor (CSF), tumor necrosis factor (TNF), or an effector molecule. method.

38. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; and (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device. Includes, The introduction of the first cells in (a), the introduction of the second cells in (b), and the introduction of the polymer precursor in (c) are carried out simultaneously. method.

39. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of introducing a plurality of protein capture surfaces, each coupled to a plurality of protein affinity reagents, into the fluid engineering device, wherein the plurality of protein affinity reagents are configured to bind to a plurality of proteins secreted by the first cell; (e) a step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively co-encapsulating (i) the first cell, (ii) the second cell, and (iii) at least one of the plurality of protein-trapping surfaces in a chamber comprising a polymer matrix wall in the fluidic device; and (f) A step of measuring the binding of the plurality of proteins secreted by the first cell to the plurality of protein affinity reagents, thereby detecting the plurality of proteins secreted by the first cell. Methods that include...

40. The method according to claim 39, wherein each of the plurality of protein capture surfaces contains a single type of protein affinity reagent from among the plurality of protein affinity reagents.

41. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device; and (e) Steps to detect the activation of the first cell. Methods that include...

42. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in a chamber containing a polymer matrix wall in the fluidic device; and (e) The step of removing any additional cells not enclosed by the chamber from the fluidic device. Methods that include...

43. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; and (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in a chamber containing a polymer matrix wall in the fluidic device, wherein the polymer matrix wall contains a hydrogel. Methods that include...

44. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; and (d) A step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in a chamber containing a polymer matrix wall in the fluidic device; and (e) Step of disassembling the chamber at least partially. Methods that include...

45. (a) A step of introducing a first cell into a fluid engineering device; (b) A step of introducing a second cell into the fluid engineering device; (c) A step of introducing a polymer precursor into the fluid engineering device; (d) the step of determining the location of at least the first cell within the fluid engineering device; and (e) and (d) are followed by a step of selectively applying light to the fluidic device using a virtual photomask to polymerize the polymer precursor, thereby selectively encapsulating the first and second cells in the fluidic device. Methods that include...

46. The method of claim 45, wherein the step of determining the location is performed using a detector.

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