Flow cells and methods for analyzing a biological component

Abstract

Disclosed herein are fluidic devices that facilitate gas diffusion between biological samples and gas-filled compartments or an external atmosphere to stabilize dissolved gas levels within the biological samples. The fluidic devices can include a gas-permeable spacer separating the biological sample-containing channel from the gas-filled compartments or external atmosphere to facilitate rapid gas exchange within the biological sample. Further disclosed herein are methods of using the fluidic devices to incubate and determine characteristics of the samples, including methods for measuring cytokine secretion from individual cells.

Description

Attorney Docket No. 59528-735601FLOW CELLS AND METHODS FOR ANALYZING A BIOLOGICAL COMPONENT CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 719,635 filed November 12, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Flow cells are broadly utilized for a diverse range of cellular assays and preparation steps. Many flow cells can have volumes ranging from nanoliters to individual milliliters. Under certain circumstances, cells can rapidly deplete nutrients, imposing relatively short timeframes on their viability. In particular, cells can deplete oxygen and generate waste gases during incubation and assay timescales, potentially generating hypoxic or anoxic conditions that can lower survival and permanently alter cell phenotypes.

[0003] In some instances, flow cells can utilize media refreshment to counteract Ch-depletion, this process imposes additional constraints on live cell assays. Media replacement is a non-selective and disruptive process that resets the local and extended environment surrounding cells, and therefore limits the study of cell secretions, cell-cell interactions, and cell microenvironments. Cells generate complex microenvironments with distinct chemical and structural signatures. These microenvironments commonly include chemical (e.g., pH, nutrient, and waste) gradients that influence cell behavior and viability, as well as physical structures that impact cell motility, adhesion, and morphology. Many cells also exhibit transient binding with nearby cells, for example as a part of activation and killing processes. Media flow can disrupt or limit microenvironment formation, remove cell secretions, and disrupt intercellular interactions, preventing assays from maintaining cells in physiologically representative states and environments. Accordingly, new methods and flow cell designs are needed to accurately study cell behavior.SUMMARY

[0004] In one embodiment, the present disclosure provides a method for analyzing one or more cells in a flow cell comprising: inputting the one or more cells into a first channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel; incubating the one or more cells in a liquid in the first channel; diffusing a first gasAttorney Docket No. 59528-735601from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel.

[0005] In some aspects, the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the second channel. In further aspects, after the inputting the liquid into the first channel, the liquid is quiescent during the incubating the one or more cells. In additional aspects, the top layer and the bottom layer are optically transparent or translucent. In certain aspects, the top layer and the bottom layer are oxygen impermeable and liquid impermeable.

[0006] In particular aspects, the first channel is coupled to a fluidic manifold. In select aspects, the first channel is sealed. In some aspects, the second channel is coupled to a gas manifold or is open to an atmosphere outside of the fluidic device. In certain aspects, the first channel and the second channel are separated by about 0.5 to 3 millimeters. In one aspect, the first channel comprises a width of about 2.5 to 15 millimeters; the first channel comprises a height of about 0.025 to 0.25 millimeters; the first channel comprises a length of about 50 to 200 millimeters; the second channel comprises a width of about 2.5 to 15 millimeters; the second channel comprises a height of about 0.025 to 0.25 millimeters; the second channel comprises a length of about 50 to 200 millimeters; or a combination thereof. In another aspect, the first channel comprises a width of about 0.1 to 15 millimeters; the first channel comprises a height of about 0.025 to 0.25 millimeters; the first channel comprises a length of about 50 to 200 millimeters; the second channel comprises a width of about 2.5 to 15 millimeters; the second channel comprises a height of about 0.025 to 0.25 millimeters; the second channel comprises a length of about 50 to 200 millimeters; or a combination thereof. In a further aspect, the channel comprises a width of about 0.1 to 2.5 millimeters.

[0007] In one aspect, the method further comprises diffusing the first gas from a third channel of the fluidic device into the first channel through another portion of the spacer layer, wherein the first channel is adjacent to the third channel, wherein the another portion of the spacer layer is located in between the first and third channels, wherein the spacer layer further includes a third cut-out region that at least partially defines the third channel. In another aspect, the third cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the third channel. In an additional aspect, the second and third channels are disposed on opposite sides of the first channel.Attorney Docket No. 59528-735601

[0008] In additional aspects, diffusing the first gas from an area outside of the fluidic device into the first channel through another portion of the spacer layer, wherein another portion of the spacer layer comprises an edge of the fluidic device, wherein the portion of the spacer layer and the another portion of the spacer layer are disposed on opposite sides of the first channel. In certain aspects, the incubating is for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours. In further aspects, the one or more cells are viable in the liquid in the first channel for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours. In an additional aspect, the liquid in the first channel is not replaced during the at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours.

[0009] In a number of aspects, the second channel is fluidically coupled to an atmosphere extrinsic to the fluidic device, and wherein following the at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours, a partial pressure of O2 in the second channel is within at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of a partial pressure of O2 in the atmosphere extrinsic to the fluidic device.

[0010] In particular aspects, the method further comprises diffusing a second gas from the liquid in the first channel of the fluidic device into the second channel through the portion of the spacer layer. In certain aspects, the second gas comprises a waste product of the one or more cells. In particular aspects, the second gas comprises a CO2. In select aspects, the first gas comprises a pressure of about 1 atmosphere in the second channel. In additional aspects, the first gas comprises a pressure of about 1.25 to 10 atmospheres in the second channel. In some aspects, the one or more cells consume the first gas. In select aspects, the first gas comprises O2. In additional aspects, the liquid comprises or approaches a steady-state dissolved O2 level of between about between about 0.1 and 0.5 mg / L, between about 0.1 and 1 mg / L, between about 0.1 and 2 mg / L, between about 0.1 and 4 mg / L, between about 0.1 and 6 mg / L, between about 0.1 and 8 mg / L, between about 0.1 and 10 mg / L, between about 0.1 and 12 mg / L, between about 0.25 and 0.5 mg / L, between about 0.25 and 1 mg / L, between about 0.25 and 2 mg / L, between about 0.25 and 4 mg / L, between about 0.25 and 6 mg / L,Attorney Docket No. 59528-735601between about 0.25 and 8 mg / L, between about 0.25 and 10 mg / L, between about 0.25 and 12 mg / L, between about 0.5 and 1 mg / L, between about 0.5 and 2 mg / L, between about 0.5 and 4 mg / L, between about 0.5 and 6 mg / L, between about 0.5 and 8 mg / L, between about 0.5 and 10 mg / L, between about 0.5 and 12 mg / L, between about 1 and 2 mg / L, between about 1 and 4 mg / L, between about 1 and 6 mg / L, between about 1 and 8 mg / L, between about 1 and 10 mg / L, between about 1 and 12 mg / L, between about 2 and 4 mg / L, between about 2 and 6 mg / L, between about 2 and 8 mg / L, between about 2 and 10 mg / L, between about 2 and 12 mg / L, between about 2 and 15 mg / L, between about 3 and 6 mg / L, between about 3 and 8 mg / L, between about 3 and 10 mg / L, between about 3 and 12 mg / L, between about 4 and 8 mg / L, between about 4 and 10 mg / L, between about 4 and 12 mg / mL, between about 6 and 8 mg / mL, between about 6 and 10 mg / mL, between about 6 and 12 mg / mL, or between about 8 and 12 mg / mL. In certain aspects, the one or more cells deplete O2 from the liquid at a rate of at least about 0.01 mg*L'1*h'1, a rate of at least about 0.02 mg*L'1*h'1, a rate of at least about 0.05 mg*L'1*h'1, a rate of at least about 0.1 mg*L'1*h'1, a rate of at least about 0.2 mg*L'1*h' a rate of at least about 0.5 mg*L'1*h'1, a rate of at least about 1 mg*L'1*h'1, a rate of at least about 1.5 mg*L'1*h'1, a rate of at least about 2 mg*L'1*h'1, a rate of at least about 2.5 mg*L' ^h’1, a rate of at least about 3 mg*L'1*h'1, a rate of at least about 4 mg*L'1*h'1, or a rate of at least about 5 mg*L'1*h'1. In select aspects, the spacer layer is impermeable to liquid water and permeable to gaseous O2. In one aspect, the spacer layer comprises an O2 permeability of about 10 to 500 barrers.

[0011] In certain aspects, the spacer layer comprises a siloxane moiety, a silicone moiety, an acrylate moiety, an acrylamide moiety, a polyvinyl moiety, a polyurethane, an epoxy moiety, a polyphenol, a polyester, a polyamide, a polyimide, polytetrafluoroethylene, polyethylene, polypropylene, a polycarbamate, a polycarbonate, a polyacrylic acid, a sulfonated polyester resin, or a combination thereof. In some aspects, the portion of the spacer layer located between the first and second channels comprises a width of about 0.25 to 2.5 millimeters.

[0012] In particular aspects, the method further comprises synthesizing one or more chambers that at least partially enclose the one or more cells in the first channel. In some aspects, the synthesizing comprises projecting light into the first channel with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers. In select aspects, the one or more chambers comprise a hydrogel. In further aspects, the one or more chambers extend from the bottom layer to the top layer. In certain aspects, the one or more chambers are synthesized at a position determined to contain at least a subset of the one or more cells by aAttorney Docket No. 59528-735601detector. In select aspects, the one or more cells are at least partially enclosed in the one or more chambers during the incubating. In additional aspects, the method further comprises degrading a chamber of the one or more chambers.

[0013] In particular aspects, the method further comprises loading the first channel with an assay reagent. In some aspects, the method further comprises determining a characteristic of the one or more cells.

[0014] In one aspect, the characteristic of the one or more cells comprises a proliferation rate. In an additional aspect, said determining said proliferation rate comprises counting cells at least partially enclosed by the one or more chambers.

[0015] In a further aspect, the characteristic of the one or more cells comprises a soluble factor secreted by the one or more cells. In one such aspect, said detecting said soluble factor comprises: (i) disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the one or more cells, and (ii) detecting the soluble factor bound to the capture surface. In an additional aspect, said detecting said soluble factor bound to said capture surface comprises: (i) contacting said soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. In another aspect, the method further comprises removing the soluble factor from the first channel and detecting a second soluble factor secreted by the one or more cells subsequent to said removing the soluble factor from the first channel.

[0016] In an additional aspect, the one or more cells comprise effector cells, and wherein the characteristic of the one or more cells comprises cytotoxicity. In one such aspect, the one or more chambers at least partially enclose one or more target cells with the effector cells, and said measuring said cytotoxicity comprises counting dead and / or viable cells from among the one or more target cells. In another aspect, the effector cells and the one or more target cells are viable at O2 levels in the liquid of the first channel.

[0017] In a certain aspect, the characteristic of the one or more cells comprises activation. In some such aspects, the activation is caused by: (i) contact between a second cell and a cell of the one or more cells; (ii) a soluble factor secreted by the second cell and the cell of the one or more cells; or (iii) a combination thereof. In further aspects, (i) the second cell is at least partially enclosed with the cell of the one or more cells by the chamber of the one or more chambers; (ii) the second cell and the cell of the one or more cells are each at least partially enclosed by different chambers of the one or more chambers; or (iii) the cell of the one or more cells is at least partially enclosed by a chamber of the one or more chambers and the second cell is not enclosed by a chamber of the one or more chambers. In a particular aspect,Attorney Docket No. 59528-735601the second cell and the cell of the one or more cells are each spaced apart within about 5, 10, 20, 40, 60, 80, or 100 pm within the first channel. In additional aspects, said determining said activation comprises detecting a surface marker of the one or more cells. In such aspects, said detecting said surface marker comprises contacting the one or more cells with a binding agent configured to bind to the surface marker and detecting the binding agent. In select aspects, said determining said activation comprises measuring proliferation of the one or more cells.

[0018] In certain aspects, a cell of the one or more cells are viable for at least 1, at least 2, at least 4, at least 6, at least 8, at least 12, at least 16, at least 24, at least 36, at least 48, at least 60, at least 90, at least 120, at least 150, or at least 180 hours under hypoxic conditions. In further aspects, a cell of the one or more cells is viable for less than 180, less than 150, less than 120, less than 90, less than 60, less than 48, less than 36, less than 24, less than 16, less than 12, less than 8, less than 6, less than 4, less than 2, or less than 1 hour under hypoxic conditions. In some aspects, the one or more cells comprise a cancer cell, an immune cell, a fibroblast, a neuron, or a combination thereof. In additional aspects, a cell of the one or more cells proliferates during the incubation. In some aspects, a cell of the one or more cells does not proliferate during the incubation.

[0019] In further embodiments, the present disclosure provides a method for analyzing one or more cells, the method comprising: inputting the one or more cells into a fluidic device, trapping the one or more cells, wherein the trapped one or more cells are disposed within the fluidic device; inputting a bispecific binding agent into the fluidic device, the bispecific binding agent comprising: (i) a first binding site configured to bind to a surface marker on a surface of the one or more cells, and (ii) a second binding site configured to bind to a soluble factor secreted by the one or more cells; and detecting binding of the soluble factor to the bispecific binding agent, wherein the bispecific binding agent is bound to the surface of the cell during the detecting.

[0020] In one aspect, the trapped one or more cells are each at least partially enclosed in one or more chambers, wherein each chamber comprises polymer matrix walls and a nanofluidic space. In another aspect, the trapping the one or more cells comprises inputting a polymer precursor into the fluidic device, and synthesizing one or more chambers that at least partially enclose the one or more cells in the fluidic device, wherein the synthesizing comprises projecting light into the fluidic device with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers. In one aspect, the detecting comprises measuring an aggregate signal from a chamber of the one or more chambers.Attorney Docket No. 59528-735601

[0021] In certain aspects, the trapping the one or more cells comprises enclosing the one or more cells within one or more droplets. In one such aspect, each droplet contains a first liquid and the one or more cells, wherein the one or more droplets are surrounded by a second liquid, wherein the first liquid and the second liquid are immiscible. In a further aspect, the first liquid comprises water and the second liquid comprises an oil. In a particular aspect, the second liquid comprises a fluorocarbon.

[0022] In some aspects, the detecting comprises imaging a cell of the one or more cells, wherein signal intensity of the imaged cell is proportional to an amount of the soluble factor bound to the bispecific binding agent of the imaged cell. In additional aspects, the bispecific binding agent is coupled to the one or more cells prior to the inputting the bispecific binding agent into the fluidic device. In select aspects, the surface marker is on a surface of a first cell of the one or more cells, and wherein the soluble factor is secreted by a second cell of the one or more cells. In one aspect, the method further comprises detecting activation of the first cell of the one or more cells. In a certain aspect, the first cell and the second cell are immune cells. In an additional aspect, the first cell comprises an effector cell and the second cell comprises a CD4+ T cell. In another aspect, the effector cell comprises a CD8+ T cell. In some aspects, the detecting the activation comprises measuring: i) cytotoxicity of the first cell towards a target cell; ii) a surface marker expressed by the first cell; an mRNA expressed by the first cell; iv) a soluble factor secreted by the first cell; or a combination thereof. In particular aspects, i) the cytotoxicity towards the target cell comprises an incidence, rate, efficiency, or number of killings of the target cell, and the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof; ii) the surface marker expressed by the first cell comprises CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, or a combination thereof; iii) the mRNA comprises mRNA encoding CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; iv) the soluble factor secreted by the first cell comprises an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; or v) a combination thereof. In certain aspects, the first and second cells are co-enclosed within a chamber. In select aspects, the first cell is an effector cell and the second cell is a target cell, wherein the soluble factor from the effector cell is detected. In some aspects, the first cell is an effector cell and the second cell is a target cell, and the soluble factor from the target cell is detected. In additional aspects, a third cell is co-enclosed within the chamber, the first cell is an immune cell, the second cell is an effector cell, and the third cell is a target cell, wherein the soluble factor from the immuneAttorney Docket No. 59528-735601cell is detected, the method further comprising determining whether the immune cell activates the effector cell. In some aspects, the first cell comprises a target cell of an effector cell and the second cell comprises a CD4+ T cell. In a certain aspect, the method further comprises measuring activation of the effector cell, killing of the target cell by the effector cell, or a combination thereof. In an aspect, the effector cell comprises a CD8+ T cell. In another aspect, the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof. In a particular aspect, the surface marker is on a surface of a first cell of the one or more cells and the soluble factor is secreted by the first cell of the one or more cells. In additional aspects, the method further comprises detecting activation of an effector cell in the fluidic device. In one such aspect, the detecting the activation comprises measuring: i) cytotoxicity of the effector cell towards a target cell; ii) a surface marker expressed by the effector cell; iii) an mRNA expressed by the effector cell; iv) a soluble factor secreted by the effector cell; or v) a combination thereof. In a select aspect, i) the cytotoxicity towards the target cell comprises an incidence, rate, efficiency, or number of killings of the target cell, and the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof; ii) the surface marker expressed by the effector cell comprises CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, or a combination thereof; iii) the mRNA comprises mRNA encoding CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; iv) the soluble factor secreted by the effector cell comprises an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; or v) a combination thereof. In particular aspects, the signal intensity comprises brightfield imaging intensity, fluorescence imaging intensity, or a combination thereof. In one aspect, the imaging comprises resolving multiple signals along the surface of the cell.

[0023] In further aspects, the method further comprises coupling the surface marker to the cell. In one such aspect, the coupling the surface marker to the cell comprises halotagging, sortagging, Q-tagging, liposome delivery, poly cation delivery, click chemistry, or a combination thereof. In select aspects, the coupling the surface marker to the cell comprises contacting the cell with the surface marker coupled to: i) a lipid configured to insert into a membrane of the cell; ii) an antibody configured to bind to an additional surface marker on the cell; iii) a reactive molecule configured to covalently couple to the additional surface marker on the cell. In some aspects, the lipid comprises a sterol, cholesterol, a prenyl group, a diacylglycerol group, myrisate, palmitate, stearate, or oleate. In additional aspects, theAttorney Docket No. 59528-735601antibody targets an immune cell marker selected from CD4, CD8, CCR7, CD45, CD45Ram C127, CD25, CD39, CD14, CD19, CD3, CD16, CD56, CD28, C62, CD11, CD69, or CD71. In particular aspects, the reactive molecule comprises a N-hydroxysuccinimide (NHS), a maleimide, a disulfide, an azide, or an alkenyl group. In additional aspects, the cell surface marker comprises avidin, an avidin fragment, streptavidin, a streptavidin fragment, neutravidin, a neutravidin fragment, biotin, a strep tag, 2,4-dinitrophenol, digoxigenin, or fluorescein.

[0024] In some aspects, the detecting the binding of the soluble factor to the bispecific binding agent comprises inputting an additional binding agent into the fluidic device, and detecting binding of the additional binding agent to the soluble factor bound to the bispecific binding agent. In one such aspect, the additional binding agent comprises an antibody. In further aspects, the detecting comprises detecting a detectable label coupled to the additional binding agent. In particular aspects, the detectable label comprises a fluorophore, a phosphorescent species, a dye, an oligonucleotide barcode, a chemiluminescent species, or a combination thereof. In certain aspects, the detecting comprises ligating the oligonucleotide barcode to a nucleic acid barcode coupled to a location on a surface of the fluidic device that comprises a sequence specific to the location and sequencing the oligonucleotide barcode and the sequence specific to the location. In additional aspects, the detecting comprises capturing the oligonucleotide barcode on a nucleic acid barcode coupled to a location on a surface of the fluidic device that comprises a sequence specific to the location, extending the nucleic acid barcode over the oligonucleotide barcode, and sequencing the extended nucleic acid barcode. In some aspects, the sequence specific to the location is blocked with a blocking agent prior to the inputting the additional binding agent into the fluidic device and then unblocked subsequent to the inputting the additional binding agent into the fluidic device. In additional aspects, the detecting comprises fixing the detectable label within the fluidic device, coupling a fluorescently labeled nucleic acid molecule to the oligonucleotide barcode, and detecting the fluorescently labeled nucleic acid molecule. In further aspects, a portion of the additional binding agent is not bound to a complex, the complex comprising the soluble factor bound to the bispecific binding agent, and the portion of the additional binding agent that is not bound to the complex is not removed from the fluidic device during the detecting of the detectable label coupled to the additional binding agent. In a particular aspect, the detectable label of the additional binding agent comprises a fluorophore, wherein a channel of the fluidic device comprises a height of about 0.025 to 0.25 millimeters, wherein a fluorescence signal intensity of the portion of the additional binding agent that is not bound toAttorney Docket No. 59528-735601a complex is at least ten times, at least 100 times, or at least 1000 times less than a fluorescence signal intensity of the additional binding agent bound to the complex, thereby avoiding a washing step of unbound additional binding agent before the detecting of the detectable label coupled to the additional binding agent.

[0025] In some aspects, the detecting the binding of the soluble factor to the bispecific binding agent comprises quantitating an amount of the soluble factor secreted by the cell of the one or more cells. In further aspects, the detecting the binding of the soluble factor to the bispecific binding agent is performed at multiple time points. In additional aspects, the method further comprises inputting an additional bispecific binding agent into the fluidic device, the additional bispecific binding agent comprising: (i) a first binding site configured to bind to the surface marker on the surface of the cell of the one or more cells, (ii) and a second binding site configured to bind to an additional soluble factor secreted by cell; and detecting binding of the additional soluble factor to the additional bispecific binding agent, wherein the additional bispecific binding agent is bound to the surface of the cell during the detecting the binding of the additional soluble factor to the additional bispecific binding agent. In select aspects, the method further comprises inputting an additional bispecific binding agent into the fluidic device, the additional bispecific comprising: (i) a first binding site configured to bind to an additional surface marker on the surface of the one or more cells, and (ii) a second binding site configured to bind to an additional soluble factor secreted by the cell; and detecting binding of the additional soluble factor to the additional bispecific binding agent, wherein the additional bispecific binding agent is bound to the surface of the cell during the detecting the binding of the additional soluble factor to the additional bispecific binding agent. In additional aspects, the bispecific binding agent and the additional bispecific binding agent are each bound to the surface of the cell of the one or more cells via the respective first binding sites. In another aspect, the detecting the binding of the soluble factor to the bispecific binding agent is simultaneous to the detecting the binding of the additional soluble factor to the additional bispecific binding agent. In a further aspect, the soluble factor comprises a cytokine, an immune active molecule, an interleukin, an interferon, a colony stimulating factor, a tumor necrosis factor, or a granzyme. In a particular aspect, the soluble factor comprises interferon-y (IFN-y), interferon-a (IFN-a), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL-10), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin-21 (IL-21), or interleukin-23 (IL-23), granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), TNF-a, TNF-P, granzymeAttorney Docket No. 59528-735601A, granzyme B, granzyme C, granzyme H, granzyme K, granzyme M, perforin, granulysin, or a combination thereof. In an additional aspect, the surface marker on the surface the cell of the one or more cells comprises an immune cell marker. In a certain aspect, the surface marker comprises CD4, CD8, CCR7, CD45, CD45Ram C127, CD25, CD39, CD14, CD19, CD3, CD16, CD56, CD28, C62, CD11, CD69, CD71, or a combination thereof. In another aspect, the first binding site of the additional bispecific binding agent comprises a first antibody or a fragment thereof. In a further aspect, the first binding site of the additional bispecific binding agent comprises biotin, avidin, an avidin fragment, streptavidin, a streptavidin fragment, neutravidin, a neutravidin fragment, biotin, a strep tag, 2,4-dinitrophenol, digoxigenin, or fluorescein. In an additional aspect, the second binding site of the additional bispecific binding agent comprises a second antibody or a fragment thereof. In a particular aspect, the cell is disposed within a chamber or channel of the fluidic device, wherein the channel or chamber comprises a height of about 0.025 to 0.25 millimeters.

[0026] In certain aspects, the fluidic device comprises a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form a first channel, and wherein the one or more cells and the polymer precursor are input into the first channel of the fluidic device. In additional aspects, the method further comprises incubating the one or more cells in a liquid in the first channel; diffusing a first gas from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel, wherein the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the second channel.

[0027] In one aspect, the first binding site binds to the surface marker on the surface of the cell of the one or more cells, and the second binding site binds to a soluble factor secreted by the cell. In an additional aspect, the polymer matrix walls of the one or more chambers comprise pores configured to permit passage of the bispecific binding agent. In another aspect, the polymer matrix walls of the one or more chambers comprise pores configured to permit passage of the additional binding agent.

[0028] A further embodiment of the present disclosure provides a method for analyzing one or more cells in a fluidic device comprising: inputting the one or more cells into a channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer;Attorney Docket No. 59528-735601wherein the spacer layer includes a cut-out region, wherein the cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the channel; incubating the one or more cells in a liquid in the channel; and diffusing a gas into the liquid in the channel. In some aspects, the gas diffuses through a portion of the spacer layer, and into the liquid in the channel. In some aspects, the gas diffuses: (i) through an opening in the top layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel, (ii) through an opening in the top layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel, or (iii) a combination thereof.

[0029] In some aspects, the opening in the top layer of the fluidic device comprises a hole, a slit, or a combination thereof. In further aspects, the opening in the bottom layer of the fluidic device comprises a hole, a slit, or a combination thereof.

[0030] An additional embodiment of the present disclosure provides a method for analyzing one or more cells in a two-channel fluidic device comprising: inputting the one or more cells into a first channel of the two-channel fluidic device, the two-channel fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein: the spacer layer includes a first cut-out region and a second cut-out region, the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel of the two-channel fluidic device, and the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form a second channel of the two-channel fluidic device; incubating the one or more cells in a liquid in the first channel; and diffusing a gas from into the liquid in the first channel, wherein the gas diffuses from outside of the two-channel fluidic device, through the spacer layer, and into the liquid in the first channel.

[0031] In certain aspects, the one or more cells are present at a density of about 104to about 107cells / mL. In additional aspects, the one or more cells are present at a density of about 105to about 5xl06cells / mL.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The novel features of the invention are set forth with particularity in the appended claims. Abetter understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0033] FIG. 1 is a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.Attorney Docket No. 59528-735601

[0034] FIG.2A is an illustration of a portion of a system as provided herein including an energy source, according to some embodiments.

[0035] FIG.2B is an illustration of a polymer matrix being formed around a biological component in a portion of a system as provided herein, according to some embodiments.

[0036] FIG.2C is an illustration of a method of forming a polymer matrix around a biological component in a system as provided herein, according to some embodiments.

[0037] FIG.3A is an illustration of a top view of the bottom layer of a flow cell without any spatial barcoded oligonucleotides.

[0038] FIG.3B is an illustration of a top view of the spacer layer with a cut-out region suitable for use as part of the flow cell.

[0039] FIG.3C is an illustration of a top view of the top layer of the flow cell where the top layer has an inlet and outlet opening.

[0040] FIG.3D is an illustration of a cross-sectional side view of a top layer and a bottom layer sandwiching a spacer layer to form multiple channels of a flow cell.

[0041] FIG.3E is an illustration of a cross-sectional side view of a flow cell that depicts gas flowing into a flow cell channel.

[0042] FIG. 4A is an illustration of a top view of a flow cell spacer that contains a channel capable of holding a gas, a cut-out region that is capable of forming a liquid channel, and multiple locations for openings to connect the gas channel to an external atmosphere.

[0043] FIG. 4B is an illustration of a top view of a flow cell spacer that contains a channel capable of holding a gas, a cut-out region that is capable of forming a liquid channel, and locations for openings to connect ends of the gas channel to an external atmosphere.

[0044] FIG. 4C is an illustration of a top view of a flow cell spacer that contains multiple channels capable of holding a gas disposed adjacent to multiple cut-out regions that are capable of forming liquid channels.

[0045] FIG. 4D is an illustration of a cross-sectional side view of a flow cell in which gas from a gas-filled channel diffuses into an adjacent liquid-filled channel.

[0046] FIG. 5A is an illustration of a system with a flow cell and an imaging apparatus that are capable of implementing methods of the present disclosure.

[0047] FIG. 5B is an illustration of a flow cell, including a blown-up view of a portion of a channel containing cells disposed in hydrogel chambers.

[0048] FIG. 6 is a schematic of a computer system 1501 that may be programmed or otherwise configured to perform methods described herein.Attorney Docket No. 59528-735601

[0049] FIGS. 7A-C are a series of brightfield images of a flow cell lane that included one atmosphere-exposed edge and contained media and cells. FIG. 7A is an image taken near a left outer edge of the flow cell lane. FIG. 7B is an image of a central portion of the flow cell lane. FIG. 7C is an image of a right outer edge of the flow cell lane.

[0050] FIGS. 8A-C are a series of brightfield images of a flow cell lane that included zero atmosphere-exposed edges and contained media and cells. FIG. 8A is an image taken near a left outer edge of the flow cell lane. FIG. 8B is an image of a central portion of the flow cell lane. FIG. 8C is an image of a right outer edge of the flow cell lane.

[0051] FIGS. 9A-C are a series of brightfield images of a flow cell lane that included zero atmosphere-exposed edges and contained media and cells. FIG. 9A is an image taken near a left outer edge of the flow cell lane. FIG. 9B is an image of a central portion of the flow cell lane. FIG. 9C is an image of a right outer edge of the flow cell lane.

[0052] FIGS. 10A-C are a series of brightfield images of a flow cell lane that included one atmosphere-exposed edge and contained media and cells. FIG. 10A is an image taken near a left outer edge of the flow cell lane. FIG. 10B is an image of a central portion of the flow cell lane. FIG. 10C is an image of a right outer edge of the flow cell lane.

[0053] FIGS. 11A-C are a series of brightfield images of a flow cell lane that included two atmosphere-exposed edges and contained media and cells. FIG. 11A is an image taken near a left outer edge of the flow cell lane. FIG. 11B is an image of a central portion of the flow cell lane. FIG. 11C is an image of a right outer edge of the flow cell lane.

[0054] FIGS. 12A-C are a series of brightfield images of a flow cell lane that included two atmosphere-exposed edges and contained media and cells. FIG. 12A is an image taken near a left outer edge of the flow cell lane. FIG. 12B is an image of a central portion of the flow cell lane. FIG. 12C is an image of a right outer edge of the flow cell lane.

[0055] FIG. 13A is a series of brightfield images of a flow cell lane spanning from the left edge to the right edge of the lane.

[0056] FIG. 13B is a series of fluorescence images spanning from the left edge to the right edge of a flow cell lane of cells stained with a hypoxia-sensitive dye.

[0057] FIGS. 14A-F are representative brightfield images of cells in separate lanes of an eight-lane flow cell with six cell and media-filled lanes and two gas-filled lanes (the fifth and seventh lanes from the leftmost edge of the flow cell). FIG. 14A is an image of cells in the leftmost lane of the flow cell. FIG. 14B is an image of cells in the second lane from the left edge of the flow cell. FIG. 14C is an image of cells in the third lane from the left edge of the flow cell. FIG. 14D is an image of cells in the fourth lane from the left edge of the flow cell.Attorney Docket No. 59528-735601FIG. 14E is an image of cells in the sixth lane from the left edge of the flow cell. FIG. 14F is an image of cells in the rightmost lane of the flow cell.

[0058] FIG. 15 is a fluorescence image of cells stained with a hypoxia-sensitive dye and incubated in the second leftmost lane of a flow cell.

[0059] FIGS. 16A-B are series of images of the cells stained with a hypoxia-sensitive dye and incubated in a flow cell lane for 0, 2, 4, 6, and 8 hours. FIG. 16A is a series of brightfield images of the cells. FIG. 16B is a series of fluorescence images of the cells.

[0060] FIGS. 17A-B are series of images of the cells stained with a hypoxia-sensitive dye and incubated in a flow cell lane for 0, 2, 4, 6, and 8 hours. FIG. 17A is a series of brightfield images of the cells. FIG. 17B is a series of fluorescence images of the cells.

[0061] FIGS. 18A-B are series of images of the cells stained with a hypoxia-sensitive dye and incubated in a flow cell lane for 0, 2, 4, 6, and 8 hours. FIG. 18A is a series of brightfield images of the cells. FIG. 16B is a series of fluorescence images of the cells.

[0062] FIGS. 19A-B are series of images of the cells stained with a hypoxia-sensitive dye and incubated in a flow cell lane for 0, 2, 4, 6, and 8 hours. FIG. 19A is a series of brightfield images of the cells. FIG. 19B is a series of fluorescence images of the cells.

[0063] FIG. 20 is a series of images of cells in alternating lanes of a flow cell in which every other lane is filled with gas.

[0064] FIG. 21A is a brightfield image of cells following 24 hours of incubation in wells.

[0065] FIG. 21B is a fluorescence image of cells stained with a hypoxia-sensitive dye and incubated for 24 hours in a well.

[0066] FIG. 22A is a brightfield image of cells following 24 hours of incubation in wells.

[0067] FIG. 22B is a fluorescence image of cells stained with a hypoxia-sensitive dye and incubated for 24 hours in a well.

[0068] FIG. 23A is a brightfield image of hydrogel-caged cells in a flow cell lane following 24 hours of incubation in wells.

[0069] FIG. 23B is a fluorescence image of hydrogel-caged cells stained with a hypoxiasensitive dye and incubated in a flow cell lane for 24 hours.

[0070] FIG. 24A is a brightfield image of hydrogel-caged cells in a flow cell lane following 24 hours of incubation in wells.

[0071] FIG. 24B is a fluorescence image of hydrogel-caged cells stained with a hypoxiasensitive dye and incubated in a flow cell lane for 24 hours.

[0072] FIGS. 25A-D are brightfield images of cells in separate lanes of a flow cell following 5 days of incubation without media replacement. FIG. 25A is an image of cells in theAttorney Docket No. 59528-735601leftmost lane of the flow cell. FIG. 25B is an image of cells in the second leftmost lane of the flow cell. FIG. 25C is an image of cells in the third leftmost lane of the flow cell. FIG. 25D is an image of cells in the fourth leftmost lane of the flow cell.

[0073] FIGS. 26A-B are images of cells stained with a hypoxia-sensitive dye and incubated for 0, 16, and 40 hours in the leftmost lane of a flow cell. FIG. 26A is a set of brightfield images of the cells at the indicated time points. FIG. 26B is a set of fluorescence images of the cells at the indicated time points.

[0074] FIGS. 27A-B are images of cells stained with a hypoxia-sensitive dye and incubated for 0, 16, and 40 hours in the third leftmost lane of a flow cell. FIG. 27A is a set of brightfield images of the cells at the indicated time points. FIG. 27B is a set of fluorescence images of the cells at the indicated time points.

[0075] FIGS. 28A-B are images of cells stained with a hypoxia-sensitive dye and incubated for 0, 16, and 40 hours in the second leftmost lane of a flow cell. FIG. 28A is a set of brightfield images of the cells at the indicated time points. FIG. 28B is a set of fluorescence images of the cells at the indicated time points.

[0076] FIGS. 29A-B are images of cells stained with a hypoxia-sensitive dye and incubated for 0, 16, and 40 hours in the third leftmost lane of a flow cell. FIG. 29A is a set of brightfield images of the cells at the indicated time points. FIG. 29B is a set of fluorescence images of the cells at the indicated time points.

[0077] FIGS. 30A-B are fluorescence images of cells stained with a hypoxia-sensitive dye and incubated for 40 hours in a flow cell lane. FIG. 30A is an image of cells in the leftmost lane of the flow cell. FIG. 30B is an image of cells in the second leftmost lane of the flow cell.

[0078] FIGS. 31A-B are fluorescence images of cells stained with a hypoxia-sensitive dye and incubated for 40 hours in a flow cell lane. FIG. 31A is an image of cells in the leftmost lane of the flow cell. FIG. 31B is an image of cells in the second leftmost lane of the flow cell.

[0079] FIG. 32 is a series of brightfield (top) and fluorescence (bottom) images of cells stained with a hypoxia-sensitive dye and incubated in separate lanes of a flow cell.

[0080] FIG. 33 is a set of fluorescence images of cells stained with a hypoxia-sensitive dye and incubated in various lanes of a flow cell for 12 hours.

[0081] FIG. 34 is a set of fluorescence images of cells stained with a hypoxia-sensitive dye and incubated in various lanes of a flow cell for 16 hours.Attorney Docket No. 59528-735601

[0082] FIGS. 35A-B are sets of images of cells stained with a hypoxia-sensitive dye and incubated in a flow cell lane for 0, 6, 12, or 16 hours. FIG. 35A is a series of brightfield images of the cells. FIG. 35B is a series of fluorescence images of the cells.

[0083] FIG. 36A is a schematic of a method for measuring soluble factor secretions from a cell using dye-labeled binding agents.

[0084] FIG. 36B is a schematic of a method for measuring soluble factor secretions from a cell using oligonucleotide barcode-labeled binding agents.

[0085] FIGS. 37A-D are images of cells coupled to bispecific binding agents targeted to a cell surface marker and IFN-y and enclosed by hydrogel chambers within a fluidic device. FIG. 37A is a brightfield image of the cells immediately following hydrogel chamber synthesis. FIG. 37B is a brightfield image of the cells 24 hours after hydrogel chamber synthesis. FIG. 37C is a fluorescence image of the cells stained with a fluorescent IFN-y antibody. FIG. 37D is a fluorescence image of the cells stained with fluorescent CD71 antibody.

[0086] FIGS. 38A-E are images of natural killer cells coupled to bispecific binding agents targeted to a cell surface marker and a soluble factor and enclosed by hydrogel chambers within a fluidic device. FIG. 38A is a brightfield image of the cells immediately following hydrogel chamber synthesis. FIG. 38B is a fluorescence image of the cells stained with a fluorescent antibody targeted to a soluble factor secreted by the cells taken two hours after hydrogel chamber synthesis. FIG. 38C is a fluorescence image of the cells stained with a fluorescent antibody targeted to a soluble factor secreted by the cells taken three hours after hydrogel chamber synthesis. FIG. 38D is a fluorescence image of the cells stained with a fluorescent antibody targeted to a soluble factor secreted by the cells taken sixteen hours after hydrogel chamber synthesis. FIG. 38E shows a blown-up image of the two cells resolved in the bottom right cage of FIG. 38D.

[0087] FIGS. 39A-D are images of CD8+ T cells coupled to bispecific binding agents targeted to a cell surface marker and a soluble factor and enclosed by hydrogel chambers within a fluidic device. FIG. 39A is a brightfield image of the cells immediately following hydrogel chamber synthesis. FIG. 39B is a fluorescence image of the cells stained with a fluorescent antibody targeted to a soluble factor secreted by the cells taken two hours after hydrogel chamber synthesis. FIG. 39C is a fluorescence image of the cells stained with a fluorescent antibody targeted to a soluble factor secreted by the cells taken three hours after hydrogel chamber synthesis. FIG. 39D is a fluorescence image of the cells stained with aAttorney Docket No. 59528-735601fluorescent antibody targeted to a soluble factor secreted by the cells taken sixteen hours after hydrogel chamber synthesis.

[0088] FIGS. 40A-E are images of cells coupled to bispecific binding agents targeted to a cell surface marker and a soluble factor and co-enclosed with cytokine capture beads in hydrogel chambers within a fluidic device. FIG. 40A is a brightfield image of the cells immediately following hydrogel chamber synthesis. FIG. 40B is a fluorescence image collected four hours after hydrogel chamber synthesis in the presence of a fluorescent TNF-a antibody. FIG. 40C is a fluorescence image collected seven hours after hydrogel chamber synthesis in the presence of a fluorescent TNF-a antibody. FIG. 40D is a fluorescence image collected four hours after hydrogel chamber synthesis in the presence of a fluorescent IFN-y antibody. FIG. 40E is a fluorescence image collected seven hours after hydrogel chamber synthesis in the presence of a fluorescent IFN-y antibody.

[0089] FIGS. 41A-C are images of OT1 and dendritic cells co-enclosed in hydrogel chambers within a fluidic device. FIG. 41 A is a pair of brightfield images of the cells immediately following hydrogel chamber synthesis. FIG. 41B is a pair of fluorescence images of the cells collected in the presence of a fluorescent antibody targeted to a soluble factor secreted by the OT1 cells and a bispecific capture agent coupled to the OT1 cell surfaces and targeted to the soluble factor. FIG. 41C is a pair of merged images generated using the images shown in FIGS. 41A-B.

[0090] FIGS. 42A-C are images immune and cancer cells co-enclosed in hydrogel chambers within a fluidic device. FIG. 42A is a brightfield image of the cells immediately following hydrogel chamber synthesis. FIG. 42B is a fluorescence image of the cells collected in the presence of a fluorescent antibody targeted to a surface marker expressed by the cancer cells.FIG. 42C is a fluorescence image collected in the presence of a fluorescent antibody targeted to a soluble factor secreted by the immune cells and a bispecific capture agent coupled to the immune cell surfaces and targeted to the soluble factor.

[0091] FIG. 43A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 43B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 43C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 43D is an illustration of a cross-sectional view of a fluidic device.

[0092] FIG. 44A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 44B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 44C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 44D is an illustration of a cross-sectional view of a fluidic device.Attorney Docket No. 59528-735601

[0093] FIG. 45A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 45B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 45C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 45D is an illustration of a cross-sectional view of a fluidic device.

[0094] FIG. 46A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 46B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 46C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 46D is an illustration of a cross-sectional view of a fluidic device.

[0095] FIG. 47A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 47B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 47C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 47D is an illustration of a cross-sectional view of a fluidic device.

[0096] FIG. 48A is an illustration of a top-down view of a bottom layer of a fluidic device.FIG. 48B is an illustration of a top-down view of a spacer layer of a fluidic device. FIG. 48C is an illustration of a top-down view of a top layer of a fluidic device. FIG. 48D is an illustration of a cross-sectional view of a fluidic device.

[0097] FIG. 49 is a plot of blue fluorescence intensities from ImagelT hypoxia detection reagent within cells in a variety of fluidic devices.

[0098] FIG. 50 is a plot of blue fluorescence intensities from ImagelT hypoxia detection reagent within cells in a variety of fluidic devices.DETAILED DESCRIPTION

[0099] Disclosed herein are flow cells that facilitate gas transfer to regions that contain media. Differing from conventional flow cells that can exhibit oxygen depletion during cell incubation, the presently disclosed flow cells permit gas transfer into flow cell channels, facilitating stable dissolved gas levels during extended (e.g., multi-day) assays. These flow cells are particularly amenable to assays that benefit from quiescent media, including methods for studying cell secretion, cell adhesion, and cell-cell interactions.

[0100] In one embodiment, the present disclosure provides a method for analyzing a biological component in a flow cell comprising: inputting one or more cells into a first channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel; incubating the one or more cells in a liquid in the first channel; andAttorney Docket No. 59528-735601diffusing a first gas from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel. Diffusion of the first gas into the liquid in the first channel can slow a rate of depletion of the first gas during the incubating, increasing the uniformity of incubation conditions and preventing complete or partial depletion of the first gas. In exemplary embodiments, the first gas comprises O2. However, other gases metabolized or otherwise utilized by cells such as CH4 or SH2 are usable in the presently disclosed methods.

[0101] The methods of the present disclosure are particularly applicable to assays that are disrupted by fluid flow and / or media replacement. Cellular behavior is typically challenging to study within flow cell settings, as many facets of cellular behavior and interactions are mediated by secretions, transient binding, and components of extracellular matrices that can be washed away or disrupted each time media is changed. However, owing to the small volumes of many flow cells, flow cell assays can require media replacement to maintain cell viability for the duration of the assay. Diffusion of the first gas into the liquid in the first chamber can obviate the need to replace liquid in the first channel, thereby enabling the maintenance of quiescent conditions in the first channel during the incubation and optionally during measurement of cellular characteristics. Accordingly, in many embodiments disclosed herein, after the inputting the liquid into the first channel, the liquid is quiescent during the incubating the one or more cells.

[0102] Furthermore, gas diffusion into flow cell samples can enable the maintenance of quiescent conditions over extended periods of time, allowing, for example, cells to be studied over multiple days under stable conditions. As non-limiting examples, the incubating can be for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours. This capability to facilitate extended assays can be due, at least in part, to extended cell viability in the disclosed flow cell. In many cases, the cells are viable in the liquid in the first channel for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours. Optionally, the media is not replaced or is replaced at most once during the at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, atAttorney Docket No. 59528-735601least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours. In certain methods, (e.g., with slow-growing and / or hypoxia-tolerant cells) the incubating is for at least 7 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days; the cells are viable in the liquid in the first channel for at least 7 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days; and / or the media is not replaced or is replaced at most once during the at least 7 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days.

[0103] In many cases, the extended viability of cells in the fluidic device are at least in part due to maintained O2 levels. In exemplary embodiments, a steady-state dissolved O2 level of the liquid in the first channel is greater than a steady-state dissolved O2 level achieved in a similar fluidic device lacking the second channel. For example, in some cases, the liquid comprises or approaches a steady-state dissolved O2 level of between about between about 0.1 and 0.5 mg / L, between about 0.1 and 1 mg / L, between about 0.1 and 2 mg / L, between about 0.1 and 4 mg / L, between about 0.1 and 6 mg / L, between about 0.1 and 8 mg / L, between about 0.1 and 10 mg / L, between about 0.1 and 12 mg / L, between about 0.25 and 0.5 mg / L, between about 0.25 and 1 mg / L, between about 0.25 and 2 mg / L, between about 0.25 and 4 mg / L, between about 0.25 and 6 mg / L, between about 0.25 and 8 mg / L, between about 0.25 and 10 mg / L, between about 0.25 and 12 mg / L, between about 0.5 and 1 mg / L, between about 0.5 and 2 mg / L, between about 0.5 and 4 mg / L, between about 0.5 and 6 mg / L, between about 0.5 and 8 mg / L, between about 0.5 and 10 mg / L, between about 0.5 and 12 mg / L, between about 1 and 2 mg / L, between about 1 and 4 mg / L, between about 1 and 6 mg / L, between about 1 and 8 mg / L, between about 1 and 10 mg / L, between about 1 and 12 mg / L, between about 2 and 4 mg / L, between about 2 and 6 mg / L, between about 2 and 8 mg / L, between about 2 and 10 mg / L, between about 2 and 12 mg / L, between about 2 and 15 mg / L, between about 3 and 6 mg / L, between about 3 and 8 mg / L, between about 3 and 10 mg / L, between about 3 and 12 mg / L, between about 4 and 8 mg / L, between about 4 and 10 mg / L, between about 4 and 12 mg / mL, between about 6 and 8 mg / mL, between about 6 and 10 mg / mL, between about 6 and 12 mg / mL, or between about 8 and 12 mg / mL. Such O2 levels can facilitate viability of the one or more cells, which, as non-limiting examples, may deplete O2 from the liquid at a rate of at least about 0.01 mg*L'1*h'1, a rate of at least about 0.02 mg*L'1*h'1, a rate of at least about 0.05 mg*L’1*h’1, a rate of at least about 0.1 mg*L' ^h’1, a rate of at least about 0.2 mg*L'1*h'1, a rate of at least about 0.5 mg*L'1*h'1, a rate of at least about 1 mg*L'1*h'1, a rate of at least about 1.5 mg*L'1*h'1, a rate of at least about 2Attorney Docket No. 59528-735601mg*L’1*h’1, a rate of at least about 2.5 mg*! / 1*!!’1, a rate of at least about 3 mg*! / 1*^1, a rate of at least about 4 mg*L’1*h’1, or a rate of at least about 5 mg*! / 1*^1.

[0104] The disclosed methods are amenable to hypoxia-tolerant and hypoxia-sensitive cells. In some cases, a cell of the one or more cells is viable for less than 180, less than 150, less than 120, less than 90, less than 60, less than 48, less than 36, less than 24, less than 16, less than 12, less than 8, less than 6, less than 4, less than 2, or less than 1 hour under hypoxic conditions. Alternatively or in addition thereto, a cell of the one or more cells can be viable for at least 1, at least 2, at least 4, at least 6, at least 8, at least 12, at least 16, at least 24, at least 36, at least 48, at least 60, at least 90, at least 120, at least 150, or at least 180 hours under hypoxic conditions. As used herein, the term “hypoxic conditions” can denote oxygen levels below those required for long-term cell viability. Many cells exhibit phenotypic responses to hypoxic conditions, including changes in metabolism and expression of hypoxiainducible factors. The oxygen concentration threshold at which cells begin to exhibit hypoxia responses is dependent on cell type. Nonetheless, in general, hypoxic conditions denote dissolved oxygen levels below about 3 mg / mL, about 2 mg / mL, or about 1 mg / mL, or dissolved oxygen levels following equilibration with an atmosphere that comprises less than about 5%, about 4%, about 3%, about 2%, or about 1% O2.

[0105] As discussed in further detail below, the second cut-out region of the spacer layer can be sandwiched between the bottom layer and the top layer to form the second channel.However, the second cut-out region can alternatively extend only part way through the spacer layer (e.g., from a top to a middle portion of the spacer layer) or be disposed within the spacer layer. As the top and bottom layers can be impermeable to liquids and optionally to gases, the permeability of the spacer layer can control the movement of species between channels. In many cases, the spacer layer is impermeable to liquid water and permeable to gaseous O2. More broadly, the spacer layer may be impermeable to the first liquid and permeable to the first (and, when present, second) gas. As non-limiting examples, the spacer layer can comprise an O2 permeability of about 10 to 500 barrers. Additionally, the spacer layer can comprise a higher O2 permeability, such as 500 to 2500 barrers or 1000 to 10000 barrers. Examples of spacer layer materials consistent with the present disclosure include siloxane, silicone, acrylate, acrylamide, polyvinyl, polyurethane, epoxy, polyphenol, polyester, polyamide, a polyimide, polytetrafluoroethylene, polyethylene, polypropylene, polycarbamate, polycarbonate, polyethylene terephthalate, cyanoacrylates, and combinations thereof. In many cases, the spacer, comprised of one or more materials outlined herein, is a nanoporous film (i.e., comprises sub-micron pores). As discussed further herein, the spacerAttorney Docket No. 59528-735601layer can, in certain designs, be comprised of two or more materials, such as a plastic core and highly gas permeable adhesive disposed on two opposing sides of the plastic core. The spacer layer can comprise a pressure-sensitive adhesive, such as a double-sided pressure sensitive adhesive having two silicone adhesive layers that are deposited on opposing sides of a polypropylene core middle layer. The spacer layer can include a double-sided adhesive with different adhesives on its two sides. For example, the spacer layer may include a middle carrier layer, wherein a first side of the middle carrier layer is coupled to a silicone adhesive and a second side of the carrier layer is coupled to an acrylic adhesive. The spacer layer can also include an ultraviolet light-curable (UV-curable) material (e.g., deposited on one or both sides of a carrier material) such as a UV-curable acrylic, a UV-curable epoxy resin, a UV-curable silicone adhesive, or a combination thereof. In such cases, the spacer layer may be exposed to UV light (e.g., about 100 to 5000 mW / cm2UV light for about 1 to 60 seconds) while the spacer layer is in contact with the fluidic device top and / or bottom layer.

[0106] The top and bottom layer of the flow channel can be gas and liquid impermeable, such that the top and bottom layers partition contents of the first and second channels from the surrounding atmosphere. An example of the top and bottom layer can both be optically transparent glass suitable for use in optical and fluorescent microscopy and is also gas and liquid impermeable. In such designs, the contents of the first and second channels can be controlled and limited to species actively input (e.g., pumped) therein. Alternatively, one or more channels (e.g., the second channel) can comprise one or more openings to an exterior space such as a surrounding atmosphere that enables gas to flow between the exterior space and the one or more channels. Furthermore, outer edges of the fluidic device can be gas permeable and liquid impermeable, thereby facilitating exchange between an edge exposed channel (e.g., in certain fluidic device designs, the first channel or see 405B of FIG. 3D) and the exterior space while preventing liquid or analytes from leaking out of the fluidic device or contaminants from entering the fluidic device. In many aspects, the first channel is coupled (e.g., sealingly coupled) to a fluidic manifold, is sealed (e.g., all entrances and exits to the first channel are sealed to isolate substances inside of the first channel from the surrounding atmosphere and optionally other channels; and / or the first channel comprises an inlet and an outlet, and the inlet and outlet are sealed to prevent ambient atmosphere from contacting the first channel), or a combination thereof. In further aspects, the second channel is coupled to a gas manifold or is open to an atmosphere outside of the fluidic device.

[0107] Gas flow from the second channel to the first channel can at least partially be controlled by the dimensions and composition of the spacer layer. In some aspects, the firstAttorney Docket No. 59528-735601and second channel are separated (i.e., the portion of the spacer layer through which the first gas diffuses from the second channel and into the liquid in the first channel, for example as depicted by (407A) in FIG. 3D) by about 50 to 3000 microns (e.g., a 50 pm PDMS wall or a 250 pm adhesive wall). For example, the first and second channel can be separated by about 0.5 to 1, 0.5 to 1.5, 0.5 to 2, 0.5 to 2.5, 0.5 to 3, 1 to 1.5, 1 to 2, 1 to 2.5, 1 to 3, 1.5 to 2, 1.5 to 2.5, 1.5 to 3, 2 to 2.5, 2 to 3, or 2.5 to 3 millimeters.

[0108] Gas flow between the first and second channels can also depend on channel dimensions. For example, relative rates of depletion of the first gas and flow of the first gas from the second channel to the first channel can at least partially depend the width, height, and length of the channels. As non-limiting examples, the first channel can comprise a width of about 0.5 to 2.5, 2.5 to 5, 2.5 to 7.5, 2.5 to 10, 2.5 to 15, 5 to 7.5, 5 to 10, 5 to 15, 7.5 to 15, 10 to 15, or 10 to 20 millimeters. In some cases, the first channel comprises a width of about 0.1 to about 2.5 millimeters. In some cases, the first channel comprises a width of about 0.1 to about 15 millimeters. Additionally, the first channel can comprise a height (e.g., a distance between opposing faces of the top and bottom surfaces) of about 0.025 to 0.05, 0.025 to 0.1, 0.025 to 0.15, 0.025 to 0.2, 0.025 to 0.25, 0.05 to 0.1, 0.05 to 0.15, 0.05 to 0.2, 0.05 to 0.25, 0.1 to 0.15, 0.1 to 0.2, 0.1 to 0.25, 0.15 to 0.25, or 0.2 to 0.4 millimeters. In exemplary embodiments, the first channel comprises a length of about 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 150, 100 to 200, 100 to 250, 150 to 250, or 200 to 400 millimeters.

[0109] In many aspects, the second channel comprises identical or similar dimensions as the first channel. For example, the second channel can comprise a width of about 2.5 to 5, 2.5 to 7.5, 2.5 to 10, 2.5 to 15, 5 to 7.5, 5 to 10, 5 to 15, 7.5 to 15, 10 to 15, or 10 to 20 millimeters. Similarly, the second channel can comprise a height of about 0.025 to 0.05, 0.025 to 0.1, 0.025 to 0.15, 0.025 to 0.2, 0.025 to 0.25, 0.05 to 0.1, 0.05 to 0.15, 0.05 to 0.2, 0.05 to 0.25, 0.1 to 0.15, 0.1 to 0.2, 0.1 to 0.25, 0.15 to 0.25, or 0.2 to 0.4 millimeters. Additionally, the second channel can comprise a length of about 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 150, 100 to 200, 100 to 250, 150 to 250, or 200 to 400 millimeters. In certain embodiments, dimensions of the first channel are within about 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% of the dimensions of the second channel. Furthermore, in many embodiments, second channel is parallel to the first channel over a majority of its length.

[0110] Gas flow between the first and second channels can further depend on spacer layer width. In some cases, the portion of the spacer layer located between the first and second channels comprises a width of about 0.05 to 2.5 millimeters, for example about 0.05 to 0.25, about 0.05 to 0.5, about 0.05 to 1. about 0.05 to 1.5, about 0.05 to 2.5, about 0.25 to 0.5,Attorney Docket No. 59528-735601about 0.25 to 1, about 0.25 to 1.5, about 0.25 to 2.5, about 0.5 to 1, about 0.5 to 1.5, about 0.25 to 2.5, about 0.5 to 1, about 0.5 to 1.5, about 0.5 to 2.5, about 1 to 1.5, about 1 to 2.5, or about 1.5 to 2.5 millimeters.[oni] The fluidic device can include multiple liquid and / or gas-containing channels. For example, the method can be performed in series or in parallel in a plurality of first and second channels disposed within a single fluidic device. Similarly, the fluidic device can include multiple gas-filled channels adjacent to the first channel. For example, in some aspects the method further comprises diffusing the first gas from a third channel of the fluidic device into the first channel through another portion of the spacer layer. In many such aspects, the first channel is adjacent to the third channel, and the another portion of the spacer layer is located in between the first and third channels. The spacer layer can also further include a third cutout region that at least partially defines the third channel. As with the second channel, the third cut-out region of the spacer layer can be sandwiched between the bottom layer and the top layer to form the third channel, or can be disposed through a subset of the height of the spacer layer. Optionally, the second and third channels are disposed on opposite sides of the first channel. For example, the first channel can be disposed between identical second and third channels within a single fluidic device (e.g., flow cell).

[0112] In many cases, the second channel is fluidically coupled to an atmosphere that is extrinsic to the fluidic system. This arrangement can include opening the second channel to a space outside of the fluidic device or connecting the second channel to a manifold that is open to the atmosphere. The second channel can be passively coupled to the atmosphere. Alternatively, the atmosphere can be continually or periodically refreshed in (e.g., pumped through) the second channel. Accordingly, the pressure of the first gas in the second channel can be similar to that of the extrinsic atmosphere (e.g., about 1 atmosphere) or elevated (e.g., about 1.25 to 10 atmospheres). In each of the above cases, a partial pressure of O2 in the second channel can be within at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of a partial pressure of O2 in the atmosphere extrinsic to the fluidic device (e.g., following at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours of incubating the one or more cells in the first channel). The atmosphere extrinsic to the fluidic device is typically an ambient atmosphere, that is it comprises a pressure of about 760 millimeters mercury (mm Hg, about 1 atm) and an O2 concentration of about 160 mm Hg (a mole fraction of about 0.21). However, the atmosphere extrinsic to theAttorney Docket No. 59528-735601fluidic device may have a pressure of greater than or less than about 760 mm Hg, an O2 mole fraction of greater than or less than 0.21, or a combination thereof.

[0113] The second channel can also act as a conduit for removing a second gas from the liquid in the first channel. For example, the method can comprise diffusing a second gas from the first channel of the fluidic device into the second channel through the portion of the spacer layer. In particular examples, the method comprises diffusing the second gas from the liquid in the first channel of the fluidic device into the second channel through the portion of the spacer layer. The second gas can comprise a waste product of the one or more cells, such as CO2, which can become toxic to the one or more cells at high concentrations (e.g., generated during the incubating).

[0114] FIGS. 3A-E illustrate an example of a fluidic device consistent with the present disclosure. These figures depict a flow cell comprised of a bottom layer (400), a spacer layer (402), and top layer (404). The bottom layer (400) and the top layer (404) can both independently be a glass or plastic material. In some cases, the top layer (404) is optically transparent or translucent. In some cases, the bottom layer (400) is optically transparent or translucent. In many cases, the top layer (404) and the bottom layer (400) are optically transparent or translucent. The spacer layer (402) can be a double-sided pressure sensitive adhesive with one or more cut-out regions (405A, 405B, 406). In this design, the one or more cut-out regions of the spacer layer are sandwiched between the bottom layer and the top layer to form one or more channels. In various embodiments, the spacer layer (402) includes a core plastic (e.g., PET) layer with pressure sensitive adhesive coating on its top and / or bottom sides that contact the top (404) and bottom (400) layers.

[0115] Referring to FIG. 3B, a peripheral portion (407) of the spacer layer (402) provides a boundary for one or more cut-out regions (405 A, 405B, 406) with defined widths (406A) and lengths (406B). In many designs disclosed herein, the spacer layer (402) adheres to the top (404) and bottom (400) layers, such that a cut-out region or plurality of cut-out regions (405A, 405B, 406) define a channel or a plurality of channels. The cut-out region may be formed, for example, by cutting (e.g., kiss cutting) or dicing the spacer layer (402), either before the spacer layer is bonded to the bottom or layer (400) or top layer (404) or after the spacer layer (402) is bonded to one of the bottom layer (400) or the top layer (404). The spacer layer (402), bottom layer (400), and / or top layer (404) can be water (or optionally more generally liquid) impermeable such that a first aqueous sample in a first cut-out region (e.g., 406) is isolated from a second aqueous sample in a second cut-out region (e.g., 405A).The one or more cut-out regions (405A, 405B, 406) can thus define flow cell channelsAttorney Docket No. 59528-735601with defined dimensions. In some cases, the top layer (404) is oxygen impermeable and liquid impermeable. In some cases, the bottom layer (400) is oxygen impermeable and liquid impermeable. In some cases, the top layer (404) and the bottom layer (400) are oxygen impermeable and liquid impermeable.

[0116] A channel defined by a cut-out region (405A, 405B, 406) can be operably coupled to an inlet (408) and / or an outlet (410) through which, for example, a gas, liquid, sample, or reagent may flow. For example, the top layer (404) can include an inlet (408) configured to receive a liquid and an outlet (410) configured to output the liquid. In various embodiments, the inlet (408) and outlet (410) can both be represented as through holes in the top layer (404). However, the inlet (408) and / or outlet (410) can be disposed within other components of the flow cell, such as the bottom layer (400) or spacer layer (402). With respect to FIG.3D, the top layer (404) can have a height of about 0.7 mm and the bottom layer can have a height of about 0.5 mm. The first cut-out region (406) can have a height (406C) defined by the distance between opposing faces of the top layer (404) and the bottom layer (400). The spacer layer can have a height ranging from about 50 microns to about 200 microns, and preferably, from about 70 microns to about 130 microns. The cut-out region can have an area based on a dimension of about 10.3 cm length x about 0.7 cm width.

[0117] Referring again to FIGS. 3A-D as examples of flow cell designs consistent with the present disclosure, a first channel defined by a cut-out region (e.g., 406) can be fluidically isolated from other channels, such that liquid disposed in the first channel does not flow or diffuse into an adjacent channel defined by an adjacent cut-out region (e.g., 405 A, 405B) or exchange with a liquid in the adjacent channel. In many flow cell designs disclosed herein, fluidic isolation is at least in part achieved by liquid impermeable peripheral portions (407), which can include materials that do not transmit fluid or solutes between separate cut-out regions. Such designs can allow individual samples and reagents to be isolated within a particular channel. For example, an analyte released by a cell in a first cut-out (405 A) portion may be prevented from entering a second cut-out region (406) by a peripheral portion (407) disposed therebetween. Similarly, a liquid (e.g., cell medium) added to a first cut-out region (406) may be prevented from transferring into a second cut-out region (405A, 405B).Alternatively, a peripheral portion (407) may be partially permeable to solvent molecules (e.g., H2O, ethanol) but prevent larger molecules (e.g., saccharides) and biological species from permeating between cut-out regions (405A, 405B, 406).

[0118] However, in many designs disclosed herein, the spacer layer (402) (and therefore one or more peripheral portions (407) permit gas diffusion and essentially permit no liquidAttorney Docket No. 59528-735601diffusion between two or more cut-out regions (405A, 405B, 406). This principle is illustrated in FIG. 3E, which depicts the flow cell side-view of FIG. 3D wherein a first cutout region 406 is filled with a fluid (e.g., cell media) and one or more adjacent cut-out regions (405A, 405B) are filled with a first gas. While the fluid in cut-out region 406 is prevented from diffusing into the one or more adjacent cut-out regions (405 A, 405B) by peripheral portions (407), gas from the one or more adjacent cut-out regions (405 A, 405B) can permeate through one or more peripheral portions (407) into the first cut-out region (406), enabling dissolved gas (e.g., O2) levels to continually refresh in the fluid disposed within the first cutout region (406). Similarly, a gas dissolved in the fluid disposed within the first cut-out region (406), such as CO2, may transfer into one or more adjacent cut-out regions (405 A, 405B). The directional flow of one or more gases between the fluid disposed within the first cut-out region (406) to one or more adjacent cut-out regions (405 A, 405B) may be driven by concentrations of the one or more gasses in the fluid and partial pressures of the one or more gases in the one or more adjacent cut-out regions (405A, 405B). For example, CO2 buildup in the fluid during cell culturing may promote CO2 transfer from the fluid into the one or more adjacent cut-out regions (405 A, 405B) comprising atmospheric levels of CO2, while O2 flow into the fluid from the one or more adjacent cut-out regions (405 A, 405B) may be promoted by O2 depletion in the fluid during cell culturing.

[0119] The one or more cut-out regions (405 A, 405B) may be open or operably coupled to a gas line or surrounding (e.g., ambient) atmosphere, such that gas within the one or more cutout regions (405A, 405B) is continually or periodically refreshed. In many designs, such as the flow cell depicted in FIG. 3E, gas does not flow through the top (404) or bottom (400) layers. While FIG. 3E illustrates an embodiment in which a first subset of otherwise identical cut-out regions are filled with a liquid (see 406) and the remaining cut-out regions (see 405 A and 405B) are filled with gas, alternate designs that dispose a gas-filled cut-out region at least partially adjacent to a liquid-filled cut-out region are contemplated herein.

[0120] For example, in FIG. 4A, which depicts a spacer layer (402) similar to the spacer layer of FIG. 3B, a peripheral portion (407) that is adjacent to and at least partially defines a first cut-out region (406) includes a second cut-out region (405C). In FIG. 4A, this second cut-out region (405C) can be a narrow channel cut into the peripheral portion (407).However, this second cut-out region can also adopt other structural forms such as trenches, serpentine chambers, or series of wells. Furthermore, this second cut-out portion can extend through the full height of the peripheral portion (407) or through a portion of the peripheral portion (407) (e.g., extend from a top of the peripheral portion to a middle of the peripheralAttorney Docket No. 59528-735601portion or define a channel of which at least a portion is fully enclosed by the peripheral portion). As with the design depicted in FIG. 3E, gas can diffuse from the second-cut out region (405C), through the peripheral portion (407), and into the first cut-out region (406). The second cut-out region (405C) can be coupled to one or more openings (409) to the outside of the spacer. While the one or more openings (409) can be disposed within the spacer layer (402), these openings (409) can also be disposed through a top (404) or bottom layer (400) of a flow cell to connect the second cut-out region (405C) to a surrounding atmosphere, gas manifold, or the like. As depicted in FIG. 4A, the one or more openings (409) can be disposed at regular or irregular intervals along a length of the second cut-out region (405C). Alternatively, as depicted in FIG. 4B, the one or more openings (409) can be disposed at opposing ends of the second cut-out region (405C). As depicted in FIG. 4C, the substrate can comprise a plurality of second cut-out regions (405C) disposed adjacent to plurality of first cut-out regions (406). The second cut-out region can be open to atmosphere or coupled to a gas manifold.

[0121] A second cut-out region (e.g., 405B in FIG. 3E or 405C in FIG. 4D) capable of accommodating a gas during operation of the flow cell can have similar dimensions as a first cut-out region capable of accommodating liquid during operation of the flow cell. For example, a second cut-out region can comprise a cross-sectional area that is within about 25%, within about 20%, within about 15%, within about 10%, within about 5%, within about 3%, within about 2%, or within about 1% of a cross-sectional area of a first cut-out region (e.g., 405A, 405B, and 406). Alternatively, the second cut-out region can be smaller than a first cut-out region. For example, the second cut-out region can comprise a cross-sectional area that is about 0.5% to 5%, about 0.5% to 10%, about 0.5% to 20%, about 0.5% to 30%, about 0.5% to 40%, about 0.5% to 50%, about 0.5% to 75%, about 5% to 10%, about 5% to 20%, about 5% to 30%, about 5% to 40%, about 5% to 50%, about 5% to 75%, about 10% to 20%, about 10% to 30%, about 10% to 40%, about 10% to 50%, about 10% to 75%, about 20% to 30%, about 20% to 40%, about 20% to 50%, about 20% to 75%, about 30% to 40%, about 30% to 50%, about 30% to 75%, about 40% to 50%, about 40% to 75%, or about 50% to 75% of a cross-sectional area of the first cut-out region. The size of the second cut-out region may be tailored for specific O2 consumption or waste gas production rates anticipated during an assay. For example, a first cut-out region that contains several thousands of cells enclosed in hydrogel cages may be replenished of O2 more readily (and therefore by a smaller volume second cut-out region) than a first cut-out region loaded with 106cells / mL.Attorney Docket No. 59528-735601

[0122] FIG. 4D depicts a possible side-view of a flow cell that contains a second cut-out region (405C) disposed within a peripheral portion (407) of a spacer layer (402) and adjacent to a first cut-out region (406). In this depiction, the first-cut out region (406) is filled with a liquid. Gas (e.g., O2) from the second cut-out region (405C) can diffuse through the peripheral portion (407) and into the first cut-out region (406). As illustrated in this figure, the second cut-out region can have smaller dimensions (e.g., height, width, and / or cross-sectional area) than the first cut-out region (406). One or more openings (409) from the second cut-out region (405C) can extend through a top layer (404) of the flow cell (and optionally through at least a portion of the spacer layer (402)) in a manner that fluidically couples the second cut-out region to an atmosphere outside of the flow cell and / or allows the second cut-out region to couple to a gas manifold, for example to control a pressure or gas composition within the second cut-out region (405C). In such embodiments, the second cutout region (405C) may be held at a pressure greater than that of a surrounding atmosphere. For example, a pressure of the second cut-out region (405C) may be elevated above about 1.1 atm, 1.25 atm, 1.5 atm, 2 atm, 3 atm, 5 atm, or 10 atm.

[0123] In some embodiments, multiple second cut-out regions (405C) can be positioned adjacent to multiple first cut-out regions (406). FIG. 4D shows a side view of a flow cell configuration in which the first cut-out region (406) is filled with a liquid medium and the adjacent second cut-out region (405C) contains gas. Gas may diffuse from the second cut-out region (405C) through the peripheral portion (407) and into the liquid within the first cut-out region (406). The second cut-out region (405C) may have smaller dimensions (e.g., height, width, and / or cross-sectional area) than the first cut-out region (406). One or more openings (409) can extend through the top layer (404) and optionally through part of the spacer layer (402), fluidically coupling the second cut-out region (405C) to an external atmosphere or gas manifold. This arrangement can allow control over gas composition or pressure within the second cut-out region (405C), which may be maintained at a pressure greater than ambient.

[0124] As with the embodiment shown in FIG. 3E, gas (e.g., O2) can diffuse from the second cut-out region (405C), through the peripheral portion (407), and into the first cut-out region (406). The second cut-out region (405C) may also be in fluidic communication with one or more openings (409) that connect the second cut-out region to the exterior of the flow cell. These openings (409) may be formed within the spacer layer (402) itself, or they may extend through a top layer (404) and / or bottom layer (400) of the flow cell to provide connection to an external atmosphere, gas manifold, or other gas source. The openings (409) can be distributed along the length of the second cut-out region (405C), either at regular or irregularAttorney Docket No. 59528-735601intervals, or located at one or both terminal ends of the second cut-out region, as illustrated in FIGS. 4A-C

[0125] FIGS. 43A-D illustrate an additional fluidic device design that includes a second cutout region (4308) that is open at both ends of the spacer layer (4302). FIG. 43A depicts the bottom layer (4300) of the fluidic device, which may be a clear or translucent slide as disclosed elsewhere herein. The spacer layer (4302) depicted in FIG. 43B may include peripheral portions (4307) that at least partially define first cut-out regions (4306) and a second cut-out regions (4308). In the depicted configuration, the second cut-out regions (4308) extend to at least one end of the spacer layer (4302), terminating at an opening (4308A, 4308B). When the spacer layer (4302) is coupled to the bottom layer (4300) and top layer (4304), the openings (4308A, 4308B) may be open to atmosphere outside of the fluidic device, and may thus allow gas to exchange between the second cut-out regions (4308) and surrounding atmosphere. In some implementations of the fluidic device, the second cut-out regions (4308) extend across the full length of the spacer layer (4302), such that both ends of the second cut-out region (4308) terminate at openings (4308 A, 4308B) to surrounding atmosphere.

[0126] The top layer (4304) depicted in FIG. 43C may include holes (4310). In the fully fabricated fluidic device, wherein the bottom layer (4300) and top layer (4304) are coupled to opposite sides of the spacer layer (4302), the holes (4310) may open to the first cut-out regions (4306) and may serve as inlets and / or outlets to channels defined by the first cut-out regions (4306) and portions of the bottom layer (4300) and the top layer (4304).

[0127] FIG. 43D provides a cross-sectional view of the fully fabricated fluidic device, wherein the bottom layer (4300) and top layer (4304) are coupled to opposite sides of the spacer layer (4302). The first cut-out regions (4306) are separated from the second cut-out regions (4308) by the peripheral portions (4307) of the spacer layer (4302). The spacer layer (4302) may be gas permeable, and may therefore allow gas to exchange between the first cutout regions (4306) and second cut-out regions (4308).

[0128] FIGS. 44A-D depict an additional fluidic device design consistent with the present disclosure. FIGS. 44A-C are illustrations of top-down views of a bottom layer (4400, FIG.44A), a spacer layer (4402, FIG. 44B), and a top layer (4404, FIG. 44C). As shown in FIG.44D, which is an illustration of a cross-sectional view of the fluidic device, the bottom layer (4400) and top layer (4404) couple to opposite sides of the spacer layer (4402). The bottom layer (4400) and top layer (4404) may be transparent or translucent, water-impermeable slides. The spacer layer (4402) may include first cut-out regions (4406) that are bounded byAttorney Docket No. 59528-735601peripheral portions (4407) of the spacer layer (4402), and second cut-out regions (4408) that are adjacent to the first cut-out regions (4406). The second cut-out regions (4408) may extend to one end of the spacer layer (4402), and terminate in openings (4408A) at that end of the spacer layer (4402). The top layer (4404) may include holes (4410) that align to the first cutout regions (4406). The holes (4410) can serve, for example, as inlets and / or outlets to channels defined by the first cut-out regions (4406) and the bottom layer (4400) and top layer (4400, 4404).

[0129] An additional fluidic device design is illustrated in FIGS. 45A-D. FIGS. 45A-C are illustrations of top-down views of a bottom layer (4500, FIG. 45A), a spacer layer (4502, FIG. 45B), and a top layer (4504, FIG. 45C) that may be combined to form the fluidic device. The bottom layer (4500) and top layer (4504) may be transparent or translucent, water-impermeable slides. As shown in FIG. 45D, which is an illustration of a cross-sectional view of the fluidic device, the bottom layer (4500) and top layer (4504) may couple to opposite sides of the spacer layer (4502). The spacer layer (4502) may include first cut-out regions (4506) that are bounded by peripheral portions (4507) of the spacer layer (4502), and second cut-out regions (4508) that are adjacent to the first cut-out regions (4506). The second cut-out regions (4508) may be open through an end of the spacer layer (4502) or, as shown in FIG. 45B, may be enclosed within the spacer layer (4502). The top layer (4504) may include holes (4510) that align to the first cut-out regions (4506) and that function as inlets, outlets, or other ports into channels that are at least partially defined by the first cut-out regions (4506). The top layer (4504) may also include slits (4511) that align to the second cut-out regions (4508). The top layer (4504) may contain one slit (4511) per second cut-out region (4508), or a plurality of slits (4511) per second cut-out region (4508). For example, as shown in FIG. 45C, the top layer (4504) may include two slits (4511) per second cut-out region (4508) in the spacer layer (4502). When the top layer (4504) is coupled to the spacer layer (4502), the slits (4511) may allow gas to flow into and out of the second cut-out regions. The slits (4511) through the top layer (4504) may be formed using a blade, a CO2 laser, or another method suitable for cutting plastic or glass. The spacer layer (4502) may be gas permeable, such that gas may diffuse between the first and second cut-out regions (4506, 4508).

[0130] FIGS. 46A-D illustrate a further fluidic device design that is consistent with the present disclosure. FIGS. 46A-C are illustrations of top-down views of a bottom layer (4600, FIG. 46A), a spacer layer (4602, FIG. 46B), and a top layer (4504, FIG. 46C) that may be combined to form the fluidic device. FIG. 46D is an illustration of a cross-sectional view of the fluidic device, showing the bottom layer (4600) and the top layer (4604) coupled toAttorney Docket No. 59528-735601opposite sides of the spacer layer (4602). The spacer layer (4602) may include first cut-out regions (4606) that are bounded by peripheral portions (4607) of the spacer layer (4602). The top layer (4604) may include holes (4610) that align to the first cut-out regions (4606). The holes may function as inlets, outlets, or other ports into channels that are at least partially defined by the first cut-out regions (4506). The top layer (4604) may also include slits (4611) that align to peripheral portions (4607) of the spacer layer. As shown in FIG. 46D, the slits (4611) may extend through the top layer (4504) to the spacer layer (4502), thereby exposing peripheral portions (4607) of the spacer layer (4602) to atmosphere. The spacer layer (4602) may be gas-permeable. Accordingly, gas may diffuse through peripheral portions (4607) of the spacer layer (4602), between the slits (4611) through the top layer (4611) and the first cutout regions (4606) that define channels of the fluidic device.

[0131] FIGS. 48A-D illustrate a further fluidic device design that is similar to the fluidic device depicted in FIGS. 46A-D. FIGS. 48A-C are illustrations of top-down views of a bottom layer (4800, FIG. 48A), a spacer layer (4802, FIG. 48B), and a top layer (4804, FIG.48C) that may be combined to form the fluidic device. FIG. 48D is an illustration of a cross-sectional view of the fluidic device, showing the bottom layer (4800) and the top layer (4804) coupled to opposite sides of the spacer layer (4802). In this design, the top layer includes holes (4811) that extend through the top layer (4804) and expose sections of the peripheral portions (4807) to surrounding atmosphere.

[0132] A fluidic device may include two channels. It was surprisingly determined herein that two channel fluidic devices, wherein each channel includes at least one edge adjacent to an outer peripheral portion of a gas-permissive spacer layer, can limit hypoxia development in cellular samples. For example, FIG. 50 shows that 1.2xl06cell / mL biological samples exhibit similar hypoxia levels after 24 hour incubations in 2-channel fluidic devices and in fluidic devices with glass slits that facilitate oxygen diffusion into sample-containing channels.

[0133] An example of a 2-channel fluidic device is depicted in FIGS. 47A-D. These figures are illustrations of a bottom layer (4700, FIG. 47A), spacer layer (4702, FIG. 47B), top layer (4704, FIG. 47C), and cross-sectional view (FIG. 47D) of the fluidic device. The bottom layer (4700) and top layer (4704) may be wider than the spacer layer (4702). Such a design may allow the fluidic device to rest within the same holders or housings that are used for fluidic devices with greater numbers of channels (e.g., 4, 5, 6, 8, 12, etc.). Alternatively, the bottom layer (4700) and top layer (4704) may have identical or similar widths as the spacer layer (4702). The bottom layer (4700) and top layer (4704) may each consist of a singleAttorney Docket No. 59528-735601slide, or may be formed from multiple slides (depicted as 4700A, 4700B, 4700C for the bottom layer (4700) and 4704A, 4704B, 4704C for the top layer (4704)) that may collectively rest within a housing structure and / or holder. The spacer may include two first cut-out regions (4706) defined by peripheral portions (4707) of the spacer layer (4702). The first cut-out regions may be parallel and / or similar or identical in size. The top layer (4704) may include holes (4710) that open to the first cut-out regions (4706), such that when the bottom layer (4700) and top layer (4704) are coupled to the spacer layer (4702), the holes (4710) serve as inlets and / or outlets to channels defined by the first cut-out regions (4706) and the bottom and top layers (4700, 4704).

[0134] A method disclosed herein can include synthesizing one or more chambers that at least partially enclose the one or more cells in the first channel. In many embodiments disclosed herein, the one or more chambers are comprised of one or more polymer matrices formed with the fluidic space. As used herein, the term "Polymer matrix" generally refers to a phase material (e.g. continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the open interstitial space within the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also comprise non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term "polymer matrix" may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels comprising glass fibers. A polymer matrix may comprise a polymerized form of a polymer precursor, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon (i.e., light), magnetic energy, or any other suitable energy. As used herein, the term "polymer precursor" includes monomers (e.g., that are polymerized to produce a polymer matrix) and / or crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices (e.g., porogen).

[0135] In some embodiments, such chambers have annular-like cross-sections. As used herein, the term "annular-like cross-section" means a cross section topologically equivalent to an annulus. In some embodiments, the inner space, or interior, of a chamber has an inner diameter from 5 pm to 500 pm and a volume in the range of from about 1 nanoliter to 100 nanoliters, or from about 1 nanoliter to 10 nanoliters. In some embodiments, the polymer matrix wall has a thickness of at least 1 pm (micrometer). In some embodiments, the heightAttorney Docket No. 59528-735601of a chamber with an annular-like cross section have a value in the range of from 10 gm to 500 pm, or in the range of from 50 gm to 250 gm. In some embodiments, a polymer matrix wall having an annular-like cross-section has an aspect ratio (i.e., height / width) of 1 or less. In some embodiments, aspect ratio and polymer matrix wall thickness are selected to maximize chamber stability against forces, such as reagent flow through the channel, washings, and the like. A wall of the chamber may have a thickness from 1 pm to 100 pm, for example from 1 pm to 5 pm, 1 pm to 10 pm, 1 pm to 20 pm, 5 pm to 10 pm, 5 pm to 20 pm, or 10 pm to 20 pm.

[0136] In particular embodiments, the one or more chambers comprise a hydrogel. As used herein, the term “hydrogel” can refer to a polymeric material that is not water soluble or is poorly water soluble but can contain water (e.g., at least 10% by weight) when fully hydrated.

[0137] In many embodiments, the polymer precursor is light activatable. For example, synthesizing one or more chambers can comprise projecting light into the first channel with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers. As discussed in further detail below, a wide variety of photo-synthesizable gels and degradable gels are available for implementing the systems and methods described herein. Guidance for selecting such gels for desired properties including, but not limited to, biocompatibility, gelation speed, degradation speed, and like properties, is provided in the following references, which are incorporated by reference: Kharkar et al, Chem. Soc. Rev., 42: 7335-7372 (2013); Kharkar et al, Polymer Chem., 6(31): 5565-5574 (2015); Neumann et al, Acta Biomater., 39: 1-11 (2016); DeForest et al, Nature Chemistry, 3(12): 925-931 (2012);Bowman et al, U.S. patent 9631092; LeValley et al, ACS Appl. Bio. Mater., 3(10): 6944-6958 (2020); Kabb et al, ACS Appl. Mater. Interfaces, 10: 16793-16801 (2018); Fairbanks et al, Macromolecules, 44: 2444-2450 (2011); Fairbanks et al, Adv. Mater., 21(48): 5005-5010 (2009); Sugiura et al, U.S. patent publication US2016 / 0177030; Shih et al, Biomacromolecules, 13(7): 2003-2012 (2012); and the like. In some embodiments, photosynthesized gels are formed using a photo-initiator for radical polymerization. In some embodiments, photo-initiators comprise Irgacure 2959, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), or Eosin-Y (e.g. see Choi et al, Biotechniques, 66(1): 40-53 (2019)). In some embodiments, one or more polymer precursors comprise hyaluronic acid, chitosan, heparin, alginate, polyethylene glycol (PEG), multi-arm PEG, polyethylene glycol)-b-poly(propylene oxide)-b-poly(ethylene glycol) (PEG-PPO-PEG), poly(lactic acid-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic acid-co-glycolic acid) (PLGA-PEG-Attorney Docket No. 59528-735601PLGA), and poly(vinyl alcohol). In some embodiments, polymer precursors comprise PEG or multi-arm PEG. In some embodiments, the polymer precursors comprise an enzymatically degradable cross-linker. In some embodiments, such enzymatically degradable cross-linker is degradable by an esterase or a peptidase. In some embodiments, polymer precursors comprise a photo-degradable cross-linker. In some embodiments, such photo-degradable cross-linker comprises a nitrobenzyl group. In some embodiments, such photo-degradable cross-linker comprises a coumarin moiety.

[0138] FIGS. 2A-C illustrate an exemplary method for forming a chamber enclosing one or more biological components in a fluidic device disclosed herein. FIG. 2A shows a portion of a system as provided herein (e.g., comprising a fluidic device as disclosed herein) including an energy source. FIG. 2B shows a polymer matrix being formed around a biological component in a portion of a system as provided herein. FIG. 2C shows a method of forming a polymer matrix around a biological component in a system as provided herein. As illustrated in FIGS. 2A-2C, in some embodiments, the one or more chambers extend from the bottom layer to the top layer of a fluidic device.

[0139] With continued reference to FIG. 2A, the first channel 200 of the system may include a first surface 201 provided by the bottom layer and a second surface 202 provided by the top layer of the fluidic device. The energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise one or more non-emitting portions (e.g., a non-emitting portion 204). The nonemitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. For example, the energy source may comprise an LED array in which individual LEDs can be selectively activated (e.g., act as an energy emitting portion 205) to create light projections with specified patterns. In some embodiments, the fluidic channel may be coupled to or disposed on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the first fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the first fluidic channel. The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on atAttorney Docket No. 59528-735601least a portion of a surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0140] A channel of a fluidic device (e.g., the first channel) can comprise one or more polymer precursors for forming chambers. In some embodiments, the one or more polymer precursors are added to the fluidic device along with the one or more cells. In various embodiments, the one or more cells and the one or more polymer precursors can be premixed and then added to the fluidic device at the same time; the one or more cells and the one or more polymer precursors can be added to the fluidic device at the same time; the one or more cells can be added to the fluidic device first and then the one or more polymer precursors can be added to the fluidic device second; or the one or more polymer precursors can be added to the fluidic device first and then the one or more cells can be added to the fluidic device second. Such precursors may be selected from a wide variety of compounds including, but not limited to, 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-isopropyl acrylamide) (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, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol 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, PEGthiol / PEG-acrylate, acrylamide / N,N'-bis(acryloyl)cystamine (BACy), or PEG / PPO. In some embodiments, the following precursors and crosslinker may be used to form chambers with degradable polymer matrix (hydrogel) walls. Polymer precursors may be formed by using any hydrogel precursor and crosslinkers of Table 1 A (columns 1 and 3, respectively). The resulting polymer matrices may be degraded with the indicated degradation agents in Table 1 A (column 4).Representative crosslinkers useful for polymer synthesis are listed in Tables 1 A and IB.Attorney Docket No. 59528-735601Table 1ATable IBAttorney Docket No. 59528-735601<>>Attorney Docket No. 59528-735601"" >Attorney Docket No. 59528-735601<"&<< >Attorney Docket No. 59528-735601<&Attorney Docket No. 59528-735601<

[0141] A polymer precursor can further comprise additional reagents that affect polymerization and polymer matrix properties. As examples, a polymer precursor can include a crosslinker, a porogen, a viscosity-modifying agent, an acid, a base, a catalyst, a salt, a photoinitiator, or a combination thereof. As another example, polymer precursors can include a crosslinker, a porogen, a photoinitiator, and a buffer.Attorney Docket No. 59528-735601

[0142] As used herein, the term “crosslinker” denotes a species with two or more polymerizable groups. For example, where the polymerizable group is an ethylenically unsaturated group, a crosslinker would contain two or more ethylenically unsaturated groups. In another example, a crosslinker can contain three or more reactive centers for bifunctional polymer synthesis (e.g., the three methoxy groups of trimethoxybenzene in the context of polyester synthesis).

[0143] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix. A porogen can be dispersed with the reactants before the polymerization process of forming the polymer matrix. Porogens typically diffuse out of polymer matrices following polymerization, leaving pores in the regions that they occupied. Porogen size, concentration, hydrophobicity, and hydrophilicity can thus influence pore density and pore size in polymer matrices. Examples of porogens consistent with the present disclosure include particles (e.g., polymeric, ceramic, metal, metal oxide, or hydrogel particles), polymers such as polyethylene glycol and alginate, and vesicles such as liposomes or micelles.

[0144] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of photoinitiators utilizable in the present methods include acetophenone, anisoin, anthraquinone, anthraquinone-2-sulfonic acid, benzil, benzoin, benzophenone, 3,3’,4,4’-benzophenonetetracarboxylic dianydride, 4-benzoylbiphenyl, 2-benzyl-2-(dimethylamino)-4’-morpholinobutyrophenone, dibenzosuberenone, 2,2-diethoxyacetophenone, 2-ethylanthraquinone, ferrocene, 2-isopropylthioxanthone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, methyl-2-benzoylbenzoate, and thiooxanthen-9-one.

[0145] In some embodiments, the generation of a polymer matrix within said fluidic 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 at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In some embodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymerAttorney Docket No. 59528-735601matrix comprises between about 1 and 3 seconds of illumination, between about 1 and 5 seconds of illumination, between about 1 and 10 seconds of illumination, between about 1 and 15 seconds of illumination, between about 1 and 20 seconds of illumination, between about 1 and 30 seconds of illumination, between about 1 and 50 seconds of illumination, between about 2 and 4 seconds of illumination, between about 3 and 5 seconds of illumination, between about 3 and 10 seconds of illumination, between about 3 and 15 seconds of illumination, between about 3 and 20 seconds of illumination, between about 3 and 30 seconds of illumination, between about 3 and 50 seconds of illumination, between about 5 and 10 seconds of illumination, between about 5 and 15 seconds of illumination, between about 5 and 20 seconds of illumination, between about 5 and 30 seconds of illumination, between about 5 and 50 seconds of illumination, between about 10 and 20 seconds of illumination, between about 10 and 30 seconds of illumination, between about 10 and 50 seconds of illumination, between about 20 and 30 seconds of illumination, or between about 20 and 50 seconds of illumination. In some embodiments, the generation of a polymer matrix within said fluidic device is performed using a spatial light modulator (SLM) (i.e. a spatial energy modulation element that is capable of generating desired light intensity pattern spatially). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid crystal based.

[0146] Optionally, a first chamber of the one or more chambers can be disposed inside of a second chamber of the one or more chambers. This design can be utilized to separately partition two species (e.g., a cell and a reagent or two cells) within close proximity. This design can also be used to control the timing with which two species are contacted. For example, a method can include forming a first chamber around a cell, flowing a bead (or other assay reagent incapable of diffusing into the first chamber) adjacent to the cell, forming a second chamber surrounding the first chamber and enclosing the bead, and selectively degrading the first chamber to allow the cell and bead to come into contact within the second chamber.

[0147] In some embodiments, a functional group can be coupled to one or more chambers. Some non-limiting examples of functional group may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, an alkene, an alkyne, an azide, or a combination thereof. In some cases, the functionalized chamber may be used to capture aAttorney Docket No. 59528-735601biomolecule enclosed therein, thereby trapping the biomolecule in proximity to a biological component (e.g., a cell) enclosed within the chamber. The biomolecule may be produced by the biological component (e.g., secretome from a cell). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. The functionalized surface may increase surface area covered by a reagent, a molecular sensor, or any molecule of interest (e.g., an antibody).

[0148] With continued reference to FIG. 2A, the polymer matrix 208, 209, or at least a portion of the polymer matrix 208, 209, may be coupled to the first surface 201, the second surface 202, or both surfaces 201, 202. In certain embodiments, the polymer matrix, or at least a portion of the polymer matrix, may be coupled to a third surface, a fourth surface, a fifth surface, etc. as appropriate. In various embodiments, the polymer matrix 208, 209 may extend from the first surface 201 to the second surface 202 (e.g., through at least a portion of a lumen of the first channel 200 or a cavity of a chamber) such that the polymer matrix surrounds, or substantially surrounds, the biological component 50. In some embodiments, two or more biological components (e.g., biological components 50, 51 of FIG. 2C) that are in close physical proximity may be separated (e.g., by agitating or shaking the fluidic device). The fluidic device may be agitated or shaken by physical movement, use of a sonic pulse, changing a flow in the channel, or any other suitable method of agitation. A polymer matrix may then be formed that surrounds (or partially surrounds) the biological components that are separated. FIG. 2B shows polymer matrices 208, 209 formed surrounding the biological component 50 after being separated from the biological component 51. FIG. 2C shows a process, according to various embodiments, of separating the two biological components 50, 51, which are in close proximity. That is, by agitating or shaking the fluidic device the biological components 50, 51 can be separated. In some embodiments, separation of the biological components is achieved through fluidic pressure, flow pulsation, dielectrophoresis, optothermal flow, or some combination thereof. In some cases, separation of the biological components is achieved through acoustic vibration. FIG. 2C also shows a polymer matrix being formed to generate a compartment 222 surrounding the biological component 50 after the separation of the biological components 50, 51.

[0149] A fluidic device disclosed herein can include a detector that is configured to detect one or more locations of one or more biological components contained within a channel. In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In various embodiments, a mask may beAttorney Docket No. 59528-735601generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 203 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. The mask may inhibit or prevent the energy source 203 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. In some embodiments, the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the camera may be coupled to, connected to, or in communication with the energy source 203. For example, the camera (not shown) may be in electrical communication with the energy source 203. In some embodiments, the energy source 203 may comprise the camera. In various embodiments, the energy source 203 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0150] In some embodiments, one or more chambers has sufficiently large pores to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) therethrough. Simultaneously, one or more chambers can have sufficiently small pores to allow movement or transfer of a reagent and / or biological component (e.g., DNA, RNA, a protein, a cell, etc.). In some embodiments, the pores have a diameter from 5 nm to 100 nm. In some embodiments, the pores have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores have a diameter smaller than 5 nm.

[0151] In particular embodiments, pores of the one or more chambers are formulated to encapsulate sufficiently large genetic material, nucleic acids with greater than 300 base pairs, but to allow smaller materials, such as reverse transcriptases and 50 base pair nucleic acid primers to pass through the pores, thereby passing in and out of the hydrogel structures. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like. In some embodiments, the average diameter of pores of a chamber prevent passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater; or having a molecular weight of 50 kDa or greater; or having a molecular weight of 75 kDa or greater; or having a molecular weight of 100 kDa or greater;Attorney Docket No. 59528-735601or having a molecular weight of 150 kDa or greater. In some embodiments, DNA or RNA retained have lengths that are sequencable using conventional sequencing-by-synthesis techniques. For example, such DNA or RNA comprise at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the pores may have an average diameter from 5 nm to 100 nm.

[0152] The pore sizes of the one or more chambers may be modulated using a chemical reagent, or by applying heat, electrical field, light, or another suitable stimulus. In other words, a chamber may comprise a tunable property (e.g., the pore size). In some cases, one or more chambers comprises a thermoresponsive or temperature-responsive polymer. A thermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM)) may phase separate from a solution upon heating or upon cooling (e.g., polymer showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix may comprise polymer which may collapse at high temperature in order to, for example, control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that may be used to form hydrogel / polymer matrix with tunable properties may include Poly(N-vinyl caprolactam), Poly(N-ethyl oxazoline), Poly(methyl vinyl ether), Poly(acrylic acid- coacrylamide), or a combination thereof. A change in temperature may enlarge or contract average pore size in the polymer matrix to allow selected molecules, such as a nucleic acid molecule, a protein, or any biomolecule or molecule smaller than the adjusted pore size to be released from a hydrogel chamber.

[0153] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” “flow channel,” or “reaction chamber”) may receive or be configured to receive a biological sample. FIG. 1 shows a simplified schematic cross-sectional side view illustration of a portion of the first channel 100 of the fluidic device disclosed herein. The first channel 100 may comprise a first surface 101 and a second surface 102. In some embodiments, the first surface 101 and the second surface 102 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 1). For example, the first surface can be provided by the bottom layer and the second surface can be provided by the top layer that at least partially define the first channel. In some embodiments, a middle spacer layer of double-sided adhesive with a cut-out portion can be used to position the first surface 101 and second surface 102 in a facing relationship to at least partly form the flow channel. As a non-limiting example, the cut-out portion can have a dimension of about 0.7 cm width and about a 10 cm length. In some embodiments, the first surface and second surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout theAttorney Docket No. 59528-735601channel, for example, where chambers are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part on the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 101 may be a lower surface. In certain embodiments, the second surface 102 may be an upper surface. The first channel 100 may receive a biological sample comprising one or more biological components 50, 51. The first channel 100 may receive one or more polymer precursors. As illustrated in FIG. 1, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 101, the second surface 102, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 101 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the second surface, 102 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In some embodiments, the first surface and / or second surface can be optically transmissive so that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flow cell, and the measurement of the analyte and biological components.

[0154] In certain cases, a channel may have a cross-sectional area that is rectangular, circular, semi-circular, or oval. Accordingly, the channel may have a single, internal surface. In some cases, a channel may have a triangular, square, rectangular, polygonal, or other cross-section. Accordingly, the channel may have three or more internal surfaces. One or more of the internal surfaces may be couple or receive, or be configured to couple or receive, the one or more biological components.

[0155] The first surface 101, the second surface 102, or both surfaces 101, 102 may be functionalized, for example with a coating. As a non-limiting example, a surface coating mayAttorney Docket No. 59528-735601be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or more moi eties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep-tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer. In particular embodiments, the surface coating comprises a material for which adherent cells have a binding affinity, such as fibronectin or laminin.

[0156] In some cases, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein.. In some embodiments, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes between any of the two numbers mentioned herein. In some cases, a barcode (or a portion / subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). In particular, a barcode may comprise a sequence that is unique (“spatially-addressed”) to a spot or region along a surface of the fluidic device. Each spot may comprise a plurality of nucleic acid barcodes that share this “spatially-addressed” sequence. Accordingly, the spot or region of origin of a barcode may be determined based on its spatially-addressed sequence. A spot or region that contains barcodesAttorney Docket No. 59528-735601with common “spatially-addressed” sequences may comprise an area of about 10 to 105pm2along the first or second surface of the fluidic device. The first or second surface of the fluidic device may comprise about 50 to 500, about 50 to 1000, about 50 to 5000, about 50 to 104, about 50 to 5xl04, about 50 to 105, about 50 to 5xl05, about 50 to 106, about 50 to 5xl06, about 50 to 107, about 100 to 500, about 100 to 1000, about 100 to 5000, about 100 to 104, about 100 to 5xl04, about 100 to 105, about 100 to 5xl05, about 100 to 106, about 100 to 5xl06, about 100 to 107, about 500 to 1000, about 500 to 5000, about 500 to 104, about 500 to 5xl04, about 500 to 105, about 500 to 5xl05, about 500 to 106, about 500 to 5xl06, about 500 to 107, about 1000 to 5000, about 1000 to 104, about 1000 to 5xl04, about 1000 to 105, about 1000 to 5xl05, about 1000 to 106, about 1000 to 5xl06, about 1000 to 107, about 5000 to 104, about 5000 to 5xl04, about 5000 to 105, about 5000 to 5xl05, about 5000 to 106, about 5000 to 5xl06, about 5000 to 107, about 104to 5xl04, about 104to 105, about 104to 5xl05, about 104to 106, about 104to 5xl06, about 104to 107, about 5xl04to 105, about 5xl04to 5xl05, about 5xl04to 106, about 5xl04to 5xl06, about 5xl04to 107, about 105to 5xl05, about 105to 106, about 105to 5xl06, about 105to 107, about 5xl05to 106, about 5xl05to 5xl06, about 5xl05to 107, about 106to 5xl06, about 106to 107, or about 5xl06to 107spots or regions that contain barcodes with unique “spatially-addressed” sequences along one or more surfaces. Similarly, the fluidic device may comprise one or more fluidic channels, each of which may comprise about 50 to 107spots or regions that contain barcodes with unique “spatially-addressed” sequences along one or more surfaces.

[0157] A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0158] In some embodiments, the fluidic device may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA or RNA sequencing). In some embodiments,Attorney Docket No. 59528-735601the fluidic device may be a microfluidic device. In certain embodiments, the fluidic device may be a nanofluidic or a microfluidic device.

[0159] In some embodiments, a method disclosed herein may include, in a first channel of a fluidic system comprising a liquid medium, synthesizing one or more chambers that at least partially enclose one or more cells, and diffusing gas from a second channel of the fluidic system into the first channel through a spacer in contact with the first and second channels; incubating the one or more cells in the first channel. In certain aspects, synthesizing the one or more chambers comprises projecting light into the first channel with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to form polymer matrix walls of the one or more chambers.

[0160] The one or more cells and polymer precursors can optionally be mixed outside of the fluidic system before they are loaded into the first channel. In some embodiments, cells are delivered to the first channel, where they are dispersed over a surface of the first channel. For example, the one or more cells may be randomly disposed on thesurface. Alternatively, the one or more cells may be randomly disposed across or between structures or regions for which the one or more cells comprise a binding affinity. As an example, the one or more cells may localize to regions that comprise fibronectin or laminin, and partition away from portions of the first surface composed of glass.

[0161] In some embodiments, the one or more cells are adherent with respect to a surface of the first channel such that upon settling and contact the cells remain on the surface, even in the presence of fluid movement. In other embodiments, the one or more cells are nonadherent with respect to the surface such that although they may settle on the first surface they may move or return to a suspension in the presence of fluid movement. One of ordinary skill in the art will recognize that a first surface may be treated (for example, by the attachment of appropriate capture elements) to retain non-adherent cells, either over the entire first surface (or polymer matrix walls or second surface) or selected portions thereof.

[0162] In various embodiments, a method of incubating one or more cells can include loading a channel (e.g., (100) in FIG. 1) with the one or more cells and one or more polymer precursors. The one or more polymer precursors can be polymerized to form one or more chambers within the channel that at least partially enclose the one or more cells. For example, after the location of the one or more cells are identified with a detector, a spatial energy modulated element may project light into the first channel such that the projected light causes cross-linking of the one or more polymer precursors to form polymer matrix walls of the one or more chambers. Exemplary chamber designs include circular, elliptical, and polygonalAttorney Docket No. 59528-735601shapes that fully enclose inner spaces (e.g., do not include a discontinuity or break).Furthermore, in many aspects, the one or more chambers extend from the bottom layer (400) to the top layer (404) of a flow cell, such that species enclosed within a chamber of the one or more chambers is isolated or partially isolated from species that are extrinsic to the one or more chambers or enclosed within other chambers. As used herein, “partially isolated” can denote that a chamber prevents a subset of species (e.g., cells and large nucleic acids) from entering or leaving a chamber enclosing a species but allows other species (e.g., small molecule analytes) to enter and leave the chamber.

[0163] Further disclosed herein are assays that may be performed on cells and cellular components contained within a flow cell. Such a method can include: inputting one or more cells into a first channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel; incubating the one or more cells in a liquid in the first channel; diffusing a first gas from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel; and determining a characteristic of the one or more cells. Optionally, the method can include loading the first channel with an assay reagent (e.g., an assay reagent that enables the characteristic of the one or more cells to be determined). The method can include determining a single characteristic of the one or more cells or a plurality of characteristics of the one or more cells. When a plurality of cellular characteristics are determined, they may be determined simultaneously (e.g,. concurrent stains for surface proteins and secreted cytokines), sequentially (e.g., a stain for a surface protein followed by cell lysis and mRNA sequencing), or a combination thereof. In this way, two or more cellular characteristics (e.g., ability to adhere to a surface, proliferation rate, cytotoxicity, activation, transcriptome, surface protein expression, etc.) can be correlated.

[0164] “ Cells” that may be cultured and assayed by methods and systems described herein may comprise any biological cells including, but not limited to, vertebrate, non-vertebrate, eukaryotic, mammalian, microbial, protozoan, prokaryotic, bacterial, insect, or fungal cells. In some embodiments, mammalian cells are assayed by methods and systems described herein. In particular, any population of mammalian cells which may be, or have been, induced, treated, modified or genetically altered (i.e. genetically engineered) for use inAttorney Docket No. 59528-735601a medical, industrial, environmental, or remedial process, may be analyzed by methods and systems described herein. In some embodiments, “cells” as used herein comprise genetically modified mammalian cells. In some embodiments, “cells” comprise stem cells that have been induced to differentiate. In some embodiments, “cells” refer to cells modified by CRISPR Cas9 techniques. In some embodiments, “cells” refer 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 special interest are cytotoxic T lymphocytes engineered for therapeutic applications, such as cancer therapy. In particular aspects, the one or more cells comprise a cancer cell, an immune cell, a fibroblast, a neuron, or a combination thereof. In further aspects, a cell of the one or more cells proliferates during the incubation. In another aspect, a cell of the one or more cells does not proliferate during the incubation.

[0165] As used herein, the phrase “determining a characteristic of the one or more cells” and the term “assay” refer to processes for detecting or measuring a cellular characteristic or property of one or more cells. Typically process steps of an assay comprise a chemical, biochemical or molecular reaction (such as a cleavage of a bond, specific binding of complementary components, enzymatic reactions, dissolution of complementary components, or the like) or a change of physical state (such as an increase or decrease in temperature, change in energy level, or the like) and result in the generation of a signal (or signals) from which the presence, absence or magnitude of a quantity related to a cell may be inferred. The nature of the signal produced by an assay may vary widely and can include, but is not limited to, an electrical signal, an optical signal, a chemical signal, or a material signal. A material signal comprises the production of a material that comprises information that can be extracted. For example, a material signal may be the amplification of a polynucleotide whose length, quantity, composition, or nucleotide sequence is indicative of a cellular characteristic. For example, a barcode oligonucleotide may be a material signal. Characteristics or properties of cells that are detected or measured may vary widely and include, but are not limited to, cytotoxicity, viability, proliferation capacity under selected conditions, size, shape, motility, types and profiles of cell surface, or cell membrane proteins, types and profiles of secreted proteins, production of metabolites, transcriptome, gene copy numbers, gene or allele identity, chromatin accessibility profiles, vector copy numbers for engineered or infected cells, and the like. Examples of characteristics that can be measured in the systems and assays of the present disclosure include, but are not limited to, those disclosed in Table 2.Attorney Docket No. 59528-735601Table 2Attorney Docket No. 59528-735601

[0166] In particular embodiments, one or more cellular characteristics determined in a method is selected from cytotoxicity, proliferative capacity or proliferation rate, activation status, cellular identity, purity, gene expression profile, transcriptome, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), or a combination thereof. Additional assays may include culture contamination assays including, but not limited to, viral, bacterial, yeast, mold, or mycoplasma assays, endotoxin assays, and cellular morphology assays.

[0167] An assay can include one or more assay components, which may be provided with or as part of a channel include, but are not limited to, capture elements such as capture oligonucleotides, primers for captured nucleic acid amplification, antibodies, and detectable labels. In some embodiments, such assay components may be attached to any one of a first surface, a second surface or a polymer matrix wall exclusively, or on combinations of such surfaces, either exclusively, or in combination with other reagents. In some embodiments, assay components that may be provided after synthesizing gel chambers include, but are not limited to, lysing reagents, transcription reagents, reverse transcription reagents, antibodies, polymerases, primers, beads, and the like. In some embodiments, cellular or assay components may be attached or captured by capture elements on a polymer matrix wall.

[0168] In some embodiments, incubating under assay conditions may comprise only a single step of an assay comprising more than one step, such as, for example, a step of detecting a signal (or in the case of a material signal, generating a sequencing-ready nucleic acid), or such step of incubating under assay conditions may comprise a plurality of steps of a multi-step assay. In some embodiments, cells may be treated or subjected to assay steps prior to loading into a channel of a fluidic device, so that the step of incubating may comprise only a single step of a multi-step assay, such as signal generation and / or signal collection. In otherAttorney Docket No. 59528-735601embodiments, the step of incubating may comprise the implementation of an assay step or part of an assay, such as, cDNA synthesis, second strand synthesis, capture of an assay component or a cellular component, or the like. In some embodiments, assay conditions may comprise a series of steps each with different conditions (e.g. temperature, pH, presence or absence of particular reagents, such as, primers or an enzyme, e.g., a ligase, a polymerase, a transposase, or the like). For example, such steps may comprise loading primers so that they diffuse across chamber walls and anneal to target sites on a template strand in a sample in the chamber, extending the annealed primers, heating the channel so that the extended primers melt from its template strand, capturing the melted extended primers by capture agents attached to the first surface, and so on.

[0169] In some aspects, determining a characteristic of the one or more cells includes determining a proliferation rate. It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. For example, chambers enclosing 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 or retard the growth of the cells, e.g. in comparison to controls not exposed to the agent. Thus, in the case of the treated cells, a negative “proliferation rate” may be possible because the final numbers of cells counted in the chambers may be less than the original numbers; or a signal monotonically related to cell number may decline in value.

[0170] Proliferation rate can be determined by counting cells at least partially enclosed by the one or more chambers generated during an assay. For example, the one or more cells can be counted periodically (e.g., with fluorescence or brightfield imaging) following at least partial enclosure within the one or more chambers to determine a rate of change in the number of cells. Separate proliferation rates can be determined for each cell or collection of cells enclosed by a unique chamber or collection of chambers. For example, an assay can include separately enclosing between 10 and 50, between 10 and 100, between 10 and 500, between 10 and 1000, between 10 and 5,000, between 10 and 10,000, between 10 and 50,000, between 50 and 100, between 50 and 500, between 50 and 1,000, between 50 and 5,000, between 50 and 10,000, between 50 and 50,000, between 50 and 100,000, between 100 and 500, between 100 and 1,000, between 100 and 5,000, between 100 and 10,000, between 100 and 50,000, between 500 and 1,000, between 500 and 5,000, between 500 and 10,000, between 500 and 50,000, between 1,000 and 5,000, between 1,000 and 10,000, between 1,000 and 50,000, between 5,000 and 10,000, between 5,000 and 50,000, or between 10,000 and 50,000 cells in distinct chambers and individually counting the changes of the number of cells in eachAttorney Docket No. 59528-735601distinct chamber. Alternatively or in addition thereto, a proliferation assay may include coenclosing two or more cells within a single chamber or set of chambers and measuring their proliferation rate or rates. In some cases, the two or more cells include cells of different types and the counting includes separately counting the different cell types disposed within the single chamber or set of chambers.

[0171] In some embodiments comprising a proliferation assay, each chamber has the same shape and area, for example an annular-like shape with an interior area selected from the range of .001 to .01 mm2or in the range of .001 to 1.0 mm2. In some embodiments, each chamber has the same shape and area for each different type of cell being assayed, for example, cytotoxic T lymphocytes may be confined in chambers having one area whereas helper T lymphocytes may be confined in chambers having another area. After a desired number of chambers are synthesized, cells are incubated for a period under growth conditions, after which, for example, cells in each chamber are counted to give a measure of proliferative capacity for each cell. In some embodiments, after synthesizing chambers to enclose selected cells, non-selected cells may be removed by a washing step or by other changes of reagents in the channel. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. The desired number of single cells enclosed by chambers depends on statistical confidence desired in the measured values. If a subpopulation of interest is present as only a small fraction of a total population then a larger number chambers is required. In some embodiments in which mammalian cells are assayed the number of hydrogel chambers synthesized around single cells may be greater than 100; or greater than 1000; or greater than 10,000; or the number may be in the range of from 100 to 100,000; or in the range of from 1000 to 100,000. After counts are recorded for each chamber, further assays may be conducted on the clonal populations within the chambers to identify the cell types, for example, by an assessment of cell surface proteins, cell protein secretions, transcriptome, or the like. This approach is particularly useful for assessing populations of immune cells, especially engineered immune cells.

[0172] In some aspects, determining a characteristic of the one or more cells includes detecting a surface marker of the one or more cells. As used herein, the term “surface marker” denotes species that are expressed on the surface of a cell. Exemplary surface markers include surface proteins such as G protein-coupled receptors (GPCRs), ion channels,Attorney Docket No. 59528-735601engineered receptors (e.g., chimeric antigen receptors), and cluster of differentiation (CD) molecules (e.g., CD3, CD4, CD5, CD6, CD7, CD8, etc.), as well as carbohydrates (e.g., sialic acids), glycolipids, and the like. However, in particular aspects of the present disclosure, the surface markers are proteins.

[0173] Surface marker detection can include contacting the one or more cells with a binding agent configured to bind to the surface marker and detecting the binding agent, thereby detecting the surface marker of the one or more cells. A single surface marker or plurality of surface markers can be detected in a single assay. More precisely, the binding agent can include a single binding agent configured to bind to a single surface marker or a plurality of binding agents configured to bind to a plurality of surface markers. Multiple surface markers can be detected simultaneously, for example by contacting the one or more cells with multiple antibodies that bind different surface markers and are coupled to distinct detectable labels such as fluorophores that are simultaneously detectable on separate imaging channels. Alternatively or in addition thereto, two or more surface markers can be detected sequentially, for example by contacting a cell with a first antibody that binds to a first surface marker, detecting the first antibody, binding the cell with a second antibody that binds to a second surface marker, and detecting the second antibody. In particular cases, detecting a plurality of surface markers includes detecting relative expression levels of the plurality of surface markers.

[0174] Exemplary binding agents include antibodies; antibody fragments such as singlechain antibody molecules, scFvs, Fab domains, diabodies, nanobodies, minibodies, linear antibodies, and cross-Fab fragments; aptamers; and affimers. A binding agent may be coupled to a detectable label such as a fluorescent label, an oligonucleotide label, a colorimetric label, an enzymatic label (e.g., pyrophosphatase), or a combination thereof. It is noted that a binding agent such as an antibody can be coupled to a cell prior to or following the cell’s introduction into a channel and / or encapsulation within a chamber.

[0175] In some aspects, determining a characteristic of the one or more cells includes detecting a soluble factor secreted by the one or more cells. Soluble factors are typically dispersed or removed from a fluidic device by media refreshment, preventing soluble factor quantitation and cell-level analysis. The fluidic devices and methods of the present disclosure, which allow cells to be maintained under quiescent conditions for extended periods of time, facilitate soluble factors capture on or adjacent to individual cells over extended periods of time, enabling higher accuracy and sensitivity for soluble factor detection. Such a method can include disposing a capture surface comprising an affinity reagent that binds the soluble factorAttorney Docket No. 59528-735601adjacent to the one or more cells and detecting the soluble factor bound to the capture surface. Disposing the capture surface adjacent to the one or more cells can denote enclosing or at least partially enclosing the capture surface with the one or more cells within the one or more chambers, and optionally removing non-enclosed capture surfaces from the first channel of the fluidic system. The capture surface can be loaded into the first channel at a controlled density, for example 1 capture surface per about 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 mm2of the first channel.

[0176] In an exemplary embodiment, the capture surface comprises a bead. As used herein, the term “bead” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide-based 30 to 10000 pm particle. However, further capture surfaces, including nanotubes, nucleic acid nanostructures, and antibody Fc domains. The capture surface affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof. Under certain circumstances, due to the geometrical dimensions of the hydrogel chamber, there is a limit to how many beads can be captured within the chamber and at the same time allow room for the targeted cells for capture in the chambers. In addition, putting more than one type of antibody modified beads makes it even more challenging for packing antibody detection beads into the chamber. It is also worthwhile to note that the relatively expensive antibody modified beads that are not captured by the chambers and remain in an interstitial space in between chambers are often washed away and wasted.

[0177] Soluble factor detection may be performed with a bispecific binding agent capable of simultaneously binding to a cell and to a soluble factor secreted by the cell. The bispecific binding agent can be coupled to a target cell of interest and then used to capture soluble factors secreted by the cell. In this way, the bispecific binding agent may couple the soluble factor to the surface of the cell. The soluble factor may then be detected, for example by coupling a detectable binding agent such as a fluorescent antibody to the soluble factor coupled to the surface of the cell, and measuring the detectable binding agent. Unlike antibody modified beads that reside randomly within the chamber for capturing secretions that happen to diffuse to the beads, bispecific antibodies are coupled to the cell surface allowing for a more efficient capture of secretions emanated from the cell surface.

[0178] For example, a method for analyzing a cell can include inputting one or more cells into a fluidic device, trapping the one or more cells, wherein the trapped one or more cells are disposed within the fluidic device; inputting a bispecific binding agent into the fluidic device, the bispecific binding agent comprising: a first binding site configured to bind to a surfaceAttorney Docket No. 59528-735601marker on a surface of the one or more cells, and a second binding site configured to bind to a soluble factor secreted by the one or more cells; and detecting binding of the soluble factor to the bispecific binding agent, wherein the bispecific binding agent is bound to the surface of the cell during the detecting. Similarly, a method for analyzing a cell can include inputting one or more cells into a first channel of a fluidic device, inputting a polymer precursor into the first channel; synthesizing one or more chambers that at least partially enclose the one or more cells in the first channel, wherein the synthesizing comprises projecting light into the first channel with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers; inputting a bispecific binding agent into the first channel, the bispecific binding agent comprising: (i) a first binding site configured to bind to a surface marker on a surface of a cell of the one or more cells, and a second binding site configured to bind to a soluble factor secreted by the cell; and detecting binding of the soluble factor to the bispecific binding agent, wherein the bispecific binding agent is bound to the surface of the cell during the detecting.

[0179] An example of such a method is outlined in FIG.36A, which depicts a cell (1001) that comprises a first cell surface marker (1002) and a second cell surface marker (1003). The cell may be contacted with one or more bispecific binding agents (1004A, 1004B, 1004C) that selectively bind to the first cell surface marker (1002) and not to the second surface marker (1003). Similarly, the bispecific binding agents (1004A, 1004B, 1004C) may not bind to a third cell surface marker (1006) of an additional cell (1005) that is optionally present, for example co-enclosed with the cell (1001) in a chamber or disposed within a fluidic channel with the cell (1001). The bispecific binding agents (1004A, 1004B, 1004C) may comprise first binding sites that target the first cell surface marker (1002) of the cell (1001), but may comprise distinct second binding sites that target distinct soluble factors secreted by the cell (1001). In the example depicted in FIG. 36A, a first bispecific binding agent (1004A) targets the first cell surface marker (1002) and a first soluble factor (1011 A), a second bispecific binding agent (1004B) targets the first cell surface marker (1002) and a second soluble factor (101 IB), and a third bispecific binding agent (1004C) targets the first cell surface marker (1002) and a third soluble factor (1011C). Accordingly, when the cell (1001) secretes (1010) soluble factors (1011), the binding agents (1004A, 1004B, 1004C) may capture the first, second, and third soluble factors (1011 A, 101 IB, 1011C) on the surface of the cell (1001). In a subsequent detection step (1020), the soluble factors (1011A, 1011B, 1011 C) may be con-Attorney Docket No. 59528-735601tacted with detectable binding agents (1021 A, 1021B, 1021C) that enable detection and optionally quantitation of the soluble factors (1011 A, 101 IB, 1011C) on the surface of the cell (1001). The detectable binding agents (1021 A, 1021B, 1021C) may each comprise a unique dye, fluorophore, or other detectable species that allows separate detection and optional quantitation of the soluble factors (1011 A, 101 IB, 1011C). While not depicted in FIG. 36A, an additional binding agent may be coupled to the second surface marker (1003) of the cell (1001), for example to identify its cell type. In various embodiments, the detectable binding agent has a fluorescent detectable label and is inputted into a fluidic device and incubated under static flow conditions. The detectable binding agent can have a concentration range of about 10-1000 nM (e.g., about 10-100 nM concentration for real-time detection and about 100 nM - 1000 nM for one-time labeling). Initially, the fluorescent signal from the detectable binding agent in the flow cell and not bound to cells should be relatively small. Due to the relatively low concentration of inputted detectable binding agent and the relatively low height of the flow cell, the fluorescent intensity is relatively small. However, as the detectable binding agent couples to cells via the bispecific antibody, the fluorescent detectable label concentrates at the cell surface increasing the fluorescent intensity at that cell. Surprisingly, the measurement of the fluorescent intensity at various cells can be measured without washing out the initial input of the detectable binding agent making the protein secretion assay simple with one less wash step and allows for secretions to accumulate in the fluidic device without inputting additional detectable binding agent or using detectable binding agent at a higher concentration. Moreover, for the situation where the cells in the fluidic channel consume significant amounts of oxygen during the incubation step under quiescent conditions, the flow cell can be configured to have an adjacent channel for allowing oxygen diffusion through the spacer layer.

[0180] A similar method is depicted in FIG. 36B. This example depicts the cell (1001), cell surface markers (1002, 1003), optional additional cell (1005), bispecific binding agents (1004A, 1004B, 1004C), secretion step (1010), and soluble factors (1011 A, 101 IB, 1011C) of FIG. 36A. However, in this example, the soluble factors (1011A, 1011B, 1011C) coupled to the surface of the cell (1001) are contacted (1030) with binding agents (1031 A, 1032A, 1033 A) that contain oligonucleotide barcodes (103 IB, 1032B, 1033C) that may be detected and optionally quantified to determine whether the soluble factors (1011 A, 101 IB, 1011C) were secreted by the cell (1001) and to optionally quantify the amount of each soluble factor (1011A, 1011B, 1011C) was secreted by the cell.Attorney Docket No. 59528-735601

[0181] The bispecific agent may comprise a first binding site configured to bind to a surface marker on a surface of a cell of the one or more cells and a second binding site configured to bind to a soluble factor secreted by the cell. The first binding site may target a surface marker expressed by a particular type of cell, such that the bispecific binding agent is targeted to that cell type. For example, the first binding site may specifically bind to a surface marker associated with a specific type of cell, immune cell marker such as CD4, CD8, CCR7, CD45, CD45Ram C127, CD25, CD39, or a combination thereof.

[0182] In a further exemplary embodiment, the soluble factor comprises a cytokine such as interferon -y (IFN-y) and interferon-a (IFN-a), an interleukins such as interleukin- 1 (IL-1), in-terleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL-10), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin-21 (IL-21), or interleukin-23 (IL-23), a colony stimulating factor (CSFs) such as granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or a tumor necrosis factors (TNF) such as TNF-a or TNF-p. In another embodiment, the secreted factor comprises an effector molecule such as a granzyme.

[0183] A soluble factor bound to a capture surface (e.g., an affinity reagent of a capture surface) can be detected by contacting the soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. Multiple soluble factors can be detected in a single assay by providing a capture surface or plurality of capture surfaces that comprise a plurality of affinity reagents configured to bind the plurality of soluble factors, contacting the plurality of soluble factors bound to the capture surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of soluble factors, and detecting a plurality of labels coupled to the plurality of antibodies. In this way, 2, 3, 4, 5, 6, or more soluble factors can be detected in a single assay. For example, an assay may utilize 2, 3, 4, 5, 6, or more beads that each include a different affinity reagent configured to bind to a different soluble factor and a commensurate number of antibodies configured to bind to the soluble factors and optionally containing distinguishable detectable labels (e.g., different fluorophores or oligonucleotide barcodes). Alternatively or in addition thereto, a second soluble factor can be detected by removing the soluble factor from the first channel and detecting a second soluble factor secreted by the one or more cells subsequent to said removing the soluble factor from the first channel.

[0184] In some aspects, determining a characteristic of the one or more cells includes sequencing at least a portion of a transcriptome of the one or more cells. Such a method can include projecting light into the first channel with a spatial energy modulating element suchAttorney Docket No. 59528-735601that the projected light causes cross-linking of one or more polymer precursors to at least partially form one or more chambers at least partially enclosing the one or more cells in a region of the first channel comprising one or more capture elements for capturing messenger RNAs from the one or more cells enclosed therein. The one or more capture elements can be coupled to a surface of the first channel, such as a top surface of the bottom layer or a bottom surface of the top layer. The first channel may be loaded with a lysing reagent so that messenger RNAs of the one or more cells are released and captured by the capture elements, and with reverse transcription reagents to copy the captured oligonucleotide labels to produce complementary DNAs thereof. The complementary DNAs may then be released from the first channel (e.g., cleaved from a surface of the top or bottom layer) and sequenced. It is understood that a sequencing step may comprise additional steps in particular embodiments including, but not limited to, tagmentation, adding adaptors, cleaving the cDNA to form appropriate lengths for sequencing, and the like. In some embodiments, an additional step may be implemented for depolymerizing or degrading the polymer matrix walls of the chambers after mRNA capture. Reverse transcription reagents comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murine leukemia virus (MMLV)), dNTPs, optional RNase inhibitor, buffer. The sequencing step may be carried out at the sites of the captured mRNAs (in situ) or cDNAs may include a spatial barcode and be eluted and sequenced on a separate sequencing instrument (“external” sequencing). For in situ sequencing, further steps may include (i) amplifying the complementary DNAs, e.g. by bridge amplification, or like method, (ii) sequencing the amplified complementary DNAs, e.g. by a sequencing-by-synthesis technique, and (iii) determining relative expression of the mRNAs for the cells of each of the chambers. For external sequencing, further steps may include (i) providing capture elements comprising spatial barcodes, (ii) synthesizing cDNAs comprising spatial barcodes, and (iii) eluting and sequencing the cDNAs and correlating each cDNA with a chamber location by its spatial barcode.

[0185] In a further aspect, the one or more cells comprises an effector cell, and determining the characteristic of the one or more cells comprises measuring a cytotoxicity of the effector cell. Such a method may be performed by at least partially enclosing one or more target cells with the effector cell, and counting dead cells, viable cells, or a combination thereof from among the one or more target cells. This method is particularly suitable to the quiescent methods of the present disclosure, as effector cell killing is often mediated by a combination of intercellular interactions (e.g., CD8 T Cell FasL binding to target cell Fas) and secretionsAttorney Docket No. 59528-735601that can be disrupted by media replacement. Accordingly, in many aspects disclosed herein, the effector cell and the one or more target cells are viable at O2 levels in the liquid first channel. As non-limiting examples, the target cells may comprise a sample of tumor cells of a patient, or target cells may be from a cell line, e.g. tumor cell line, such as, hepatic tumor cell line, SK-HEP-1, Chava et al, J. Vis. Exp., 2020 Feb 22: (156): 10.3791 / 60714. Examples of effector cells include Tel cells, Tc2 cells, Tc9 cells, Tcl7 cells, Tc22 cells, natural killer cells. In some embodiments, the effector cell is engineered for a therapeutic purpose. For example, the effector cell may express a chimeric antigen receptor that confers cytotoxicity against a particular cancer.

[0186] A cytotoxicity assay may include loading the effector cell and target cells into the first channel (either simultaneously or sequentially). In some aspects, the target cells are loaded into channel where they optionally adhere to a surface prior to effector cell loading. In other aspects, the effector cell is loaded into the channel prior to target cell loading. The effector cell may be at least partially enclosed with the target cells inside one or more chambers. The target cells and effector cells are then incubated. The incubation may be performed in the presence of a vital dye that generates an optical signal in response to a characteristic of viable cells or dead cells to facilitate dead cell enumeration. Alternatively or in addition thereto, the target cells may be pre-treated with a dye that internalizes into the target cells prior to loading into the first channel. In addition to target cell killing, effector cells can be identified based on other cellular characteristics disclosed herein, such as their secretome, transcriptome, or the like.

[0187] In some aspects, the characteristic of the one or more cells comprises activation. Cellular activation can be measured by detected using numerous assays disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation, morphology, or a combination thereof. As non-limiting examples, these methods are broadly amenable to detecting activation caused by contact between a second cell and a cell of the one or more cell, a soluble factor secreted by the second cell and the cell of the one or more cells, and combinations thereof. Accordingly, the efficiency with which the activation can be enhanced by the quiescent conditions enabled by the present methods, which limit soluble factor removal and disruptions of intercellular interactions. In a particular aspect of the present disclosure, determining activation comprises detecting a surface marker of the one or more cells. For example, the method can comprise contacting the one or more cells with a binding agent configured to bind to the surface marker and detecting the binding agent.Attorney Docket No. 59528-735601

[0188] In many activation assays, the second cell is at least partially enclosed with the cell of the one or more cells by a chamber of the one or more chambers. However, in some aspects, the cell of the one or more cells and the second cell are enclosed by different chambers of the one or more chambers (e.g., adjacent chambers in sufficient proximity for secreted proteins from the second cell to reach the cell of the one or more cells); or the cell of the one or more cells is at least partially enclosed by a chamber of the one or more chambers and the second cell is not enclosed by a chamber of the one or more chambers. Alternatively, the second cell can be enclosed within a chamber while the cell of the one or more cells is not enclosed within a chamber. In each of the above chamber schemes, the second cell and the cell of the one or more cells can be within 5, 10, 20, 40, 60, 80, or 100 pm within the first channel.

[0189] FIG. 5A is an example system for carrying out the above method. Flow cell (500) is a component of a fluidic device that provides channels for carrying out a variety of 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. However, as detailed elsewhere herein, systems of the present disclosure can utilize flow cells with fewer or greater numbers of channels.

[0190] The system of FIG. 5A includes an optical system (521) for photosynthesizing chambers at locations of cells or other analytes in the channels (502, 504, 506, and 508) of the flow cell (500) and for collecting images and other optical signals. The optical system (521) includes a light source (522) that generates a light beam (523) of appropriate wavelength light (e.g. UV light) for synthesizing chambers (e.g., hydrogel chambers) in the flow cell (500). The light beam (523) that passes through an appropriate photo-mask or beam-shaping or beam steering (Galvo) system (524) for shaping a beam to synthesize a desired structure or structures in a channel. In some embodiments, this beam shaping system (524) includes a digital micromirror device (DMD). In other embodiments, a physical photomask may be employed. Reflected light from DMD (524) is shaped using conventional optics, e.g. collimating optics (528), and is directed through objective lens system (534) into channel 2 segment (510). In exemplary embodiments, the light is directed by one or more dichroic mirrors (530 and 531).

[0191] Chamber position, shape and polymer matrix wall thickness is determined at least in part from cell position information determined from images collected by detector (532). Objective (534) and flow cell (500) move relative to one another in the xy-directions (536) to photosynthesize chambers at any position in any of the channels. In some embodiments, the flow cell (500) moves and optical system (521) is stationary. The system may utilize lightAttorney Docket No. 59528-735601from a light source (599), such as a homogenized light condenser, that is positioned on an opposite side of the flow cell as the optical system and directs light through the flow cell (500) to the objective (534). To achieve this functionality, the light source positioned on the opposite side of the flow cell (599) can be configured to move in tandem with the optical system (521), or the light source (599) and optical system can be stationary and the flow cell (500) can be moved to the region illuminated by the light source (599) and from which light is collected by the objective (534). In some embodiments, objective (534) may also direct light beam (527) from light source (529) to targets, such as cells, on first surface (514) and collect optical signals, such as fluorescent signals, from assays taking place on first surface (514). Optical signal collection can also be carried out with a separate objective.Information collected by detector (532), particularly cellular positions in their respective channels, is employed by computer (538) and / or subsidiary controllers to direct DMD (524) and translation devices controlling the relative positions of objective (534) and flow cell (500) to synthesize hydrogel chambers of the appropriate shape and size at the appropriate locations.

[0192] FIG. 5B provides a blown-up view of the exemplary channel segment (510) of the flow cell of FIG. 5A. On 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 polymer matrix walls of the hydrogel chambers is selected to be impermeable to the cells, but permeable to assay reagents. Thus, reagents may be introduced to, and removed from, the interiors of the hydrogel chambers by flowing (520) them through the channels, but cells (518) are retained in hydrogel chamber (516).

[0193] One of ordinary skill in the art would recognize that optical systems with different configurations than those of FIG. 5A and 5B may be employed for carrying out these functions. In some embodiments, a plurality of DMD-objective subsystems for synthesizing hydrogel structures may be employed to increase the speed of synthesis by synthesizing multiple structures simultaneously.Computer Systems

[0194] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 6 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameterAttorney Docket No. 59528-735601using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0195] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0196] The CPU 1505 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 1510. The instructions can be directed to the CPU 1505, which can subsequently program or otherwise configure the CPU 1505 to implement methods of the present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0197] The CPU 1505 can be part of a circuit, such as an integrated circuit. One or more other components of the system 1501 can be included in the circuit. In some cases, the circuit may be an application specific integrated circuit (ASIC).Attorney Docket No. 59528-735601

[0198] The storage unit 1515 can store files, such as drivers, libraries, and saved programs. The storage unit 1515 can store user data, e.g., user preferences and user programs. The computer system 1501 in some cases can include one or more additional data storage units that are external to the computer system 1501, such as located on a remote server that may be in communication with the computer system 1501 through an intranet or the Internet.

[0199] The computer system 1501 can communicate with one or more remote computer systems through the network 1530. For instance, the computer system 1501 can communicate with a remote computer system of a user (e.g., a laptop, a personal computer, a tablet, or a mobile phone). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 1501 via the network 1530.

[0200] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software.During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0201] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0202] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the softwareAttorney Docket No. 59528-735601may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0203] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0204] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter.Attorney Docket No. 59528-735601Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0205] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 1505. The algorithm can, for example, identify a biological component, detect a barcode, generate a spatial modulating element (e.g., a mask), provide energy from an energy source, detect or measure a local parameter using a sensor, etc.

[0206] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.EXAMPLES EXAMPLE 1Cell Growth And Viability In Flow Cell Channels

[0207] This example covers cell growth and viability in flow cell channels exposed to varying oxygen levels. The cells were incubated in a flow cell with eight lanes (“lanes 1-8” from left-to-right) cut into a spacer, such that the eight lanes were separated by gas-permeable spacers, and the outer edges of the outer lanes (“lanes 1 and 8”) were enclosed by the spacer. The spacer was a pressure sensitive dual-sided adhesive that contained a top silicone adhesive layer, a middle clear polypropylene carrier layer, and a lower silicone adhesive layer. Cells were incubated in lanes 1, 2, 3, 4, 6, and 8, while lanes 5 and 7 were evacuated and left open to the surrounding atmosphere. Accordingly, two lanes utilized for incubation (“lanes 6 and 8”) included two edges adjacent to ambient atmosphere, two lanes utilized for incubation (“lanes 1 and 4”) included a single edge adjacent to ambient atmosphere, and two lanes (“lanes 2 and 3”) were enclosed by other lanes filled with media.

[0208] As a first step, each lane of the eight-lane flow cell was coated with fibronectin by flowing 25 pg / mL fibronectin in phosphate-buffered saline through the eight lanes of the flow cell for one hour at room temperature. The lanes were then washed with media. Immortalized mouse dendritic cells (MutuDC1940, ABM Cat. No. T0528) were dissociated with nonenzyme dissociation buffer and adjusted to 3xl06cell / mL densities. 100 pL of the cells were flowed into lanes 1, 2, 3, 4, 6, and 8 of the flow cell. Lanes 5 and 7 were evacuated and leftAttorney Docket No. 59528-735601open to the surrounding atmosphere. The cells were incubated for 18 hours in quiescent media and then imaged at lOx magnification.

[0209] Representative images from lanes 1, 2, 3, 4, 6, and 8 are shown in FIGS. 7-12, respectively. Within each figure, labels 701 indicate spacer lane boundaries and labels 702 indicate exemplary cells. FIGS. 7A, 8A, 9A, 10A, 11A, and 12A are images of left-edges of lanes 1, 2, 3, 4, 6, and 8, respectively (i.e., where lane 1 is adjacent to an outer edge of the flow cell, lane 2 is adjacent to lane 1, etc.). FIGS. 7B, 8B, 9B, 10B, 11B, and 12B are images of middle portions of lanes 1, 2, 3, 4, 6, and 8, respectively. FIGS. 7C, 8C, 9C, 10C, 11C, and 12C are images of right-edges of lanes 1, 2, 3, 4, 6, and 8, respectively (i.e., where lane 1 is adjacent to an outer edge of the flow cell, lane 2 is adjacent to lane 3, etc.). Bubbles are visible in FIGS. 10C, 11A, 11C, 12A, and 12C.

[0210] As can be seen from these figures, the dendritic cells spread well across the full widths of lanes 6 and 8 (FIGS. 11A-C and FIGS. 12A-C), indicating that these cells were exposed to sufficient oxygen levels to support their viability. Cells at the left boundary of lane 1 (adjacent to the outer edge of the flow cell, FIG 7A) and right boundary of lane 4 (FIG. 10C, adjacent to gas-filled lane 5) exhibited normal spreading. However, cells disposed in other portions of these lanes maintained small, circular morphologies, indicating low oxygen levels away from their atmosphere-exposed boundaries. Cells exhibited uniform small, circular morphologies across the widths of lanes 2 and 3, indicating low oxygen levels throughout the full volumes of these channels (FIGS. 8A-C and 9A-C).EXAMPLE 2Hypoxia Measurements On Dendritic Cells In Flow Cell Channels

[0211] This example covers hypoxia development and cell responses in flow cell channels with varying atmosphere-exposed surface areas. An eight-lane flow cell was prepared and seeded with dendritic cells according to Example 1. Prior to loading into the flow cell, the cells were exposed to Image-iT™ Green Hypoxia Reagent, which is a dye that internalizes within cells and becomes fluorescent after the cells are exposed to low oxygen levels. Cells were incubated in lanes 1, 2, 3, 4, 6, and 8, while lanes 5 and 7 were evacuated and left open to the surrounding atmosphere such that two lanes utilized for incubation (“lanes 6 and 8”) included two edges adjacent to ambient atmosphere, two lanes utilized for incubation (“lanes 1 and 4”) included a single edge adjacent to ambient atmosphere, and two lanes (“lanes 2 and 3”) were enclosed by other lanes filled with media. The cells were incubated for 18 hoursAttorney Docket No. 59528-735601under quiescent conditions and then analyzed with brightfield imaging and fluorescence imaging for Green Hypoxia Reagent.

[0212] FIGS. 13A-B are brightfield and Green Hypoxia Reagent fluorescence images, respectively, of lane 1 following 18 hours of cell incubation. Each figure includes 6 images spanning from the outer edge of the leftmost flow cell lane (leftmost image) to the boundary of this flow cell lane with the second flow cell lane (rightmost image). Labels 701 indicate spacer lane boundaries and labels 702 indicate exemplary cells within lane 1. These images show a progression from a relatively high oxygen level at the outer edge of the flow cell (leftmost image) to a relatively lower oxygen level at the boundary with lane 2 (rightmost image) that indicated hypoxia. Following this spatial progression, in FIG. 13A, the cells exhibit a transition from spread-out morphologies to smaller, circular morphologies, while in FIG. 13B, Green Hypoxia Reagent fluorescence increases approaching the lane 2 boundary.

[0213] FIGS. 14A-F are representative brightfield images of cells in lanes 1-4, 6, and 8, respectively. Exemplary cells are indicated with the label 702 in each image. As can be seen from these figures, the cells in lanes 1, 4, 6, and 8 were large and exhibited spread-out morphologies, indicating normoxic conditions in these lanes. The cells in lanes 2 and 3 were comparatively smaller and more circular than the cells in lanes 1, 4, 6, and 8. FIG. 15 is a representative Green Hypoxia Reagent fluorescence image of cells in lane 2 of the flow cell in which an exemplary cell is indicated with the label 702. Numerous cells exhibited fluorescence intensity, small size, and circular morphologies. Collectively, these results indicate that lanes 2 and 3 included low oxygen levels.EXAMPLE 3O2 Depletion In Flow Cell Cultures

[0214] This example covers hypoxia responses in cells in flow cell channels with different levels of atmospheric gas exchange. An eight-lane flow cell was prepared and seeded with Green Hypoxia Reagent-exposed dendritic cells according to Example 1. Cells were incubated in lanes 1-3 and lanes 4-8 were left open to the surrounding atmosphere. The cells were incubated for 8 or 20 hours in quiescent media, during which time the cells were periodically analyzed with brightfield imaging and fluorescence imaging for Green Hypoxia Reagent.

[0215] FIGS. 16-17 are images of cells in lane 2, wherein exemplary cells are indicated with the label 702. FIGS. 16A-B are time course series of brightfield (FIG. 16A) and fluorescence (FIG. 16B) images of the cells over the first 8 hours of incubation following seeding. FIGS.Attorney Docket No. 59528-73560117A-B are time course series of brightfield (FIG. 17A) and Green Hypoxia Reagent fluorescence (FIG. 17B) images of the cells over the first 20 hours of incubation following seeding. FIGS. 18-19 are images of cells in a portion of lane 3 that was close to the edge of lane 2. Label 702 indicates exemplary cells in these figures. FIGS. 18A-B are time course series of brightfield (FIG. 18A) and Green Hypoxia Reagent fluorescence (FIG. 18B) images of the cells over the first 8 hours of incubation following seeding. FIGS. 19A-B are time course series of brightfield (FIG. 19A) and Green Hypoxia Reagent fluorescence (FIG. 19B) images of the cells over the first 20 hours of incubation following seeding. The dendritic cells in lanes 2 and 3 began exhibiting hypoxic responses about 8 hours after seeding. The cells appeared to become unhealthy after hypoxia developed, exhibiting poor adherence and spread. The similar rates at which hypoxia developed in lane 2 and at the inner-edge of lane 3 (adjacent to lane 2) suggest that limited amounts of oxygen diffused from the outer edge (adjacent to lane 4) to the inner edge (adjacent to lane 2) of lane 3, a distance of 7 mm.EXAMPLE 4Dendritic Cell Cultures In Air-Adjacent Flow Cell Lanes

[0216] An eight-lane flow cell was prepared and seeded with dendritic cells according to Example 1. Cells were incubated in alternating lanes (1, 3, 5, and 7), and the remaining lanes (2, 4, 6, and 8) were left open to the surrounding atmosphere. The cells were incubated overnight in quiescent media and were then analyzed with brightfield imaging. Results of these analyses, taken after the overnight incubations, are shown in FIG. 20, which is a set of images of cells (of which an exemplary cell is indicated by label 702) in lanes 1, 3, 5, and 7 following overnight incubation. The bottom images in this figure are zoomed-in views portions of the top images. The cells exhibited spread and adherence indicative of suitable oxygen levels in each lane for maintaining cell viability after an overnight incubation.EXAMPLE 5Leukemic Cell Cultures In Cell Flow Lanes With Variable Atmospheric Exposure

[0217] This example covers the response and morphological development of leukaemic cells in quiescent wells and flow cells with varying degrees of atmospheric exposure. For these analyses, K562 and Jurkat cells were incubated in three distinct environments and then assessed for health and responses to hypoxia. Prior to loading, the K562 and Jurkat cells were stained with Green Hypoxia Reagent for 30 minutes and washed with media. First and second portions of the Jurkat and K562 cells were loaded at 106cell / mL densities into lanes 1-2 andAttorney Docket No. 59528-735601lanes 3-4, respectively, of two four-lane flow cells. It is worthwhile to note that lane 1 has one side of the channel that is adjacent to a single edge portion of the spacer exposed to ambient atmosphere and lane 4 has one side of the channel that is adjacent to another single edge portion of the spacer exposed to ambient atmosphere. In addition, lanes 2 and 3 each have both sides of the channel flanked by other channels filled with quiescent liquid. In the second of these flow cells, sets of the K562 and Jurkat cells were then enclosed within cylindrical hydrogel cages. Additional 200 pL portions of the K562 and Jurkat cells were loaded into wells of a 96 well plate at 106cell / mL densities. The cells were incubated for 1 or 5 days and then analyzed with brightfield imaging and fluorescence imaging for Green Hypoxia Reagent, BB2, CYAN 100, and CYAN 500 dyes.

[0218] Brightfield and Green Hypoxia Reagent fluorescence images of K562 cells following 24 hours of incubation in the wells are shown in FIGS. 21A-B, respectively. The morphologies of the cells and positive Green Hypoxia Reagent fluorescence signals indicate hypoxia in the K562 wells. Conversely, brightfield and Green Hypoxia Reagent fluorescence images of Jurkat cells following 24 hours of incubation in the wells, which are shown in FIGS. 22A-B, respectively, indicate a lack of hypoxia following the 24-hour incubations in wells.

[0219] FIGS. 23A-B are brightfield and Green Hypoxia Reagent fluorescence images, respectively, of K562 cells in lane 4 of the flow cell with cylindrical hydrogel cages following 24-hour incubations. These images evidence the development of weak hypoxia in hydrogel cages with high densities of cells. Mirroring the results from the well incubations, the Jurkat cells did not exhibit hypoxia responses following 24-hour culturing. Exemplary brightfield and Green Hypoxia Reagent fluorescence images of these Jurkat cells in lane 2 are shown in FIGS. 24A-B, respectively.

[0220] FIGS. 25A-D are brightfield images of the Jurkat cells in lanes 1 and 2 and K562 cells in lanes 3 and 4, respectively, following 5 days of incubation without media replacement. Within each image, exemplary cells are indicated with the label 702 and exemplary hydrogel cages are indicated with label 703. These images indicate that lanes 1 and 4 contained higher oxygen levels than lanes 2 and 3, as cells proliferated in lanes 1 and 4 and exhibited low viabilities in lanes 2 and 3.Attorney Docket No. 59528-735601EXAMPLE 6Ch-Depletion in Flow Cell Channels with Varying Cell Densities

[0221] This example covers O2 depletion in flow cells containing varying cell densities. K562 cells were stained with 2.5 pM Hypoxia Green Reagent for 30 minutes. The cells were then prepared at 2.5xl06and 106cell / mL densities. The 2.5xl06cell / mL K562 cell preparation was loaded into lanes 1 and 2 of a four-lane flow cell. The 106cell / mL K562 cell preparation was loaded into lanes 3 and 4 of the four-lane flow cell. cell / mL. The cells were incubated in quiescent media in the flow cells and imaged at various time points. It is worthwhile to note that lane 1 has one side of the channel that is adjacent to a single edge portion of the spacer exposed to ambient atmosphere and lane 4 has one side of the channel that is adjacent to another single edge portion of the spacer exposed to ambient atmosphere. In addition, lanes 2 and 3 each have both sides of the channel flanked by other channels filled with quiescent liquid.

[0222] FIGS. 26-29 are brightfield (FIGS. 26A, 27A, 28A, 29A) and Hypoxia Green Reagent fluorescence (FIGS. 26B, 27B, 28B, 29B) images of cells in lanes 1, 3, 2, and 3, respectively. FIGS. 27 and 29 are images of different fields within lane 3 of the flow cell. Each figure contains images of the cells taken at 0, 16, and 40 hours (from left to right in each figure). Exemplary cells are indicated with the label 702. Hypoxia was detected at 16 hours in lanes 1 and 2 (which contained 2.5xl06cells / mL) and at 40 hours in lane 3 (which contained 106cells / mL).

[0223] Additionally, inner lanes not adjacent to a gas-filled lane exhibited greater degrees of hypoxia than outer lanes exposed to a gas-filled lane or outer edge of the flow cell. FIGS. 30A-B are Hypoxia Green Reagent fluorescence images of lanes 1 and 2 at 40 hours, respectively. FIGS. 31A-B are Hypoxia Green Reagent fluorescence images of lanes 3 and 4 at 40 hours, respectively. The inner lane (lane 2 for 2.5xl06cells / mL as shown in FIG. 30B and lane 3 for 106cells / mL shown in FIG. 31 A) exhibited greater Hypoxia Green Reagent fluorescence intensity than lanes 1 and 4, indicating higher degrees of hypoxia in lanes 2 and 3.EXAMPLE 7Effect of Media Replacement On O2 Levels In Fluidic Channel

[0224] This example covers the effect of refreshing media on oxygen levels in flow cells. Dendritic cells were loaded into lanes 2-4 and 6-8 of an eight-lane flow cell. Lanes 1 and 5 ofAttorney Docket No. 59528-735601the flow cell were left empty and open to the surrounding atmosphere. The cells were then incubated in the flow cell at about 25°C for 14 hours. During these incubations, lanes 2-4 were refreshed with new media every 6 hours. No media exchange was performed in lanes 6-8. After 14 hours, the cells were analyzed with Hypoxia Green Reagent fluorescence imaging and brightfield imaging.

[0225] The results of these analyses are shown in FIG. 32. The six columns of images in this figure correspond (from left to right) to lanes 2-4 and 6-8, respectively. The top row of images are Hypoxia Green Reagent fluorescence of the cells, while the bottom row of images are brightfield images. Lanes 2-4, which were perfused with new media every 6 hours, did not exhibit hypoxia following 14 hours of incubation. The cells in lanes 6-8 were smaller and less spread than the cells in lanes 2-4, indicating hypoxic conditions in these lanes. Among these three lanes, cells in lane 7 exhibited the highest Green Hypoxia Reagent fluorescence intensities, suggesting that lane 7 contained lower oxygen levels than lanes 6 or 8.

[0226] The assay was then repeated in a new flow cell. Fluorescence and brightfield images were collected upon cell loading and then at 0, 6, 12, and 16 hours. FIGS. 33-34 are sets of Green Hypoxia Reagent fluorescence images of the flow cell lanes at 12 hours (FIG. 33) and 16 hours (FIG. 34). Mirroring the results of the first assay, lanes 6-8 (in which media was not refreshed) exhibited greater degrees of hypoxia than lanes 2-4 (in which media was refreshed every 6 hours), with lane 7 displaying the highest fluorescence intensity. Green Hypoxia Reagent fluorescence (FIG. 35A) and brightfield (FIG. 35B) images taken at 0, 6, 12, and 16 hours in lane 7 indicated that hypoxia developed in this lane after about 6 hours.EXAMPLE 8CD71 and IFN-y Co-Staining with a Mixture of Monospecific and Bispecific Binding Agents

[0227] This example covers simultaneous measurement of a surface marker and a soluble factor expressed by a chamber-enclosed cell. Natural killer (NK) cells were enclosed within hydrogel chambers in a fluidic device channel, cultured for 24 hours, and then co-stained for IFN-y and CD71.

[0228] Prior to loading into the fluidic device channel, the NK cells were centrifuged at 1300 rpm for 3 minutes and washed with cold PBS. The cells were mixed with bispecific binding agents (from Miltenyi Biotec, IFN-y Secretion Assay - Detection Kit (FITC), human, Cat No.130-090-433) that included an anti-IFN-y monoclonal antibody conjugated to cell surface specific CD45 monoclonal antibody and subjected to two additional 3 -minute, 1300 rpmAttorney Docket No. 59528-735601centrifuge cycles. The cells were then loaded into a fluidic device with a polymer precursor that was photopolymerized to form hydrogel chambers enclosing the cells. Abrightfield image of the cells immediately following chamber synthesis is shown in FIG. 37A, with examples of a cell and cage indicated by labels 3701 and 3702, respectively. The cells were incubated for 24 hours in the presence of phorbol myristate acetate (PMA) and imaged. As shown in FIG. 37B, which is a brightfield image of the same fluidic device region shown in FIG. 37A, the cells were viable in the presence of PMA and the CD45-targeted bispecific binding agent.

[0229] The cells were then contacted with fluorescently labeled IFN-y and CD71 antibodies. The CD71 antibody bound to CD71 on the surfaces of the NK cells, while the IFN-y antibodies bound to IFN-y antigen coupled to the bispecific binding agent that was also coupled to CD45 antigen on the NK cell surfaces. Fluorescence images with the IFN-y fluorophore and CD71 fluorophore are shown in FIGS. 37C and 37D, respectively. These results showed that co-staining for CD71 and IFN-y did not result in crosstalk between CD71 and IFN-y fluorescence images, as some of cells strongly fluoresced with the PE fluorophore of the CD71 antibody but did not exhibit any fluorescent intensity from the FITC fluorophore of the IFN-y antibody.EXAMPLE 9Cytokine Secretion Timelapse Using a Bispecific Binding Agent

[0230] This example covers real time imaging of cellular cytokine release. CD8+ and natural killer T cells were prepared, bound to bispecific binding agents targeted to CD45 (expressed on the CD8+ and natural killer T cell surfaces) and IFN-y, input into fluidic devices, enclosed within hydrogel chambers, and cultured in the presence of PMA as described in Example 8. Background fluorescence images were taken immediately following chamber synthesis, and are shown in FIGS. 38A and 39A for the natural killer T cells and CD8+ T cells, respectively. IFN-y secretion by the cells was then imaged at 2, 3, and 16 hours following chamber synthesis by inputting cytokine-targeted fluorescent antibodies into the fluidic devices and measuring fluorescence of individual cells, with fluorescence indicating IFN-y secretion and subsequent binding to the bispecific binding agents coupled to the cell surfaces. Fluorescence images of select chamber-enclosed natural killer T cells at 2, 3, and 16 hours are shown in FIGS. 38B-D, respectively, with exemplary cells labeled 3801 and exemplary chambers labeled 3802. Fluorescence images of select chamber-enclosed C8+ T cells at 2, 3, and 16 hours are shown in FIGS. 39B-D, respectively, with exemplary cells labeled 3901 andAttorney Docket No. 59528-735601exemplary chambers labeled 3902. As seen in these images, the majority of the natural killer and CD8+ T cells did not fluoresce at 2 hours and exhibited strong fluorescence by 16 hours, demonstrating the ability of the analyzed method to track time-dependent soluble factor release. One CD8+ T cell, labeled 3903 in FIGS. 39A-D, exhibited resolvable fluorescence intensity at 2 hours and increased fluorescence intensities at 3 and 16 hours.

[0231] Furthermore, these images show that the analyzed method is capable of resolving cytokine release at the single cell level. In FIG. 38D, the two cells resolved in the bottom right cage exhibited considerably different fluorescent intensities, with the uppermost cell 3803 exhibiting high fluorescence intensity and the lower cell 3804 exhibiting low fluorescence intensity. These intensity differences were resolved even though the two cells (3803, 3804) were co-enclosed within a single chamber and, as displayed in FIGS. 38A-D, in contact at each of the imaging timepoints. FIG. 38E shows a blown-up image of the two cells resolved in the bottom right cage of FIG. 38D.EXAMPLE 10Simultaneous Cytokine Detection Using Bispecific Binding Agents and Cytokine Beads

[0232] This example covers simultaneous cytokine secretion analysis with beads coated in cytokine-targeted antibodies and with bispecific binding agents targeted to cell surface markers and cytokines. For these analyses, a fluidic device was loaded with natural killer cells coupled to bispecific binding agents targeted to IFN-y and TNF-a and with beads coated with anti-ZFN-y and TNF-a antibodies. Cells were enclosed within hydrogel chambers within the fluidic device and incubated in the presence of PMAto stimulate cytokine secretion. IFN-y and TNF-a secretions were measured 4 and 7 hours following caging using fluorescently labeled IFN-y and TNF-a antibodies. A background fluorescence image collected directly following chamber synthesis is shown in FIG. 40A. TNF-a fluorescence images collected 4 and 7 hours following chamber synthesis are shown in FIGS. 40B and 40C, respectively. IFN-y fluorescence images collected 4 and 7 hours following chamber synthesis are shown in FIGS. 40D and 40E, respectively. In these figures, exemplary cells, beads, and chambers, are indicated by the labels 4001, 4002, and 4003, respectively. IFN-y and TNF-a secreted by the cells were captured on the surfaces of the NK cells (through the cell-targeted IFN-y and TNF-a antibodies) and on the IFN-y and TNF-a capture beads. IFN-y and TNF-a fluorescence antibody stains indicated progressively increased IFN-y and TNF-a levels on the cells and beads during the 7 hour incubation.Attorney Docket No. 59528-735601EXAMPLE 11T Cell Cytokine Secretion in the Presence of Dendritic Cells

[0233] This example covers a method for detecting cytokine secretion from CD8+ T cells coenclosed with dendritic cells within hydrogel chambers. Fresh OT1 CD8+ T cells were labeled with a bispecific binding agent targeted to IFN-y, and therefore designed to localize IFN-y secreted by the CD8+ T cells to their surfaces. Dendritic cells were primed with 1 pg / mL OVA peptide. The OT1 CD8+ T cells and dendritic cells were then co-enclosed within hydrogel chambers within a fluidic device. Brightfield images of separate chambers 4101 coenclosing dendritic cells 4102 with OT1 CD8+ T cells 4103 are shown in FIG. 41A. The cells were incubated for 24 hours and then analyzed for IFN-y secretion by fluorescently imaging the cells in the presence of a fluorescent IFN-y antibody (following a 30-minute incubation with these antibodies). Fluorescence images of the same chambers from FIG. 41A are shown in FIG. 41B. Merged images are shown in FIG. 41C. These images show that fluorescence intensity, indicative of IFN-y, was localized to OT1 CD8+ T cell surfaces.EXAMPLE 12Subcellular Cytokine Secretion Analysis

[0234] This example is directed to a method for resolving localized cytokine distribution along a cell surface. Natural killer cells were added to a fluidic device with target K562 tumor cells. Hydrogel chambers were synthesized within the fluidic device to co-enclose natural killer and K562 target cells. A brightfield image of a chamber 4201 enclosing a natural killer cell 4202 and a tumor cell 4203 is shown in FIG. 42A. As shown in FIG. 42B, the tumor cells were identified with a tumor cell-specific fluorescent antibody. IFN-y secretion was measured using a bispecific binding agent with binding regions targeted to a cell surface marker and IFN-y. IFN-y bound to cells through the bispecific binding agent were resolved using a fluorescent IFN-y antibody. As shown in FIG. 42C, fluorescence intensity was highest on the portion of the natural killer cell surface in proximity to a tumor cell (indicated by label 4204), suggesting that the interaction between the tumor and NK cell enriches IFN-y secretion along the surface between these cells.Attorney Docket No. 59528-735601EXAMPLE 13Single-Cell Hypoxia Measurements in Fluidic Devices with Gas-Filled Channels

[0235] This example covers hypoxia measurements in multiple fluidic devices with different mechanisms for gas exchange from sample-filled channels. Six fluidic devices were utilized in these analyses. All six fluidic devices included top and bottom glass layers connected to a gas-permeable spacer layer. Cut-out regions in the spacer layers, taken together with the top and bottom glass layers, defined sample channels in the fluidic devices. The first fluidic device (‘Control’) contained eight parallel sample channels separated by continuous (not interrupted by slits or channels) segments of a spacer layer as depicted in FIGS. 3A-E. The second fluidic device (‘Full Slit’) included eight parallel sample channels separated by spacer layer segments that contained gas-filled channels open to surrounding atmosphere through the ends of the fluidic device (as depicted in FIGS. 43A-D). The third fluidic device (‘Glass Holes’) included eight parallel sample channels separated by continuous spacer layer segments exposed to surrounding atmosphere through holes in the top glass layer (as depicted in FIGS. 48A-D). The fourth fluidic device (‘Glass Slit’) included eight parallel sample channels separated by continuous spacer layer segments that were exposed to surrounding atmosphere through slits in the top glass layer (as depicted in FIGS. 46A-D). The fifth fluidic device (‘2-Channel’) included two parallel sample channels separated by a continuous segment of a spacer layer as depicted in FIGS. 47A-D. The sixth fluidic device (‘Partial Slit’) included eight parallel sample channels separated by spacer layer segments with gas-filled channels open to surrounding atmosphere at one end of the fluidic device (as depicted in FIGS. 44A-D)

[0236] Bottom surfaces of fluidic device channels were coated with fibronectin by incubating room temperature, 25 pg / mL fibronectin-containing phosphate-buffered saline (PBS) in the channels for 30 minutes and then removing unbound fibronectin from the channels by washing the channels with fibronectin-free phosphate-buffered saline. Dendritic cells were then loaded into fluidic device channels at densities of either 1.2xl06or 3xl06cells / mL in media that contained 0.4 pM / mL ImagelT hypoxia detection reagent. ImagelT hypoxia detection reagent is non-fluorescent under normoxic conditions and becomes increasingly fluorescent as dissolved O2 levels decrease below about 50 pM (corresponding to dissolved O2 levels in 37 °C aqueous media under 5% O2 atmosphere). Both channels of the two-channel fluidic device were loaded with cell-containing media. Sample channel inlets and outlets were sealed with Kapton tape. The fluidic devices were then incubated under 18.6%Attorney Docket No. 59528-735601O2 atmospheres inside of CO2 incubators for either 16 or 24 hours. The fluidic devices were then removed from the incubators, and ImagelT hypoxia detection reagent fluorescence intensities were measured from individual cells within each sample channel using a fluorescence microscope at 10X objective.

[0237] The results of the fluorescence analyses are summarized in FIGS. 49-50. FIG. 49 is a box plot of single-cell ImagelT hypoxia detection reagent fluorescence intensities from the 16 hour cell incubations within (from left-to-right along the plot) the ‘Control’ fluidic device, the ‘Full Slit’ fluidic device, the ‘Glass Holes’ fluidic device, and the ‘Glass Slit’ fluidic device. Within these fluidic devices, channels 1-4, 6, and 8 had been loaded with cell-containing media (channel numbers in order from the leftmost channel to the rightmost channel), while channels 5 and 7 were left open to atmosphere. Cell densities and channel IDs are indicated below each dataset. In the ‘Control’ fluidic device, the outermost sample channels (channels 1 and 8), which contained 1.2xl06cells / mL, exhibited low hypoxia. Within this fluidic device, channel 2, which contained 1.2xl06cells / mL but was surrounded by sample containing channels, and channels 3 and 4, which contained 3xl06cells / mL, exhibited high levels of hypoxia. All sample-filled channels in the remaining three fluidic devices (‘Full Slit’, ‘Glass Holes, and ‘Glass Slit’) exhibited low degrees of hypoxia, indicating that the modifications to these fluidic devices (gas-filled spacer layer channels, glass holes, and glass slits) facilitated O2 diffusion into the sample channels.

[0238] FIG. 50 is a box plot of single-cell ImagelT hypoxia detection reagent fluorescence intensities from the 24 hour cell incubations within (from left-to-right along the plot) the ‘Two-Channel’ fluidic device, the ‘Control’ fluidic device, and the ‘Partial Slit’ fluidic device. Only the channels indicated in this plot were filled with samples. All sample-filled channels of the ‘Partial Slit’ fluidic device exhibited low levels of hypoxia. In the ‘2-Channel’ fluidic device, the channel with 1.2xl06cells / mL exhibited low levels of hypoxia, while the channel with 3xl06cells exhibited relatively high levels of hypoxia. In the ‘Control’ fluidic device, sample filled channel 5, which contained 1.2xl06cells / mL and was adjacent to a gas-filled channel, exhibited relatively low levels of hypoxia. Within the fluidic device, channel 6, which contained 1.2xl06cells / mL and was surrounded by sample-filled channels, and channel 7, which was adjacent to a gas-filled channel but contained 3xl06cells, exhibited high levels of hypoxia.

Claims

1. Attorney Docket No. 59528-7356012.CLAIMS3.WHAT IS CLAIMED IS:

1. A method for analyzing one or more cells in a fluidic device comprising:5.inputting the one or more cells into a first channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel; incubating the one or more cells in a liquid in the first channel;6.diffusing a first gas from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel.

2. The method of claim 1, wherein the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the second channel 3. The method of claim 1 or claim 2, wherein after the inputting the liquid into the first channel, the liquid is quiescent during the incubating the one or more cells.

4. The method of any one of claims 1-3, wherein the top layer and the bottom layer are optically transparent or translucent.

5. The method of any one of claims 1-4, wherein the top layer and the bottom layer are oxygen impermeable and liquid impermeable.

6. The method of any one of claims 1-4, wherein the first channel is coupled to a fluidic manifold.

7. The method of any one of claims 1-6, wherein the first channel is sealed.

8. The method of any one of claims 1-7, wherein the second channel is coupled to a gas manifold or is open to an atmosphere outside of the fluidic device.

9. The method of any one of claims 1-8, wherein the first channel and the second channel are separated by about 0.5 to 3 millimeters.Attorney Docket No. 59528-73560110. The method of any one of claims 1-9, wherein:15.a) the first channel comprises a width of about 2.5 to 15 millimeters;16.b) the first channel comprises a height of about 0.025 to 0.25 millimeters;17.c) the first channel comprises a length of about 50 to 200 millimeters;18.d) the second channel comprises a width of about 2.5 to 15 millimeters;19.e) the second channel comprises a height of about 0.025 to 0.25 millimeters;20.f) the second channel comprises a length of about 50 to 200 millimeters; or g) a combination thereof.

11. The method of any one of claims 1-10, further comprising diffusing the first gas from a third channel of the fluidic device into the first channel through another portion of the spacer layer, wherein the first channel is adjacent to the third channel, wherein the another portion of the spacer layer is located in between the first and third channels, wherein the spacer layer further includes a third cut-out region that at least partially defines the third channel.

12. The method of claim 11, wherein the third cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the third channel.

13. The method of claim 10 or claim 11, wherein the second and third channels are disposed on opposite sides of the first channel.

14. The method of any one of claims 1-13, further comprising diffusing the first gas from an area outside of the fluidic device into the first channel through another portion of the spacer layer, wherein the another portion of the spacer layer comprises an edge of the fluidic device, wherein the portion of the spacer layer and the another portion of the spacer layer are disposed on opposite sides of the first channel.

15. The method of any one of claims 1-14, wherein the incubating is for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours.

16. The method of any one of claims 1-15, wherein the one or more cells are viable in the liquid in the first channel for at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours.Attorney Docket No. 59528-73560117. The method of claim 15 or claim 16, wherein the liquid in the first channel is not replaced during the at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours.

18. The method of any one of claims 1-17, wherein the second channel is fluidically coupled to an atmosphere extrinsic to the fluidic device, and wherein following the at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 15, at least 18, at least 24, at least 30, at least 36, at least 48, at least 60, at least 72, at least 96, or at least 120 hours, a partial pressure of O2 in the second channel is within at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 99.5% of a partial pressure of O2 in the atmosphere extrinsic to the fluidic device.

19. The method of any one of claims 1-18, further comprising diffusing a second gas from the liquid in the first channel of the fluidic device into the second channel through the portion of the spacer layer.

20. The method of claim 19, wherein the second gas comprises a waste product of the one or more cells.

21. The method of claim 19 or claim 20, wherein the second gas comprises carbon dioxide (CO2).

22. The method of any one of claims 1-21, wherein the first gas comprises a pressure of about 1 atmosphere in the second channel.

23. The method of any one of claims 1-21, wherein the first gas comprises a pressure of about 1.25 to 10 atmospheres in the second channel.

24. The method of any one of claim 1-23, wherein the one or more cells consume the first gas.

25. The method of any one of claims 1-24, wherein the first gas comprises O2.

26. The method of claim 25, wherein the liquid comprises or approaches a steady-state dissolved O2 level of between about between about 0.1 and 0.5 mg / L, between about 0.1 and 1 mg / L, between about 0.1 and 2 mg / L, between about 0.1 and 4 mg / L, between about 0.1 and 6 mg / L, between about 0.1 and 8 mg / L, between about 0.1 and 10 mg / L,Attorney Docket No. 59528-73560137.between about 0.1 and 12 mg / L, between about 0.25 and 0.5 mg / L, between about 0.25 and 1 mg / L, between about 0.25 and 2 mg / L, between about 0.25 and 4 mg / L, between about 0.25 and 6 mg / L, between about 0.25 and 8 mg / L, between about 0.25 and 10 mg / L, between about 0.25 and 12 mg / L, between about 0.5 and 1 mg / L, between about 0.5 and 2 mg / L, between about 0.5 and 4 mg / L, between about 0.5 and 6 mg / L, between about 0.5 and 8 mg / L, between about 0.5 and 10 mg / L, between about 0.5 and 12 mg / L, between about 1 and 2 mg / L, between about 1 and 4 mg / L, between about 1 and 6 mg / L, between about 1 and 8 mg / L, between about 1 and 10 mg / L, between about 1 and 12 mg / L, between about 2 and 4 mg / L, between about 2 and 6 mg / L, between about 2 and 8 mg / L, between about 2 and 10 mg / L, between about 2 and 12 mg / L, between about 2 and 15 mg / L, between about 3 and 6 mg / L, between about 3 and 8 mg / L, between about 3 and 10 mg / L, between about 3 and 12 mg / L, between about 4 and 8 mg / L, between about 4 and 10 mg / L, between about 4 and 12 mg / mL, between about 6 and 8 mg / mL, between about 6 and 10 mg / mL, between about 6 and 12 mg / mL, or between about 8 and 12 mg / mL.

27. The method of claim 25 or 26, wherein the one or more cells deplete O2 from the liquid at a rate of at least about 0.01 mg*L'1*h'1, a rate of at least about 0.02 mg*L'1*h'1, a rate of at least about 0.05 mg*L'1*h'1, a rate of at least about 0.1 mg*L'1*h'1, a rate of at least about 0.2 mg*L'1*h'1, a rate of at least about 0.5 mg*L'1*h'1, a rate of at least about 1 mg*L-i*h'i, a rate of at least about 1.5 mg*L'1*h'1, a rate of at least about 2 mg*L'1*h'1, a rate of at least about 2.5 mg*L'1*h'1, a rate of at least about 3 mg*L'1*h'1, a rate of at least about 4 mg*L'1*h'1, or a rate of at least about 5 mg*L'1*h'1.

28. The method of any one of claims 1-27, wherein the spacer layer is impermeable to liquid water and permeable to gaseous O2.

29. The method of any one of claims 1-28, wherein the spacer layer comprises an O2 permeability of about 10 to 500 barrers.

30. The method of any one of claims 1-29, wherein the spacer layer comprises a siloxane moiety, a silicone moiety, an acrylate moiety, an acrylamide moiety, a polyvinyl moiety, a polyurethane, an epoxy moiety, a polyphenol, a polyester, a polyamide, a polyimide, polytetrafluoroethylene, polyethylene, polypropylene, a polycarbamate, a polycarbonate, a polyacrylic acid, a sulfonated polyester resin, or a combination thereof.Attorney Docket No. 59528-73560131. The method of any one of claims 1-30, wherein the portion of the spacer layer located between the first and second channels comprises a width of about 0.25 to 2.5 millimeters.

32. The method of any one of claims 1-31, further comprising synthesizing one or more chambers that at least partially enclose the one or more cells in the first channel.

33. The method of claim 32, wherein the synthesizing comprises projecting light into the first channel with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers.

34. The fluidic system of claim 32 or claim 33, wherein the one or more chambers comprise a hydrogel.

35. The fluidic system of any one of claims 32-34, wherein the one or more chambers extend from the bottom layer to the top layer.

36. The method of any one of claims 32-35, wherein the one or more chambers are synthesized at a position determined to contain at least a subset of the one or more cells by a detector.

37. The method of any one of claims 32-36, wherein the one or more cells are at least partially enclosed in the one or more chambers during the incubating.

38. The method of any one of claims 32-37, further comprising degrading a chamber of the one or more chambers.

39. The method of any one of claims 1-38, further comprising loading the first channel with an assay reagent.

40. The method of any one of claims 1-38, further comprising determining a characteristic of the one or more cells.

41. The method of claim 40, wherein the characteristic of the one or more cells comprises a proliferation rate.

42. The method of claim 41, wherein said determining said proliferation rate comprises counting cells at least partially enclosed by the one or more chambers.

43. The method of any one of claims 40-42, wherein the characteristic of the one or more cells comprises a soluble factor secreted by the one or more cells.Attorney Docket No. 59528-73560144. The method of claim 43, wherein said detecting said soluble factor comprises:56.(i) disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the one or more cells, and57.(ii) detecting the soluble factor bound to the capture surface.

45. The method of claim 44, wherein said detecting said soluble factor bound to said capture surface comprises:59.(i) contacting said soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and60.(ii) detecting the labeled antibody.

46. The method of any one of claims 43-45, further comprising removing the soluble factor from the first channel and detecting a second soluble factor secreted by the one or more cells subsequent to said removing the soluble factor from the first channel.

47. The method of any one of claims 40-46, wherein the one or more cells comprise effector cells, and wherein the characteristic of the one or more cells comprises cytotoxicity.

48. The method of claim 47, wherein the one or more chambers at least partially enclose one or more target cells with the effector cells, and said measuring said cytotoxicity comprises counting dead and / or viable cells from among the one or more target cells.

49. The method of claim 47 or claim 48, wherein the effector cells and the one or more target cells are viable at O2 levels in the liquid of the first channel.

50. The method of any one of claims 40-49, wherein the characteristic of the one or more cells comprises activation.

51. The method of claim 50, wherein the activation is caused by:66.(i) contact between a second cell and a cell of the one or more cells;67.(ii) a soluble factor secreted by the second cell and the cell of the one or more cells; or (iii) a combination thereof.

52. The method of claim 51, wherein:69.(i) the second cell is at least partially enclosed with the cell of the one or more cells by the chamber of the one or more chambers;70.(ii) the second cell and the cell of the one or more cells are each at least partially enclosed by different chambers of the one or more chambers; or Attorney Docket No. 59528-73560171.(iii) the cell of the one or more cells is at least partially enclosed by a chamber of the one or more chambers and the second cell is not enclosed by a chamber of the one or more chambers.

53. The method of claim 51 or claim 52, wherein the second cell and the cell of the one or more cells are each spaced apart within about 5, 10, 20, 40, 60, 80, or 100 pm within the first channel.

54. The method of any one of claims 50-53, wherein said determining said activation comprises detecting a surface marker of the one or more cells.

55. The method of claim 54, wherein said detecting said surface marker comprises contacting the one or more cells with a binding agent configured to bind to the surface marker and detecting the binding agent.

56. The method of any one of claims 50-55, wherein said determining said activation comprises measuring proliferation of the one or more cells.

57. The method of any one of claims 1-56, wherein a cell of the one or more cells are viable for at least 1, at least 2, at least 4, at least 6, at least 8, at least 12, at least 16, at least 24, at least 36, at least 48, at least 60, at least 90, at least 120, at least 150, or at least 180 hours under hypoxic conditions.

58. The method of any one of claims 1-57, wherein a cell of the one or more cells is viable for less than 180, less than 150, less than 120, less than 90, less than 60, less than 48, less than 36, less than 24, less than 16, less than 12, less than 8, less than 6, less than 4, less than 2, or less than 1 hour under hypoxic conditions.

59. The method of any one of claims 1-58, wherein the one or more cells comprise a cancer cell, an immune cell, a fibroblast, a neuron, or a combination thereof.

60. The method of any one of claims 1-59, wherein a cell of the one or more cells proliferates during the incubation.

61. The method of any one of claims 1-59, wherein a cell of the one or more cells does not proliferate during the incubation.

62. A method for analyzing one or more cells, the method comprising:82.inputting the one or more cells into a fluidic device, Attorney Docket No. 59528-73560183.trapping the one or more cells, wherein the trapped one or more cells are disposed within the fluidic device;84.inputting a bispecific binding agent into the fluidic device, the bispecific binding agent comprising:85.(i) a first binding site configured to bind to a surface marker on a surface of the one or more cells, and86.(ii) a second binding site configured to bind to a soluble factor secreted by the one or more cells; and87.detecting binding of the soluble factor to the bispecific binding agent, wherein the bispecific binding agent is bound to the surface of the one or more cells during the detecting.

63. The method of claim 62, wherein the trapped one or more cells are each at least partially enclosed in one or more chambers, wherein each chamber comprises polymer matrix walls and a nanofluidic space.

64. The method of claim 63, wherein the trapping the one or more cells comprises inputting a polymer precursor into the fluidic device, and synthesizing one or more chambers that at least partially enclose the one or more cells in the fluidic device, wherein the synthesizing comprises projecting light into the fluidic device with a spatial energy modulating element such that the projected light causes cross-linking of one or more polymer precursors to at least partially form the one or more chambers.

65. The method of claim 64, wherein the detecting comprises measuring an aggregate signal from a chamber of the one or more chambers.

66. The method of claim 62, wherein the trapping the one or more cells comprises enclosing the one or more cells within one or more droplets.

67. The method of claim 66, wherein each droplet contains a first liquid and the one or more cells, wherein the one or more droplets are surrounded by a second liquid, wherein the first liquid and the second liquid are immiscible.

68. The method of claim 67, wherein the first liquid comprises water and the second liquid comprises an oil.

69. The method of claim 67 or claim 68, wherein the second liquid comprises a fluorocarbon.Attorney Docket No. 59528-73560170. The method of any one of claims 62-69, wherein the detecting comprises imaging a cell of the one or more cells, wherein signal intensity of the imaged cell is proportional to an amount of the soluble factor bound to the bispecific binding agent of the imaged cell.

71. The method of any one of claims 62-70, wherein the bispecific binding agent is coupled to the one or more cells prior to the inputting the bispecific binding agent into the fluidic device.

72. The method of any one of claims 62-71, wherein the surface marker is on a surface of a first cell of the one or more cells, and wherein the soluble factor is secreted by a second cell of the one or more cells.

73. The method of claim 72, further comprising detecting activation of the first cell of the one or more cells.

74. The method of claim 72 or claim 73, wherein the first cell and the second cell are immune cells.

75. The method of any one of claims 72-74, wherein the first cell comprises an effector cell and the second cell comprises a CD4+ T cell.

76. The method of claim 75, wherein the effector cell comprises a CD8+ T cell.

77. The method of any one of claims 73-76, wherein the detecting the activation comprises measuring:102.i) cytotoxicity of the first cell towards a target cell;103.ii) a surface marker expressed by the first cell;104.iii) an mRNA expressed by the first cell;105.iv) a soluble factor secreted by the first cell; or106.v) a combination thereof.

78. The method of claim 77, wherein:108.i) the cytotoxicity towards the target cell comprises an incidence, rate, efficiency, or number of killings of the target cell, and the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof; Attorney Docket No. 59528-735601109.ii) the surface marker expressed by the first cell comprises CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, or a combination thereof;110.iii) the mRNA comprises mRNA encoding CD25, CD38, CD69, CD71, HLA- DR, Ki-67, LFA-lhigh, CD2, VLA-4, an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; iv) the soluble factor secreted by the first cell comprises an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; or111.v) a combination thereof.

79. The method of any one of claims 72-78, wherein the first and second cells are coenclosed within a chamber.

80. The method of claim 72, wherein the first cell is an effector cell and the second cell is a target cell, wherein the soluble factor from the effector cell is detected.

81. The method of claim 72, wherein the first cell is an effector cell and the second cell is a target cell, wherein the soluble factor from the target cell is detected.

82. The method of any one of claims 79-81, wherein a third cell is co-enclosed within the chamber, the first cell is an immune cell, the second cell is an effector cell, and the third cell is a target cell, wherein the soluble factor from the immune cell is detected, the method further comprising determining whether the immune cell activates the effector cell.

83. The method of claim 72, wherein the first cell comprises a target cell of an effector cell and the second cell comprises a CD4+ T cell.

84. The method of claim 83, further comprising measuring activation of the effector cell, killing of the target cell by the effector cell, or a combination thereof.

85. The method of claim 83 or claim 84, wherein the effector cell comprises a CD8+ T cell.

86. The method of any one of claims 83-85, wherein the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof.Attorney Docket No. 59528-73560187. The method of any one of claims 62-71, wherein the surface marker is on a surface of a first cell of the one or more cells and the soluble factor is secreted by the first cell of the one or more cells.

88. The method of claim 87, further comprising detecting activation of an effector cell in the fluidic device.

89. The method of claim 88, wherein the detecting the activation comprises measuring:123.i) cytotoxicity of the effector cell towards a target cell;124.ii) a surface marker expressed by the effector cell;125.iii) an mRNA expressed by the effector cell;126.iv) a soluble factor secreted by the effector cell; or127.v) a combination thereof.

90. The method of claim 89, wherein:129.i) the cytotoxicity towards the target cell comprises an incidence, rate, efficiency, or number of killings of the target cell, and the target cell comprises a cancer cell, a bacterial cell, an infected cell, or a combination thereof;130.ii) the surface marker expressed by the effector cell comprises CD25, CD38, CD69, CD71, HLA-DR, Ki-67, LFA-lhigh, CD2, VLA-4, or a combination thereof;131.iii) the mRNA comprises mRNA encoding CD25, CD38, CD69, CD71, HLA- DR, Ki-67, LFA-lhigh, CD2, VLA-4, an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; iv) the soluble factor secreted by the effector cell comprises an interferon, a tumor necrosis factor, a granzyme, a perforin, a granulysin, or a combination thereof; or132.v) a combination thereof.

91. The method of claim 70, wherein the signal intensity comprises brightfield imaging intensity, fluorescence imaging intensity, or a combination thereof.

92. The method of claim 91, wherein the imaging comprises resolving multiple signals along the surface of the cell.Attorney Docket No. 59528-73560193. The method of any one of claims 62-92, further comprising coupling the surface marker to the cell.

94. The method of claim 93, wherein the coupling the surface marker to the cell comprises halotagging, sortagging, Q-tagging, liposome delivery, polycation delivery, click chemistry, or a combination thereof.

95. The method of claim 93 or claim 94, wherein the coupling the surface marker to the cell comprises contacting the cell with the surface marker coupled to:138.i) a lipid configured to insert into a membrane of the cell;139.ii) an antibody configured to bind to an additional surface marker on the cell; iii) a reactive molecule configured to covalently couple to the additional surface marker on the cell.

96. The method of claim 95, wherein the lipid comprises a sterol, cholesterol, a prenyl group, a diacylglycerol group, myrisate, palmitate, stearate, or oleate.

97. The method of claim 95, wherein the antibody targets an immune cell marker selected from CD4, CD8, CCR7, CD45, CD45Ram C127, CD25, CD39, CD14, CD19, CD3, CD 16, CD56, CD28, C62, CD11, CD69, or CD71.

98. The method of claim 95, wherein the reactive molecule comprises a N- hydroxysuccinimide (NHS), a maleimide, a disulfide, an azide, or an alkenyl group.

99. The method of claim 95, wherein the cell surface marker comprises avidin, an avidin fragment, streptavidin, a streptavidin fragment, neutravidin, a neutravidin fragment, biotin, a strep tag, 2,4-dinitrophenol, digoxigenin, or fluorescein.

100. The method of any one of claims 62-99, wherein the detecting the binding of the soluble factor to the bispecific binding agent comprises inputting an additional binding agent into the fluidic device, and detecting binding of the additional binding agent to the soluble factor bound to the bispecific binding agent.

101. The method of claim 100, wherein the additional binding agent comprises an antibody.

102. The method of claim 100 or claim 101, wherein the detecting comprises detecting a detectable label coupled to the additional binding agent.Attorney Docket No. 59528-735601103. The method of claim 102, wherein the detectable label comprises a fluorophore, a phosphorescent species, a dye, an oligonucleotide barcode, a chemiluminescent species, or a combination thereof.

104. The method of claim 103, wherein the detecting comprises ligating the oligonucleotide barcode to a nucleic acid barcode coupled to a location on a surface of the fluidic device that comprises a sequence specific to the location and sequencing the oligonucleotide barcode and the sequence specific to the location.

105. The method of claim 103, wherein the detecting comprises capturing the oligonucleotide barcode on a nucleic acid barcode coupled to a location on a surface of the fluidic device that comprises a sequence specific to the location, extending the nucleic acid barcode over the oligonucleotide barcode, and sequencing the extended nucleic acid barcode.

106. The method of claim 104 or claim 105, wherein the sequence specific to the location is blocked with a blocking agent prior to the inputting the additional binding agent into the fluidic device and then unblocked subsequent to the inputting the additional binding agent into the fluidic device.

107. The method of claim 103, wherein the detecting comprises fixing the detectable label within the fluidic device, coupling a fluorescently labeled nucleic acid molecule to the oligonucleotide barcode, and detecting the fluorescently labeled nucleic acid molecule.

108. The method of any one of claim 100-107, wherein a portion of the additional binding agent is not bound to a complex, the complex comprising the soluble factor bound to the bispecific binding agent, wherein the portion of the additional binding agent that is not bound to the complex is not removed from the fluidic device during the detecting of the detectable label coupled to the additional binding agent.

109. The method of claim 108, wherein the detectable label of the additional binding agent comprises a fluorophore, wherein a channel of the fluidic device comprises a height of about 0.025 to 0.25 millimeters, wherein a fluorescence signal intensity of the portion of the additional binding agent that is not bound to a complex is at least ten times, at least 100 times, or at least 1000 times less than a fluorescence signal intensity of the additional binding agent bound to the complex, thereby avoiding a washing step of unboundAttorney Docket No. 59528-735601153.additional binding agent before the detecting of the detectable label coupled to the additional binding agent.

110. The method of any one of claims 62-109, wherein the detecting the binding of the soluble factor to the bispecific binding agent comprises quantitating an amount of the soluble factor secreted by the cell of the one or more cells.

111. The method of 62-110, wherein the detecting the binding of the soluble factor to the bispecific binding agent is performed at multiple time points.

112. The method of 62-111, further comprising inputting an additional bispecific binding agent into the fluidic device, the additional bispecific binding agent comprising:157.(i) a first binding site configured to bind to the surface marker on the surface of the cell of the one or more cells, and158.(ii) a second binding site configured to bind to an additional soluble factor secreted by cell; and159.detecting binding of the additional soluble factor to the additional bispecific binding agent, wherein the additional bispecific binding agent is bound to the surface of the cell during the detecting the binding of the additional soluble factor to the additional bispecific binding agent.

113. The method of any one of claims 62-111, further comprising inputting an additional bispecific binding agent into the fluidic device, the additional bispecific comprising:161.(i) a first binding site configured to bind to an additional surface marker on the surface of the one or more cells, and162.(ii) a second binding site configured to bind to an additional soluble factor secreted by the cell; and163.detecting binding of the additional soluble factor to the additional bispecific binding agent, wherein the additional bispecific binding agent is bound to the surface of the cell during the detecting the binding of the additional soluble factor to the additional bispecific binding agent.

114. The method of claim 112 or 113, wherein the bispecific binding agent and the additional bispecific binding agent are each bound to the surface of the cell of the one or more cells via the respective first binding sites.Attorney Docket No. 59528-735601115. The method of any one of claims 112-114, wherein the detecting the binding of the soluble factor to the bispecific binding agent is simultaneous to the detecting the binding of the additional soluble factor to the additional bispecific binding agent.

116. The method of any one of claims 62-115, wherein the soluble factor comprises a cytokine, an immune active molecule, an interleukin, an interferon, a colony stimulating factor, a tumor necrosis factor, or a granzyme.

117. The method of any one of claims 62-116, wherein the soluble factor comprises interferon -y (IFN-y), interferon-a (IFN-a), interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL- 10), interleukin- 13 (IL- 13), interleukin- 15 (IL- 15), interleukin-21 (IL- 21), or interleukin-23 (IL-23), granulocyte-macrophage colony stimulating factor (GM- CSF), granulocyte colony stimulating factor (G-CSF), TNF-a, TNF-P, granzyme A, granzyme B, granzyme C, granzyme H, granzyme K, granzyme M, perforin, granulysin, or a combination thereof.

118. The method of any one of claims 62-117, wherein the surface marker on the surface the cell of the one or more cells comprises an immune cell marker.

119. The method of claim 118, wherein the surface marker comprises CD4, CD8, CCR7, CD45, CD45Ram C127, CD25, CD39, CD14, CD19, CD3, CD16, CD56, CD28, C62, CD11, CD69, CD71, or a combination thereof.

120. The method of any one of claims 112-119, wherein the first binding site of the additional bispecific binding agent comprises a first antibody or a fragment thereof.

121. The method of any one of claims 112-119, wherein the first binding site of the additional bispecific binding agent comprises biotin, avidin, an avidin fragment, streptavidin, a streptavidin fragment, neutravidin, a neutravidin fragment, biotin, a strep tag, 2,4-dinitrophenol, digoxigenin, or fluorescein.

122. The method of any one of claims 112-121, wherein the second binding site of the additional bispecific binding agent comprises a second antibody or a fragment thereof.

123. The method of any one of claims 112-122 wherein the cell is disposed within a chamber or channel of the fluidic device, wherein the channel or chamber comprises a height of about 0.025 to 0.25 millimeters.Attorney Docket No. 59528-735601124. The method of any one of claims 62-123, wherein the fluidic device comprises a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a first cut-out region, wherein the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form a first channel, and wherein the one or more cells and the polymer precursor are input into the first channel of the fluidic device.

125. The method of claim 124, further comprising incubating the one or more cells in a liquid in the first channel; diffusing a first gas from a second channel of the fluidic device into the liquid in the first channel through a portion of the spacer layer, wherein the first channel is adjacent to the second channel, wherein the portion of the spacer layer is located in between the first and second channels, wherein the spacer layer further includes a second cut-out region that at least partially defines the second channel, wherein the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the second channel.

126. The method of claim 62, wherein the first binding site binds to the surface marker on the surface of the cell of the one or more cells, and the second binding site binds to a soluble factor secreted by the cell.

127. The method of any one of claims 63-65, wherein the polymer matrix walls of the one or more chambers comprise pores configured to permit passage of the bispecific binding agent.

128. The method of claim 100, wherein the polymer matrix walls of the one or more chambers comprise pores configured to permit passage of the additional binding agent.

129. A method for analyzing one or more cells in a fluidic device comprising:179.inputting the one or more cells into a channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a cut-out region, wherein the cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the channel;180.incubating the one or more cells in a liquid in the channel; and181.diffusing a gas:182.(i) through an opening in the top layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel,183.(ii) through an opening in the bottom layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel, or Attorney Docket No. 59528-735601184.(iii)a combination thereof.

130. The method of claim 129, wherein:186.(i) the opening in the top layer of the fluidic device comprises a hole, a slit, or a combination thereof;187.(ii) the opening in the bottom layer of the fluidic device comprises a hole, a slit, or a combination thereof; or188.(iii) a combination thereof.

131. A method for analyzing one or more cells in a two-channel fluidic device comprising:190.inputting the one or more cells into a first channel of the two-channel fluidic device, the two-channel fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein:191.the spacer layer includes a first cut-out region and a second cut-out region, the first cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the first channel of the two-channel fluidic device, and192.the second cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form a second channel of the two-channel fluidic device;193.incubating the one or more cells in a liquid in the first channel; and194.diffusing a gas from into the liquid in the first channel, wherein the gas diffuses from outside of the two-channel fluidic device, through the spacer layer, and into the liquid in the first channel.

132. The method of any one of claims 1-131, wherein the one or more cells are present at a density of about 104to about 107cells / mL.

133. The method of claim 132, wherein the one or more cells are present at a density of about 105to about 5xl06cells / mL.

134. A method for analyzing one or more cells in a fluidic device comprising:198.inputting the one or more cells into a channel of a fluidic device, the fluidic device comprising a top layer, a bottom layer, and a spacer layer; wherein the spacer layer includes a cut-out region, wherein the cut-out region of the spacer layer is sandwiched between the bottom layer and the top layer to form the channel; Attorney Docket No. 59528-735601199.incubating the one or more cells in a liquid in the channel; and200.diffusing a gas into the liquid in the channel.

135. The method of claim 134, wherein the gas diffuses through a portion of the spacer layer, and into the liquid in the channel.

136. The method of claim 134, wherein the gas diffuses:203.(i) through an opening in the top layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel,204.(ii) through an opening in the bottom layer of the fluidic device, through a portion of the spacer layer, and into the liquid in the channel, or (iii) a combination thereof.

137. The method of claim 136, wherein:206.(i) the opening in the top layer of the fluidic device comprises a hole, a slit, or a combination thereof;207.(ii) the opening in the bottom layer of the fluidic device comprises a hole, a slit, or a combination thereof; or208.(iii) a combination thereof.

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