Systems and methods for analysis of cells

Abstract

Provided herein are methods and systems for analyzing adherent cells. In an aspect, provided herein is a fluidic device for processing a biological component, comprising: a surface, wherein the surface comprises (i) one or more oligonucleotides configured to associate with a biomolecule, and (ii) a cell adherent support, wherein the cell adherent support is bound to at least a portion of the one or more oligonucleotides.

Description

SYSTEMS AND METHODS FOR ANALYSIS OF CELLSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 642,382, filed May 3, 2024, which application is incorporated herein by reference in its entirety for all purposes.SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 59528-726_601_SL.xml. The date of creation of the Sequence Listing is May 1st, 2025, and the size of the file is 24,934 bytes.BACKGROUND

[0003] Surfaces of fluidic devices or flow cells can be functionalized with oligonucleotides that hybridize to cells or cellular components (including nucleic acids). Using the same surface for other applications (coupling to adherent cells or grafting antibodies, for example) can be challenging due to the oligonucleotide spotting. Under certain circumstances, cells in a suspension state may not reliably transition to an adherent state on oligonucleotide coated surfaces.SUMMARY

[0004] Described herein are systems, devices, and methods for modifying barcoded surfaces, or the capture oligonucleotide themselves, to enable adhesion and spreading of adherent cells.

[0005] In an aspect, provided herein is a fluidic device for processing a biological component, comprising: a surface, wherein the surface comprises (i) one or more oligonucleotides configured to associate with a biomolecule, and (ii) a cell adherent support, wherein the cell adherent support is bound to at least a portion of the one or more oligonucleotides.

[0006] In some cases, the one or more oligonucleotides each comprise a barcode sequence. In some cases, the one or more oligonucleotides each comprise a spatial location tag corresponding to a unique location of the oligonucleotide on the fluidic device. In some cases, the one or more oligonucleotides comprise a binding sequence configured to couple to the biomolecule. In some cases, the binding sequence comprises a polyT sequence, and wherein the biomolecule comprises a messenger ribonucleic acid (mRNA) comprising a poly adenylated (poly A) tail. In some cases, the one or more oligonucleotides are distributed amongst one or more reaction sites on the surface, and each reaction site has a density of the one or more oligonucleotides from about 2 x 102to about 5 x 107oligonucleotides / pm2. In some cases, the biomolecule comprises acomplementary binding sequence configured to couple to the binding sequence of the one or more oligonucleotides.

[0007] In some cases, the one or more oligonucleotides are treated with one or more cell adhesive proteins. In some cases, the one or more oligonucleotides comprise a binding sequence, wherein a poly A-streptavidin is coupled to the binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin. In some cases, the one or more oligonucleotides comprise: (i) a first binding sequence, wherein a poly A-streptavidin is coupled to the first binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin; and (ii) a second binding sequence, wherein the second binding sequence is configured to associate with the biomolecule.

[0008] In some cases, the cell adhesive protein is biotinylated. In some cases, the cell adhesive protein comprises laminin. In some cases, the cell adhesive protein comprises fibronectin. In some cases, the cell adhesive protein comprises poly-l-lysine (PLL). In some cases, the cell adhesive protein comprises poly-d-lysine (PDL). In some cases, the cell adhesive protein comprises vitronectin. In some cases, the cell adhesive protein comprises collagen. In some cases, the cell adhesive protein comprises gelatin. In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a RGD peptide (arginine-glycine-aspartic acid). In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a cell adhesive peptide such as, for example, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. A peptide reactive polymer can be a N-hydroxysuccinimide siloxane polymer, N-hydroxysuccinimide polymer, epoxy functionalized polymer, aldehyde functionalized polymer, or a combination thereof.

[0009] In some cases, the cell adherent support comprises one or more cell adherent biomolecules. In some cases, the one or more cell adherent biomolecules comprise a protein. In some cases, the protein comprises poly-l-lysine (PLL). In some cases, the protein comprises poly-d-lysine (PDL). In some cases, the protein comprises laminin and poly-l-ornithine (PLO). In some cases, the protein comprises fibronectin. In some cases, the protein comprises vitronectin. In some cases, the protein comprises collagen. In some cases, the protein comprises gelatin. In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a RGD peptide (arginine-glycine-aspartic acid). In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a cell adhesive peptide such as, for example, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. A peptide reactive polymer can be a N-hydroxysuccinimide siloxane polymer, N-hydroxysuccinimide polymer, epoxy functionalized polymer, aldehyde functionalized polymer, or a combination thereof. In some cases, the one or more cell adherent biomolecules comprise streptavidin. In some cases, the one or more cell adherent biomolecules comprise biotinylated fibronectin. In some cases, the one or more cell adherent biomolecules comprise biotinylated laminin. In some cases, the one or more cell adherent biomolecules comprise one of biotinylated RGD peptide, biotinylated DGEA (SEQ ID NO: 14), biotinylated IKVAV (SEQ ID NO: 15), biotinylated YIGSR (SEQ ID NO: 16), biotinylated KQAGDV (SEQ ID NO: 17), biotinylated CKKQRFRHRNRKG (SEQ ID NO: 18), biotinylated LIGRKK (SEQ ID NO: 19), biotinylated SPPRRARV (SEQ ID NO: 20), biotinylated NGRAHA (SEQ ID NO: 21), biotinylated ID APS (SEQ ID NO: 22), biotinylated REDV (SEQ ID NO: 23), biotinylated KRSR (SEQ ID NO: 24), biotinylated VGVAPG (SEQ ID NO: 25), biotinylated CNYYSNS (SEQ ID NO: 26), and combinations thereof. In some cases, a density of the one or more cell adherent biomolecules on the cell adherent support is 200 to about 5 x 107cell adherent biomolecules / pm2.

[0010] In some cases, the cell adherent support comprises one or more cell adherent particles. In some cases, the one or more cell adherent particles comprise a polymer particle at least partially coated with a particle cell adherent coating. In some cases, the particle cell adherent coating comprises a fibronectin coating. In some cases, the particle cell adherent coating comprises poly-l-ornithine. In some cases, the particle cell adherent coating comprises a laminin coating. In some cases, the particle cell adherent coating further comprises poly-l-ornithine. In some cases, the particle cell adherent coating comprises poly-l-lysine. In some cases, the particle cell adherent coating comprises poly-d-lysine. In some cases, the particle cell adherent coating comprises vitronectin. In some cases, the particle cell adherent coating comprises collagen. In some cases, the particle cell adherent coating comprises gelatin. In some cases, the particle cell adherent coating comprises one of RGD peptide, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), biotinylated YIGSR (SEQ ID NO: 16), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. The particle cell adherent coating can further comprise a functional group such as N- hydroxysuccinimide moieties, epoxide moieties, aldehyde moieties, or a combination thereof. Insome cases, a density of the one or more cell adherent particles on the surface is from about 10 to about 2500 particles / mm2.

[0011] In some cases, the surface comprises one or more flow channels, wherein a flow channel of the one or more flow channels comprises one or more arrays, wherein an array of the one or more arrays comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to a spatial location of the array of the one or more arrays. In some cases, each array comprises greater than 500 reaction sites. In some cases, each array comprises greater than 1000 reaction sites. In some cases, each array comprises greater than 1500 reaction sites. In some cases, the surface comprises at least 5 flow channels. In some cases, the surface comprises at least 10 flow channels. In some cases, a flow channel of the one or more flow channels comprises at least 5 arrays. In some cases, a flow channel of the one or more flow channels comprises at least 10 arrays. In some cases, a flow channel of the one or more flow channels comprises at least 15 arrays. In some cases, the one or more reaction sites are separated by a pitch distance of about 100 to 150 pm. In some cases, each reaction site has a diameter of about 50 to 100 pm.

[0012] In some cases, the fluidic device further comprises a hydrogel chamber. In some cases, wherein the hydrogel chamber is formed upon polymerization of one or more polymer precursors. In some cases, the fluidic device is in optical communication with a spatial energy modulation element configured to selectively direct energy to the fluidic device. In some cases, the fluidic device further comprises a detector in optical communication with the fluidic device.

[0013] In some cases, the biomolecule comprises genetic material. In some cases, the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some cases, the genetic material comprises messenger RNA (mRNA). In some cases, the fluidic device further comprises a cell. In some cases, the biomolecule comprises a component of the cell. In some cases, the component of the cell comprises messenger ribonucleic acid (mRNA).

[0014] In some cases, the cell comprises an adherent cell. In some cases, the cell adherent support is configured to couple to said adherent cell. In some cases, the cell adherent support is functionalized for association with an additional biomolecule. In some cases, the additional biomolecule comprises a secreted protein. In some cases, the additional biomolecule comprises a surface protein.

[0015] In some cases, the cell adherent support is configured to enable adhesion and spreading of adherent cells. In some cases, the surface is a bottom surface of the fluidic device. In some cases, the bottom surface is optically transparent, allowing for imaging of the fluidic device. In some cases, a top surface of the fluidic device is optically transparent, allowing for imaging ofthe fluidic device. In some cases, the surface further comprises an antibody layer configured to capture one or more proteins.

[0016] In another aspect, provided herein is a method for processing one or more adherent cells, comprising: (a) introducing a sample comprising the one or more adherent cells to a fluidic device, wherein the fluidic device comprises a surface, wherein the surface (i) is functionalized for association with a biomolecule and (ii) comprises a cell adherent support; (b) encapsulating an adherent cell of the one or more adherent cells in a hydrogel chamber; and (c) releasing the biomolecule from the adherent cell such that the biomolecule associates with the surface. In some embodiments, the releasing the biomolecule from the adherent cell includes lysing the adherent cell. In some embodiments, the lysing is performed with a lysing agent (e.g., SDS, Triton-X, Triton-XlOO, Tween 20, Sarkosyl). In some embodiments, before (c), the method further includes analyzing the encapsulated adherent cell using cell morphology measurements, cell interactions, secretions, surface receptors, mobility within the hydrogels, calcium ion flux measurements, metabolite measurements. In some embodiments, before (c), but after the analyzing the encapsulated adherent cell, the method further includes fixing and permeabilizing the adherent cell. For example, the fixing may include introducing glutaraldehyde, formaldehyde, and paraformaldehyde to the fluidic device and the permeabilizing may include introducing one of Triton, digitonin, saponin, and a combination thereof

[0017] In some cases, the surface is a bottom surface of the fluidic device. In some cases, the bottom surface is optically transparent, allowing for imaging of the fluidic device. In some cases, a top surface of the fluidic device is optically transparent, allowing for imaging of the fluidic device.

[0018] In some cases, the adherent cell is coupled to the cell adherent support. In some cases, the adherent cell is coupled to the cell adherent support prior to (b) and (c). In some cases, the adherent cell is coupled to the cell adherent support subsequent to (b) and prior to (c). In some cases, the releasing in (c) comprises lysing the adherent cell to release the biomolecule.

[0019] In some cases, the surface is functionalized with one or more oligonucleotides. In some cases, the one or more oligonucleotides each comprise a barcode sequence. In some cases, the one or more oligonucleotides each comprise a spatial location tag corresponding to a unique location of the oligonucleotide on the surface. In some cases, the one or more oligonucleotides comprise a binding sequence configured to couple to the biomolecule. In some cases, the binding sequence comprises a polyT sequence, and wherein the biomolecule comprises a messenger ribonucleic acid (mRNA) comprising a polyadenylated (poly A) tail. In some cases, the one or more oligonucleotides are distributed amongst one or more reaction sites on thesurface, and each reaction site has a density of the one or more oligonucleotides from about 2 x 102to about 5 x 107oligonucleotides / pm2.

[0020] In some cases, the biomolecule comprises a complementary binding sequence configured to couple to the binding sequence of the one or more oligonucleotides. In some cases, the one or more oligonucleotides are distributed amongst one or more reaction sites on the surface, and each reaction site has a density of the one or more oligonucleotides from about 2 x 102to about 5 x 107oligonucleotides / pm2.

[0021] In some cases, the one or more oligonucleotides are treated with one or more cell adhesive proteins. In some cases, the one or more oligonucleotides comprise a binding sequence, wherein a poly A-streptavidin is coupled to the binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin. In some cases, the one or more oligonucleotides comprise: (i) a first binding sequence, wherein a poly A-streptavidin is coupled to the first binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin; and (ii) a second binding sequence, wherein the second binding sequence is configured to associate with the biomolecule. In some cases, the cell adhesive protein is biotinylated. In some cases, the cell adhesive protein comprises laminin. In some cases, the cell adhesive protein comprises fibronectin. In some cases, the cell adhesive protein comprises poly- 1-lysine (PLL). In some cases, the cell adhesive protein comprises poly-d-lysine (PDL). In some cases, the cell adhesive protein comprises vitronectin. In some cases, the cell adhesive protein comprises collagen. In some cases, the cell adhesive protein comprises gelatin. In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a RGD peptide (arginine-glycine-aspartic acid). In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a cell adhesive peptide such as, for example, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. A peptide reactive polymer can be a N-hydroxysuccinimide siloxane polymer, N- hydroxysuccinimide polymer, epoxy functionalized polymer, aldehyde functionalized polymer, or a combination thereof.

[0022] In some cases, the cell adherent support comprises one or more cell adherent biomolecules. In some cases, the one or more cell adherent biomolecules comprise a protein. In some cases, the protein comprises poly-l-lysine (PLL). In some cases, the protein comprises poly-d-lysine (PDL). In some cases, the protein comprises laminin and comprises poly-1- ornithine (PLO). In some cases, the protein comprises fibronectin. In some cases, the proteincomprises vitronectin. In some cases, the protein comprises collagen. In some cases, the protein comprises gelatin. In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a RGD peptide (arginine-glycine-aspartic acid). In some cases, the cell adherent support comprises a peptide reactive polymer coupled to a cell adhesive peptide such as, for example, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. A peptide reactive polymer can be a N-hydroxy succinimide siloxane polymer, N-hydroxysuccinimide polymer, epoxy functionalized polymer, aldehyde functionalized polymer, or a combination thereof. In some cases, the one or more cell adherent biomolecules comprise streptavidin. In some cases, the one or more cell adherent biomolecules comprise biotinylated fibronectin. In some cases, the one or more cell adherent biomolecules comprise biotinylated laminin. In some cases, the one or more cell adherent biomolecules comprise one of biotinylated RGD peptide, biotinylated DGEA (SEQ ID NO: 14), biotinylated IKVAV (SEQ ID NO: 15), biotinylated YIGSR (SEQ ID NO: 16), biotinylated KQAGDV (SEQ ID NO: 17), biotinylated CKKQRFRHRNRKG (SEQ ID NO: 18), biotinylated LIGRKK (SEQ ID NO: 19), biotinylated SPPRRARV (SEQ ID NO: 20), biotinylated NGRAHA (SEQ ID NO: 21), biotinylated ID APS (SEQ ID NO: 22), biotinylated REDV (SEQ ID NO: 23), biotinylated KRSR (SEQ ID NO: 24), biotinylated VGVAPG (SEQ ID NO: 25), biotinylated CNYYSNS (SEQ ID NO: 26), and combinations thereof. In some cases, a density of the one or more cell adherent biomolecules on the cell adherent support is 200 to about 5 x 107cell adherent biomolecules / mm2.

[0023] In some cases, the cell adherent support comprises one or more cell adherent particles. In some cases, the one or more cell adherent particles comprise a polymer particle at least partially coated with a particle cell adherent coating. In some cases, the particle cell adherent coating comprises a fibronectin coating. In some cases, the particle cell adherent coating comprises poly-l-ornithine. In some cases, the particle cell adherent coating comprises a laminin coating. In some cases, the particle cell adherent coating further comprises poly-l-ornithine. In some cases, the particle cell adherent coating comprises poly-l-lysine. In some cases, the particle cell adherent coating comprises poly-d-lysine. In some cases, the particle cell adherent coating comprises vitronectin. In some cases, the particle cell adherent coating comprises collagen. In some cases, the particle cell adherent coating comprises gelatin. In some cases, the particle cell adherent coating comprises one of RGD peptide, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), biotinylated YIGSR (SEQ ID NO: 16), KQAGDV (SEQ ID NO: 4),CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), and combinations thereof. The particle cell adherent coating can further comprise a functional group such as N- hydroxysuccinimide moieties, epoxide moieties, aldehyde moieties, or a combination thereof. In some cases, a density of the one or more cell adherent particles on the surface is from about 10 to about 2500 particles / mm2.

[0024] In some cases, the surface is functionalized with one or more oligonucleotides, and wherein the one or more cell adherent particles rest on top of the one or more oligonucleotides. In some cases, the surface comprises one or more flow channels, wherein a flow channel of the one or more flow channels comprises one or more arrays, wherein an array of the one or more arrays comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises: (a) a first oligonucleotide sequence unique to a spatial location of the reaction site within the array; and (b) a second oligonucleotide unique to a spatial location of the array of the one or more arrays. In some cases, each array comprises greater than 500 reaction sites. In some cases, each array comprises greater than 1000 reaction sites. In some cases, each array comprises greater than 1500 reaction sites. In some cases, the surface comprises at least 5 flow channels. In some cases, the surface comprises at least 10 flow channels. In some cases, the surface comprises at least 15 flow channels. In some cases, the surface comprises at least 30 arrays. In some cases, a flow channel of the one or more flow channels comprises at least 5 arrays. In some cases, a flow channel of the one or more flow channels comprises at least 10 arrays. In some cases, the one or more reaction sites are separated by a pitch distance of about 100 to 150 pm. In some cases, each reaction site has a diameter of about 50 to 100 pm.

[0025] In some cases, the hydrogel chamber is formed upon polymerization of one or more polymer precursors. In some cases, the fluidic device is in optical communication with a spatial energy modulation element configured to selectively direct energy to the fluidic device. In some cases, the method comprises a detector in optical communication with the fluidic device.

[0026] In some cases, the biomolecule comprises genetic material. In some cases, the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some cases, the genetic material comprises messenger RNA (mRNA). In some cases, the fluidic device further comprises a cell. In some cases, the biomolecule comprises a component of the cell. In some cases, the component of the cell comprises messenger ribonucleic acid (mRNA).

[0027] In some cases, the cell comprises an adherent cell. In some cases, the cell adherent support is configured to couple to said adherent cell. In some cases, the cell adherent support is functionalized for association with an additional biomolecule. In some cases, the additionalbiomolecule comprises a secreted protein. In some cases, the additional biomolecule comprises a surface protein. In some cases, the surface further comprises an antibody layer configured to capture one or more proteins. In some cases, the proteins comprise cytokines.

[0028] In some cases, the method further comprises, prior to (c), performing one or more assays on the adherent cell. In some cases, the one or more assays comprises a fluorescence assay. In some cases, the one or more assays comprises a secretion assay. In some cases, the adherent cell secretes one or more cytokines. In some cases, the one or more assays comprise measuring one or more surface markers of the adherent cell. In some cases, the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof. In some cases, the one or more assays comprises a morphological assay.

[0029] In another aspect, provided herein is a method for processing a cell, comprising: (a) introducing a sample comprising a plurality of cells to a fluidic device, wherein the one or more cells settle on a surface of the fluidic device, wherein the surface comprises one or more reaction zones, and wherein a reaction zone of the one or more reaction zones: (i) is functionalized for association with a biomolecule, and (ii) comprises a cell adherent support, wherein the plurality of cells settle on the surface; (b) incubating the one or more cells on the surface such that the one or more cells transform to an adherent state; (c) encapsulating a cell of the one or more cells and at least a portion of the reaction zone in a hydrogel chamber; and (d) lysing the cell to release messenger ribonucleic acid (mRNA) molecules, wherein the mRNA molecules settle to the surface.

[0030] In some cases, the surface is further functionalized with one or more protein capture antibodies. In some cases, prior to (d), one or more proteins are secreted from the cell. In some cases, the method further comprises capturing the secreted proteins with the protein capture antibodies. In some cases, the cell comprises an adherent cell. In some cases, the cell adherent support is configured to couple to said adherent cell. In some cases, (b) occurs prior to (c). In some cases, (c) occurs prior to (b).

[0031] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, where only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0032] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTIONS OF THE DRAWINGS

[0033] FIG. 1 illustrates an embodiment of a fluidic device with barcoded oligonucleotides on a bottom surface to capture mRNA released from a cell.

[0034] FIG. 2 illustrates an embodiment of a fluidic device with barcoded oligonucleotides on a bottom surface that have been post-treated with cell adhesive proteins.

[0035] FIG. 3 illustrates an embodiment of a fluidic device with a bottom surface that is cospotted with oligonucleotides and cell adhesive proteins.

[0036] FIG. 4 illustrates an embodiment of a fluidic device with barcoded oligonucleotides on a bottom surface with cell adherent particles disposed thereon.

[0037] FIG. 5 illustrates an embodiment of a fluidic device with barcoded oligonucleotides and a cell adhesive coating on a bottom surface.

[0038] FIG. 6 illustrates an embodiment in which cells are caged prior to incubation and analysis.

[0039] FIGs. 7A and 7B illustrate an embodiment of FIG. 6 using fibroblast cells that are caged prior to incubation. FIG. 7A shows an image of IMR90 (fibroblast) cells that have been induced (top) and uninduced (bottom) with intracellular staining of P16 (green) and P21 (blue). FIG. 7B illustrates the process by which cells are caged, and subsequently incubated, adhered, fixed, and stained.

[0040] FIG. 8 illustrates an embodiment by which cells are incubated prior to caging and analysis.

[0041] FIGs. 9A and 9B illustrate an embodiment of FIG. 8 using adherent neurons that are incubated prior to enclosure within cages. FIG. 9A shows an image of caged adherent neurons. FIG. 9B illustrates a process by which neurons are incubated and adhered prior to enclosure within cages.

[0042] FIG. 10A illustrates a portion of a surface of a fluidic device, spanning a width of 15 fields in a given lane.

[0043] FIGs. 10B and 11 A illustrate an exploded view of one of the fields shown in FIG. 10 A.

[0044] FIG. 1 IB illustrates a zoomed in view of the field of 11 A.

[0045] FIG. 12 shows a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.

[0046] FIG. 13 shows a portion of a system as provided herein including an energy source, according to some embodiments.

[0047] FIG. 14 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0048] FIG. 15 illustrates an embodiment of a fluidic device with a mixture of barcoded mRNA capturing oligonucleotides and protein grafting oligonucleotides for various reaction zones on a bottom surface that has been post-treated with cell adhesive proteins. FIG. 15 discloses SEQ ID NO: 28.

[0049] FIG. 16 is a top-down image of a fluidic device that contains cells adhered to gelatin spots that are co-disposed with oligonucleotide barcodes on a bottom surface of the fluidic device.

[0050] FIG. 17 is a top-down image of a fluidic device that contains cells adhered to fibronectin that is coupled to oligonucleotide barcodes on a bottom surface of the fluidic device.

[0051] FIGs. 18A-F are images of fluidic devices that contain immune cells and antibody-coated surfaces. FIG. 18A is an image of a portion of a fluidic device with a bare glass bottom surface, an antibody-coated top surface, immune cells that have been activated to release cytokines configured to bind to the antibody-coated surface, and fluorescent detection antibodies configured to bind to the cytokines released by the cells. FIG. 18B is an image of a portion of a fluidic device with a bare glass top surface, an antibody-coated bottom surface, immune cells that have been activated to release cytokines configured to bind to the antibody-coated surface, and fluorescent detection antibodies configured to bind to the cytokines released by the cells. FIG. 18C is an image of a portion of a fluidic device with a bare glass top surface, an antibody- coated bottom surface, immune cells that have not been activated to release cytokines configured to bind to the antibody-coated surface, and fluorescent detection antibodies configured to bind to the cytokines released by the cells. FIG. 18D is a combined brightfield, fluorescence image of a portion of the fluidic device of FIG. 18 A. FIG. 18E is a combined brightfield, fluorescence image of a portion of the fluidic device of FIG. 18B. FIG. 18F is a combined brightfield, fluorescence image of a portion of the fluidic device of FIG. 18C.DETAILED DESCRIPTIONIntroduction

[0052] A polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of one or more biological components in a fluidic device to isolate selected biological components. A hydrogel matrix may be selectively generated to surround a component, for example by projecting light in a shape or pattern around the component to photopolymerize apolymer precursor to form polymer matrices in the shape or pattern in which light was projected into the fluidic device. One or more hydrogel or polymer matrix walls can be used to physically separate one or more biological components from one another.

[0053] In order to compartmentalize individual components of a biological sample, a polymer matrix (e.g., a hydrogel matrix) can be formed adjacent to or around at least of portion of an individual component in a fluidic device. The hydrogel matrix may be selectively generated to surround a component after the system detects the component or hydrogel matrices can be generated according to a predefined pattern in a fluidic device. The hydrogel matrix may allow reagents and smaller entities to pass while retaining the individual component of the biological sample in place. Because one or more individual components can be localized within a fluidic device (e.g., encapsulated) and the localized components be exposed to one or more reagents and / or washing solutions during and / or in between analyses, multiple assays can be performed within the compartments (e.g., simultaneously, substantially simultaneously, serially, etc.).

[0054] Different assays may be performed in different locations of the fluidic device, for example, to test effects of different treatment conditions. By having two or more components within a compartment, interactions between components can be studied as well. The polymer matrix can be degradable “on demand” allowing for controlled localization and release mechanisms. The solutions provided herein can retain spatial information of the components and generate data on a cellular, proteomic, transcriptomic, or genomic level. Since spatial information is retained, the data can be associated (e.g., linked) with phenotypic data. Further, the solutions provided herein can retain spatial information of the components and link data (e.g., phenotypic data) on a cellular, proteomic, transcriptomic, or genomic level.

[0055] Whenever the term “at least” precedes the first numerical value in a series of two or more numerical values, the term “at least” applies to each of the numerical values in that series of numerical values. For example, at least 1, 2, or 3 is equivalent to at least 1, at least 2, or at least 3.

[0056] Whenever the term “less than” precedes the first numerical value in a series of two or more numerical values, the term “less than” applies to each of the numerical values in that series of numerical values. For example, less than 3, 2, or 1 is equivalent to less than 3, less than 2, or less than 1.

[0057] The terms “coupled to,” “connected to,” and “in communication with,” as used herein, generally refer to any form of interaction between two or more entities, including mechanical, electrical, magnetic, electromagnetic, fluid, biological, and thermal interaction. Two components may be coupled to each other even though they are not in direct contact with each other.

[0058] The terms “polypeptide” and “peptide,” as used interchangeably herein, generally refer to a polymer of amino acids in which an amino acid may be linked to another amino acid by a peptide bond. In some examples, a polypeptide is a protein. The amino acid may be a naturally occurring amino acid or a non-naturally occurring amino acid (e.g., an amino acid analogue). The polypeptide can be linear or branched. The polypeptide can include modified amino acids. The polypeptide may be interrupted by non-amino acids. A polypeptide can occur as a single chain or an associated chain. The polypeptide may include a plurality of amino acids. The polypeptide may have a secondary and tertiary structure (e.g., the polypeptide may be a protein). In some examples, the polypeptide can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 1,000, 10,000, or more amino acids. The polypeptide may be a fragment of a larger polymer. In some examples, the polypeptide can be a fragment of a larger polypeptide, such as a fragment of a protein.

[0059] The term “amino acid,” as used herein, generally refers to a naturally occurring or non- naturally occurring amino acid (e.g., an amino acid analogue). The non-naturally occurring amino acid may be an engineered or synthesized amino acid.

[0060] The term “sample,” as used herein, generally refers to a chemical or biological sample containing a biological component. The biological component may comprise a cell, a nucleic acid, a microbiome, a protein, a combination of cells, a metabolite, a combination thereof, or any other suitable component of a biological sample. For example, a sample can be a biological sample including one or more cells. For another example, a sample can be a biological sample including one or more polypeptides. The biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool, and tears. The biological sample can be a fluid or tissue sample (e.g., skin sample). In some instances, the sample may be derived from a homogenized tissue sample (e.g., brain homogenate, liver homogenate, or kidney homogenate). In certain embodiments, the sample may include a specific type of cell (e.g., a neuronal cell, muscle cell, liver cell, or kidney cell,). The sample may comprise or be acquired from a diseased cell or tissue (e.g., a tumor cell or a necrotic cell), In some embodiments, the sample may include or may be from a disease- associated inclusion (e.g., a plaque, a biofilm, a tumor, or a non-cancerous growth). In certain embodiments, the sample may include or may be obtained from a cell-free bodily fluid, such as whole blood, saliva, or urine. In various embodiments, the sample can include circulating tumor cells. In some cases, the sample may include or may be an environmental sample (e.g., soil, waste, or ambient air), industrial sample (e.g., samples from any industrial processes), or a food sample (e.g., dairy product, vegetable product, or meat product). The sample may be processedprior to loading into a microfluidic device. For example, the sample may be processed to purify a certain cell type or polypeptide and / or to include reagents.

[0061] As used herein, the term “polymer matrix” generally refers to a phase material (e.g., continuous phase material) that comprises at least one polymer. In some embodiments, the polymer matrix refers to the at least one polymer as well as the interstitial space not occupied by the polymer. A polymer matrix may be composed of one or more types of polymers. A polymer matrix may include linear, branched, and crosslinked polymer units. A polymer matrix may also contain non-polymeric species intercalated within its interstitial spaces not occupied by polymer chains. The intercalated species may be solid, liquid, or gaseous species. For example, the term “polymer matrix” may encompass desiccated hydrogels, hydrated hydrogels, and hydrogels containing glass fibers. A polymer matrix may comprise one or more polymer precursors in a polymerized form, which generally refers to one or more molecules that upon activation can trigger or initiate a polymeric reaction. A polymer precursor can be activated by electrochemical energy, photochemical energy, a photon, magnetic energy, or any other suitable energy. As used herein, the term “polymer precursor” includes monomers (that are polymerized to produce a polymer matrix), porogens, and crosslinking compounds, which may include photo-initiators, other compounds necessary or useful for generating polymer matrices, and the like. A polymer matrix may be semi permeable so that cells and beads (ranging from 3 to 50 microns) are too big to pass through, but smaller reagents can pass through such as antibodies, buffering salts, cellular media, lysing agents (e.g., SDS, Triton-X, Triton-XlOO, Tween 20, Sarkosyl).

[0062] In some embodiments, as used herein, the term “local parameter” means a value of a parameter (such as, pH) in or immediately adjacent to a chamber formed by polymer matrix walls.

[0063] As used herein, the term “on demand” means an operation may be directed to individual, discrete, selected locations (e.g. a spatial location of polymer precursor solution; or a selected polymer matrix chamber). Such selection may be based on manual observation of optical signals or data collected by a detector, or such selection may be based on a computer algorithm operating on optical signals or data collected by a detector. Manual observation of optical signals or data collected by a detector can include either real-time detection or detection at a time period prior to modulating a unit of energy to polymerize polymer precursors or degrading a chamber. For example, a subset of chambers (all formed with photo-degradable polymer matrix walls) may be pre-selected for releasing and removing their contents based on position information and the values of optical signals from an analytical assay carried out in the chambers. The pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulatingelement) to degrade the polymer matrix walls of the pre-selected chambers. In another embodiment, the pre-selected chambers may be photo-degraded by selectively projecting a light beam of appropriate wavelength characteristics (for example, with the spatial energy modulating element) in the presence of a photoinitiator to degrade the polymer matrix walls of the preselected chambers. Examples of a photoinitiator includes one of lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2, 2’-azobis[2-methyl-N-(2 -hydroxy ethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin- Y, Riboflavin, and combination thereof. In another example, a plurality of chambers may be observed in real-time (e.g. via fluorescent microscopy) for detection of an analyte of interest and one or more chambers of the plurality of chambers is selected, in real-time, upon detection of the analyte of interest, for degradation.

[0064] As used herein, the term “analyte” generally refers to a discrete biological or chemical entity to be measured, detected, and / or distinguished using the methods and systems described herein. In some embodiments, an analyte may be a biological component as described herein.

[0065] As used herein, the term “morphology” generally refers to the study of the size, shape, and structure of biological components (i.e., cells) and of the relationships of constituent parts thereof. As used herein, “morphological assays” generally refer to assays used to analyze morphological features of the biological components. Morphological assays can utilize microscopy to identify the shape, structure, form, color, texture, pattern, and size of a biological component. In some embodiments, the shape is circular, oval, or oblong. In some embodiments, the size is an area, number of pixels, diameter, eccentricity (e.g., shortest diameter and longest diameter), perimeter, or texture of the biological material.

[0066] The present disclosure provides systems for compartmentalizing or isolating one or more biological components. The system can include a fluidic device containing or including one or more biological components. The fluidic device may contain or include one or more polymer precursors. In some cases, the fluidic device can comprise a first surface configured to couple or receive at least one of the one or more biological components to form a coupled biological component. The systems may also include at least one energy source, wherein the energy source is in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In various embodiments, the at least one energy source may form a polymer matrix on or adjacent to at least a portion of the one or more biological components.

[0067] In some cases, a sample may be introduced or provided to the system. In certain cases, the sample may comprise one or more biological components. In various cases, the biological components may be physically separated. In some cases, the biological components may bephysically separated but in fluidic communication with one another. In certain cases, the biological components may be in chemical communication with one another. The system may be used for single-cell analysis. In some embodiments, the system may be used for single-cell analysis on a genome level. For example, the system may be used for genome sequencing. For another example, the system may be used for deoxyribonucleic acid (DNA) sequencing. The system may be used for DNA sequencing of cell-free DNA, whole genome sequencing, whole exome sequencing, targeted sequencing, or 16S sequencing. The system may be used for studying DNA tags attached to biomolecules of interest. The biomolecules may comprise proteins, metabolites, etc. In some cases, the DNA may be a nuclear DNA or a mitochondrial DNA. The system may be used for single-cell or bulk analysis on a transcriptome level. For example, the system may be used for ribonucleic acid (RNA) sequencing. For example, the system may be used for 3’ or 5’ gene expression analysis, immune repertoire study of a cell, or full-length mRNA analysis. In some embodiments, the system may be used for single-cell analysis on a proteome level. The system may be used for functional assay(s) of a biological component. The system may be used for studying surface proteins, secreted proteins, or metabolites of a biological component. In some cases, the system may be used to measure a quality of a biological component. In some cases, the measured quality may be the size or shape of a biological component. In some cases, the system may be used to study epigenomics, DNA methylation, or chromatin accessibility in a biological component. The system may be used for other suitable assays, experiments, and processes.

[0068] In certain embodiments, the system may be used for single-cell analysis on an indirect cell-cell interaction level. For example, an effect of one or more molecules produced from a first cell on a second cell can be analyzed using the system as provided herein. Such a measurement may be conducted by enclosing the first and second cells in different chambers that are separated by about 5 to about 100 pm so that soluble factors secreted by the first cell diffuse to the second cell, and soluble factors secreted by the second cell diffuse to the first cell. In various embodiments, the system may be used for analyzing direct cell-cell interactions. For example, two or more cells (e.g., a first cell and a second cell) can be in physical contact and the effect or effects of the first cell on the second cell, or vice versa, can be analyzed using the system as disclosed herein. In some embodiments, the system may be used for drug response analysis in a biological component. In certain embodiments, the system may be used for analyzing a biological component’s response to various physiological conditions (e.g., various media, temperature, mechanical stimuli, etc.). In some embodiments the analyte is selected from a plurality of analytes in the fluidic device prior to (a).

[0069] In certain embodiments, one or more polymer precursors may be added to or included with the biological sample. One or more biological samples and one or more polymer precursors may be introduced into the system (e.g., into the fluidic device of the system). The one or more biological samples and the one or more polymer precursors may be introduced into the fluidic device in any order (e.g., in parallel, sequentially, etc.). For example, the biological sample(s) may be introduced prior to the polymer precursor(s), the polymer precursor(s) may be introduced prior to the biological sample(s), the biological sample(s) and polymer precursor(s) may be introduced simultaneously (or substantially simultaneously), or in any other suitable manner or order.

[0070] In some embodiments, a polymer precursor may include one or more hydrogel precursors, a porogen, and a photoinitiator. As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of a photoinitiator includes one of lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP), Irgacure 2959, diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO) nanoparticles, 2, 2’-azobis[2-methyl-N-(2 -hydroxy ethyl) promionamide] (VA-086), BAPO-Oli, BAPO-Ona, Eosin- Y, Riboflavin, and combination thereof.

[0071] 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. In some embodiments, a porogen can be polyethylene glycol (PEG, molecular weight from 1 kDa to 1000 kDa), 8 arm PEG, 4 arm PEG, 3 arm PEG, and combinations thereof.

[0072] The one or more polymer precursors may be stored and / or introduced separately into the system. In some cases, the one or more polymer precursors may be mixed with the one or more biological components prior to introduction into the system. In various cases, the one or more polymer precursors may be mixed with the one or more biological components after introduction into the system.

[0073] The system may comprise a fluidic device. In some embodiments, the fluidic device may include one or more polymer precursors. In other words, one or more polymer precursors may be disposed within at least a portion of the fluidic device (e.g., within at least a portion of a channel of the fluidic device). In some embodiments, the fluidic device may comprise one or more channels or chambers. In some embodiments, the fluidic device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000 channels or chambers, or any number of channels or chambers between any of the two numbers mentioned herein. In some embodiments, the fluidic device comprises more than 10,000 channels or chambers. As described herein, the fluidic device may include one or more channels. The fluidic device may also, or alternatively, include one or more chambers. The terms channel and chamber may be used interchangeably in the disclosure herein unless indicated otherwise. For example, a channel or a chamber of the fluidic device may comprise a first surface, a second surface, or more surfaces.

[0074] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” “flow channel,” or “reaction chamber,” as opposed to a chamber that is formed from polymer matrix walls within a channel) may receive or be configured to receive a biological sample. FIG. 12 shows a simplified schematic cross-sectional side view illustration of a portion of a channel 300 that may be disposed in at least a portion of a fluidic device of a system as provided herein. The fluidic device may comprise a channel 300. The channel 300 may comprise a first surface 301. Further, the channel 300 may comprise a second surface 302. In some embodiments, the first surface 301 and the second surface 302 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 12). In some embodiments, a middle spacer layer of double sided adhesive with a cut-out portion can be used to position the first surface 301 and second surface 302 in a facing relationship to at least partly form the flow channel. In some embodiments, the first surface and second surface are substantially parallel, so that the perpendicular distance between them is substantially the same throughout the channel, for example, where chambers are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part and the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range offrom twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 301 may be a lower surface. In certain embodiments, the second surface 302 may be an upper surface. The channel 300 may receive a biological sample comprising one or more biological components 50, 51. The channel 300 may receive one or more polymer precursors. As illustrated in FIG. 12, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 301, the second surface 302, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 301 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the second surface, 302 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In some embodiments, the first surface and / or second surface can be optically transmissive so that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flowcell, and the measurement of the analyte and biological components.

[0075] 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.

[0076] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may be functionalized, for example, with a coating (e.g., a surface coating). In some embodiments, the surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), a functional group to capture one or more 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 someembodiments, 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.

[0077] In some cases, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise one or more barcodes (e.g., nucleic acid barcodes). In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 500 nm2to about 100,000 pm2and preferably 500 nm2to about 5000 pm2. In some embodiments, the first surface 301, the second surface 302, or both surfaces 301, 302 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodes between any of the two numbers mentioned herein. In some cases, a barcode (or a portion / subset of barcodes) may be associated with the location of the barcode on a surface (location coordinates (e.g., x-, y-coordinates) on a surface of a channel). A barcode may be attached to or coupled to the captured biological component. In some embodiments, the barcode may be a unique identifier that distinguishes a biological component from other biological components (e.g., that identifies a first biological component versus a second biological component). In some embodiments, a barcode may comprise a nucleic acid sequence (e.g., common sequence) to capture a biological component, or used in amplification. In some embodiments, a barcode may comprise a unique identifier comprising a unique nucleic acid sequence (e.g., DNA sequence, RNA sequence, etc.), protein tag, antibody, or an aptamer. In some embodiments the barcode may comprise a fluorescent molecule. In some embodiments, a location of the captured biological component may be associated with the unique identifier to, for example, retain spatial information of a biological component.

[0078] In some embodiments, the fluidic device may be a flow cell. For example, the fluidic device may be used for sequencing (e.g., DNA or RNA sequencing). In some embodiments, the fluidic device may be a microfluidic device. In certain embodiments, the fluidic device may be a nanofluidic device.

[0079] The system disclosed herein may comprise one or more energy sources. The energy source may be in communication with the fluidic device. In some embodiments, the energy source may be in optical communication with the fluidic device. In some cases, the energysource can be used to form one or more polymer matrices in the fluidic device (e.g., on or adjacent to a surface of a channel or chamber of the fluidic device). In some embodiments, the energy source may comprise a light generating device, a heat generating device, an electrochemical reaction generating device, an electrode, or a microwave device. A polymer matrix may be formed in a channel of the fluidic device. The energy source may direct or transfer energy to a predetermined position in the fluidic device. The energy may cause or activate the one or more polymer precursors to form a polymer matrix (e.g., to polymerize) in the predetermined position.

[0080] In some embodiments, the polymer matrix may comprise a hydrogel. In some embodiments, the hydrogel may be porous enough, or have pores of a suitable size, to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) through the polymer matrix, while the hydrogel may not allow movement or transfer of the biological component (e.g., DNA, RNA, a protein, a cell, etc.) through the polymer matrix. In some embodiments, the pores may have a diameter from 5 nm to 100 nm. In some embodiments, the pores may have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores may have a diameter smaller than 5 nm. The reagent may comprise an enzyme or a primer having a size of less than 50 base pairs (bp). A primer may comprise a single-stranded DNA (ssDNA). In some embodiments, a primer may have a size from 5 bp to 50 bp. In some embodiments, a primer may have a size from 5 bp to 10 bp, 10 bp to 20 bp, from 20 bp to 30 bp, 30 bp to 40 bp, or 40 bp to 50 bp. In some embodiments, a primer may have a size of more than 50 bp. In certain cases, a primer may have a size of less than 5 bp. A reagent may comprise a lysozyme, a proteinase K, hexamers (e.g., random hexamers), a polymerase, a transposase, a ligase, a catalyzing enzyme, a deoxyribonuclease, a deoxyribonuclease inhibitor, a ribonuclease, a ribonuclease inhibitor, DNA oligos, deoxynucleotide triphosphates, buffers, detergents, salts, divalent cations, or any other suitable reagent.

[0081] FIG. 13 shows a portion of a system as provided herein including an energy source 203. The embodiment of FIG. 13 may include components that resemble components of FIG. 12 in some respects. For example, the embodiment of FIG. 13 includes a channel 200 that may resemble the channel 300 of FIG. 12. With continued reference to FIG. 13, the channel 200 of the system may include a first surface 201 and a second surface 202. In some embodiments, the energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise an energy one or morenon-emitting portions (e.g., a non-emitting portion 204). The non-emitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. In certain embodiments, the emitting portion 205 can emit energy to the fluidic device. In some embodiments, the fluidic channel may be coupled to on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the fluidic channel. In some embodiments, the emitting portion 205 may be coupled to an objective (e.g., a microscope objective or lens), where the objective may be moved to different portions of the fluidic device. The objective may provide a shape (e.g., virtual mask) to allow light to form a pattern on the fluidic device, in order to form a polymer matrix similar or complementary to the pattern. In various embodiments, the one or more polymer precursors in the fluidic device or mixed with the biological sample can absorb emitted energy 206. In some embodiments, the emitted energy 206 can form, or be sufficient to form, a polymer matrix from the one or more polymer precursors. For example, a portion of the one or more polymer precursors within the channel 200 of the fluidic device may be activated by the emitted energy and a polymerization reaction may be initiated to form a polymer matrix.

[0082] The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on at least a portion of a surface of the fluidic device. The polymer matrix may be formed similarly or complementary to the pattern of energy emission.

[0083] In some embodiments, a first polymer matrix 208 can be formed on or adjacent to a biological component 50. In certain embodiments, the first polymer matrix 208 can form a cylindrical analysis chamber or compartment 220 that separates (e.g., physically separates) the biological component 50 from other biological components (e.g., biological components 51, 52, or 53) in the fluidic device. Stated another way, the polymer matrix may compartmentalize the channel (e.g., channel 200) in cooperation with the first surface 201 and the second surface 202. In various embodiments, the polymer matrix may partially surround a biological component. For example, a polymer structure fully surrounding a biological component may form a closed structure (e.g., a hollow cylinder-shaped polymeric structure) or a partially open structure (e.g., a crescent-shaped polymeric structure). In some embodiments, two or more polymer matrices may be formed adjacent to a biological component forming a compartment separating the biologicalcomponent from other biological components. In certain embodiments, the polymer matrix may comprise or form a wall (e.g., a polymer matrix wall).

[0084] With continued reference to FIG. 13, in some cases, the energy source 203 can, or be configured to, form or produce one or more emitting portions 205 and one or more non-emitting portions 204. The systems disclosed herein may further include a spatial energy modulating element to direct energy from the energy source to one or more targeted portions of the fluidic device. For example, the spatial energy modulating element may be configured to selectively direct the energy from the energy source to form a polymer matrix in a discrete area of the fluidic device. In some embodiments, the discrete area is chosen based on the location of a biological component. In some embodiments, the area of the discrete area is less than the area of the fluidic device. In some embodiments, a biological component is captured within the discrete area. In some embodiments, the size and shape of the discrete area is adjustable according to the size, shape, or other properties of the biological component. In some embodiments, an algorithm is used to determine the shape and size of the discrete area. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. The spatial energy modulating element may be configured to selectively direct the energy by, for example, inhibiting or preventing energy from being directed to one or more portions other than the one or more targeted portions of the fluidic device. In some embodiments, the spatial energy modulating element may comprise a physical mask. In some cases, the spatial energy modulating element may comprise a virtual mask. In some cases, the spatial energy modulating element may be a spatial light modulator (SLM). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid-crystal based.

[0085] In some embodiments, the first surface 201 or the second surface 202 may comprise a detector that detects, or is configured to detect, one or more locations of one or more biological components in the fluidic device (e.g., in the channel 200). In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In some embodiments, the detector may be a microscope objective for imaging the fluidic device. In various embodiments, a mask may be generated using an image obtained from at least a portion of the fluidic device. The mask may allow or permit the energy source 203 to emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. The mask may inhibit or prevent the energy source 203 from emitting energy in or toward one or more locations or positions where one or more biological components are present on or adjacent the first surface 201. In some embodiments,the image may be obtained from a camera (e.g., a digital camera, fluorescent imaging camera, etc.). In some embodiments, the imaging is bright-field imaging, phase-contrast imaging, or fluorescence imaging, or any combination thereof. In some embodiments, the camera may be coupled to, connected to, or in communication with the energy source 203. For example, the camera (not shown) may be in electrical communication with the energy source 203. In some embodiments, the energy source 203 may comprise the camera. In various embodiments, the energy source 203 may comprise a microscope (e.g., a fluorescence microscope, a confocal microscope, lens-free imaging system, a transmission electron microscopy (TEM), a scanning electron microscope (SEM), etc.). The microscope may be used to detect one or more positions of one or more biological components (e.g., in combination with the detector).

[0086] In some embodiments, an algorithm is used to determine where a biological component or analyte is located based on the imaging. In some embodiments, the algorithm is a supervised, a self-supervised, or an unsupervised learning algorithm. In some embodiments, the objective is coupled to an energy source to emit energy to the predetermined portion in the fluidic channel.Spatially Barcoded Surface

[0087] Surfaces of fluidic devices or flow cells can be functionalized with oligonucleotides that hybridize to cells or cellular components (including nucleic acids). Using the same surface for other applications (coupling to adherent cells or grafting antibodies, for example) can be challenging due to the oligonucleotide spotting. Described herein are systems, devices, and methods for modifying capture oligonucleotide barcodes to make them cell adherent and / or cospotting oligonucleotides and cell adherent biomolecules on a surface to promote adhesion of cells to the surface. The cell adherent biomolecules may form a ‘cell adherent coating’ (or, as used herein synonymously, a ‘cell adherent support’) on the surface or a portion of the surface of the fluidic device. In reference to FIG. 3, in some embodiments, a cell adherent material 160 can be co-spotted with oligonucleotide barcodes 105 in discrete spots or regions along a fluidic device surface 101. In reference to FIG. 5, in some embodiments, a cell adherent material 130 can coat the majority or entirety of a fluidic device surface 101, wherein the fluidic device surface also contains oligonucleotide barcodes 105. In other embodiments, for example as depicted in FIG. 2, a fluidic device surface 101 may contain oligonucleotide barcodes 105, and the oligonucleotide barcodes can be functionalized with an adherent material 160 such as fibronectin.

[0088] Such embodiments may be used, for example, in applications for adherent cell attachment and growth or secreted protein capture on a surface spotted with oligonucleotides for mRNA capture. The systems and methods described herein can allow adherent cell attachmentand / or protein capture without negatively impacting the mRNA capture on oligonucleotides. In addition, under certain conditions, the mRNA capture can be more efficient because the mRNA is released and captured on the same surface due to a relatively shorter diffusion pathway. It is also worthwhile to note that the oligonucleotide reaction zones that include cell adhesive material can cause cells to aggregate mainly on the oligonucleotide reaction zone and not on the interstitial spaces (uncoated portion of the surface). This allows for a simple cage generation algorithm where cylindrical hydrogel structures can be created on a portion of or all of the reaction zones that have cells on the surface of the oligonucleotide reaction zone. Because there are few to no cells in between the oligonucleotide reaction zone, it is easier and faster to identify cells coincident with the reaction zones improving the capture of cells that were flowed into the fluidic device.

[0089] In an aspect, provided herein is a fluidic device for processing a biological component with a surface. In some cases, the surface comprises one or more oligonucleotides configured to associate with a biomolecule. The surface can further comprise a cell adherent support that is bound to at least a portion of the one or more oligonucleotides. The first surface may be a bottom surface of the fluidic device. The oligonucleotides may comprise barcodes, as described elsewhere herein. In some cases, the oligonucleotides comprise a spatial location tag corresponding to a unique location of the oligonucleotide on a surface of the fluidic device.

[0090] In some cases, the one or more oligonucleotides each further comprise a binding sequence configured to couple to a biomolecule. The binding sequence may include a polyT sequence, and the biomolecule can comprise a messenger ribonucleic acid (mRNA) comprising a polyadenylated (poly A) tail. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-streptavidin conjugate is coupled to the binding sequence. A biotinylated fibronectin can be coupled to the complementary binding sequence-streptavidin conjugate. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-streptavidin conjugate is coupled to the binding sequence. A biotinylated laminin is coupled to the complementary binding sequence-streptavidin conjugate.

[0091] In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-streptavidin conjugate is coupled to the binding sequence. A biotinylated RGD peptide can be coupled to the complementary binding sequence-streptavidin conjugate. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-streptavidin conjugate is coupled to the binding sequence. A biotinylated peptide selected from the group consisting of biotinylated RGD peptide, biotinylated DGEA (SEQ ID NO: 14), biotinylated IKVAV (SEQ ID NO: 15), biotinylatedYIGSR (SEQ ID NO: 16), biotinylated KQAGDV (SEQ ID NO: 17), biotinylated CKKQRFRHRNRKG (SEQ ID NO: 18), biotinylated LIGRKK (SEQ ID NO: 19), biotinylated SPPRRARV (SEQ ID NO: 20), biotinylated NGRAHA (SEQ ID NO: 21), biotinylated ID APS (SEQ ID NO: 22), biotinylated REDV (SEQ ID NO: 23), biotinylated KRSR (SEQ ID NO: 24), biotinylated VGVAPG (SEQ ID NO: 25), biotinylated CNYYSNS (SEQ ID NO: 26), and combinations thereof can be coupled to the complementary binding sequence-streptavidin conjugate.

[0092] In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-fibronectin conjugate is coupled to the binding sequence. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-laminin conjugate is coupled to the binding sequence. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence-RGD peptide conjugate is coupled to the binding sequence. In some cases, the one or more oligonucleotides comprise a binding sequence, and a complementary binding sequence- biotinylated peptide conjugate is coupled to the binding sequence. The biotinylated peptide can be selected from the group consisting of biotinylated RGD peptide, biotinylated DGEA (SEQ ID NO: 14), biotinylated IKVAV (SEQ ID NO: 15), biotinylated YIGSR (SEQ ID NO: 16), biotinylated KQAGDV (SEQ ID NO: 17), biotinylated CKKQRFRHRNRKG (SEQ ID NO: 18), biotinylated LIGRKK (SEQ ID NO: 19), biotinylated SPPRRARV (SEQ ID NO: 20), biotinylated NGRAHA (SEQ ID NO: 21), biotinylated ID APS (SEQ ID NO: 22), biotinylated REDV (SEQ ID NO: 23), biotinylated KRSR (SEQ ID NO: 24), biotinylated VGVAPG (SEQ ID NO: 25), biotinylated CNYYSNS (SEQ ID NO: 26), and combinations thereof.

[0093] In some cases, the one or more oligonucleotides comprise a binding sequence, and a poly A -streptavidin is coupled to the binding sequence. A biotinylated fibronectin can be coupled to the poly A -streptavidin. In some cases, the one or more oligonucleotides comprise a binding sequence, and a poly A -streptavidin can be coupled to the binding sequence. A biotinylated laminin can be coupled to the poly A -streptavidin.

[0094] FIG. 1 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface may comprise spots with one or more oligonucleotides 105. In some embodiments, the top surface 102 and the bottom surface 101 are disposed, placed, or positioned opposite of one another. The channel 100 may receive a cell 110. The channel 100 can also receive one or more polymer precursors. In some embodiments, the top surface 102 and / or the bottom surface 101 can be optically transmissive so that visible and / or UV light can transmitthrough one or both of the surface for the generation of polymeric hydrogels, imaging of the fluidic device, and measurement of the cell or components thereof. A hydrogel chamber 108 can be formed around a cell 110. In some cases, the cell 110 is encapsulated within the hydrogel chamber 108. The cell 110 can be lysed such that cellular components 120 are released from the cell into the hydrogel chamber. Cellular components can include mRNA. Once released from the cell, cellular components 120 (including mRNA) can diffuse through the channel 100 and couple to the oligonucleotides 105 on the top surface 102.

[0095] In some cases, a fluidic device can comprise a barcoded surface. The barcoded surface may be a bottom surface of the fluidic device. In some embodiments, chamber footprints containing spatial barcodes are randomly distributed and do not cover the entirety of surface. In some embodiments, a barcode array comprises a surface comprising regions, or zones, of successively smaller areas, wherein each zone comprises spatial barcodes comprising the same unique nucleotide sequence and wherein the total area of the zones comprises an area at least 70 percent of the surface, or at least 80 percent of the surface, or at least 90 percent of the surface, or at least 95 percent of the surface.

[0096] FIGs. 10A, 10B, 11 A, and 1 IB show a top view of an example of a barcoded surface. The surface may comprise one or more lanes (also sometimes referred to as a “flow channel”). In some cases, the fluidic device comprises 5 lanes to 50 lanes. In some cases, the fluidic device comprises 5 lanes to 10 lanes, 5 lanes to 20 lanes, 5 lanes to 30 lanes, 5 lanes to 50 lanes, 10 lanes to 20 lanes, 10 lanes to 30 lanes, 10 lanes to 50 lanes, 20 lanes to 30 lanes, 20 lanes to 50 lanes, or 30 lanes to 50 lanes. In some cases, the fluidic device comprises 5 lanes, 10 lanes, 20 lanes, 30 lanes, or 50 lanes. In some cases, the fluidic device comprises at least 5 lanes, 10 lanes, 20 lanes, or 30 lanes. In some cases, the fluidic device comprises at most 10 lanes, 20 lanes, 30 lanes, or 50 lanes. Each lane may be associated with a spatial barcode unique to the lane in which it is located. FIG. 10A shows a portion of a surface of a fluidic device with eight lanes.

[0097] Each lane may include one or more discrete locations referred to as a “field” or “array” with oligonucleotides disposed thereon. In some cases, each lane comprises 5 fields to 30 fields. In some cases, each lane comprises 5 fields to 10 fields, 5 fields to 15 fields, 5 fields to 20 fields, 5 fields to 30 fields, 10 fields to 15 fields, 10 fields to 20 fields, 10 fields to 30 fields, 15 fields to 20 fields, 15 fields to 30 fields, or 20 fields to 30 fields. In some cases, each lane comprises 5 fields, 10 fields, 15 fields, 20 fields, or 30 fields. In some cases, each lane comprises at least 5 fields, 10 fields, 15 fields, or 20 fields. In some cases, each lane comprises at most 10 fields, 15 fields, 20 fields, or 30 fields. Each field may be associated with a spatial barcode unique to the field in which it is located. In FIG. 10 A, each lane comprises 15 fields.

[0098] FIGs. 10B and 11 A show an exploded view of one of the fields of FIG. 10 A. Each field may include one or more discrete locations referred to as a “reaction site” or “dot” or “spot” or “discrete site” with oligonucleotides disposed thereon. In some cases, each field comprises 100 dots to 3,000 dots. In some cases, each field comprises 100 dots to 500 dots, 100 dots to 1,000 dots, 100 dots to 1,500 dots, 100 dots to 2,000 dots, 100 dots to 3,000 dots, 500 dots to 1,000 dots, 500 dots to 1,500 dots, 500 dots to 2,000 dots, 500 dots to 3,000 dots, 1,000 dots to 1,500 dots, 1,000 dots to 2,000 dots, 1,000 dots to 3,000 dots, 1,500 dots to 2,000 dots, 1,500 dots to 3,000 dots, or 2,000 dots to 3,000 dots. In some cases, each field comprises 100 dots, 500 dots, 1,000 dots, 1,500 dots, 2,000 dots, or 3,000 dots. In some cases, each field comprises at least 100 dots, 500 dots, 1,000 dots, 1,500 dots, or 2,000 dots. In some cases, each field comprises at most 500 dots, 1,000 dots, 1,500 dots, 2,000 dots, or 3,000 dots. Each reaction site may be associated with a spatial barcode unique to the dot or spot in which it is located. In FIGs. 10B and 11 A, the field includes 1536 dots. FIG. 1 IB shows a further zoomed in view of the field shown in FIGs. 10B and 11 A. FIG. 1 IB shows a field with 80 pm diameter dots and a 130 pm hexagonal pitch. As shown in FIG. 1 IB, a hydrogel chamber can be formed on the fluidic device that is at least partially vertically aligned with a dot. Based on the spatial barcoding scheme described above, the unique spatial location (lane, field, and spot) of an mRNA can be determined upon sequencing of the mRNA and oligonucleotide associated therewith.Adherent Cell Support

[0099] In an aspect, a surface of a fluidic device as described herein comprises a cell adherent support. The surface may be a bottom surface of a fluidic device. The second surface can also comprise one or more oligonucleotides.

[0100] The cell adherent support can promote adhesion, attachment, incubation, and fixation of cells to the surface or to the oligonucleotides. The cell adherent support may comprise a coating, one or more biomolecules, one or more particles, or any combination thereof.

[0101] The cell adherent support may be coupled to a surface of a fluidic device prior to fluidic device fabrication. An overview of such a method is disclosed in EXAMPLE 3. In this method, a glass slide is plasma treated to prime the slide for adherent support coating. The glass slide is then incubated in a solution containing the adherent support, dried, washed, and used for fluidic device fabrication.

[0102] The cell adherent support may also be coupled to a surface of a fabricated fluidic device. Such a method may include inputting a solution that contains the adherent support into a fluidic device, incubating the solution so that the adherent support couples to a surface of the fluidic device, and washing unbound adherent support out of the fluidic device.

[0103] Alternatively, the cell adherent support may be spotted on a surface of the fluidic device. EXAMPLE 1 overviews a method for cell adherent support spotting in which a surface of a glass slide is first coated in a polymer, and then oligonucleotide barcodes and gelatin were co-spotted into discrete circular sites along the polymer-coated surface of the glass slide to adhere to the polymer coating. A fluidic device was then fabricated using the glass slide as a bottom surface. In some cases, when the cell adherent support is capable of adhering to a glass surface, the cell adherent support can be spotted directly onto surface of a fluidic device.

[0104] FIG. 2 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface may comprise spots with one or more oligonucleotides 105. A poly A- streptavidin 150 can be coupled to a binding sequence of an oligonucleotide. The oligonucleotides can be treated with one or more cell adherent materials (e.g., protein or peptide) 160. The cell adhesive material (e.g., protein or peptide) can couple to the poly A-streptavidin of the oligonucleotide. The cell adhesive material (e.g., protein or peptide) can promote adhesion of the cell 110 to the oligonucleotides or surface bottom surface 101.

[0105] In some cases, oligonucleotides have multiple categories of binding sequences. For example, the one or more oligonucleotides can have a first binding sequence that couple to a poly A-streptavidin. The poly A-streptavidin can then couple to a cell adhesive material (e.g., protein or peptide). The one or more oligonucleotides can also have a second binding sequence configured to couple to a biomolecule (including mRNA released from a cell).

[0106] FIG. 15 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface may comprise spots with a mixture of one or more oligonucleotides 105 and one or more protein grafting oligonucleotides 1502. The protein grafting oligonucleotides 1502 can include a binding sequence and optionally a spacer sequence, wherein the binding sequence is configured to bind to a complementary binding sequence. As an example, the protein grafting oligonucleotides 1502 can be bound at an amino moiety at the 5’ end and have an optional spacer sequence of TTTTTTTTT and a binding sequence of TCTTATATGGG (SEQ ID NO: 27). A complementary binding sequence-streptavidin 1504 can be coupled to the binding sequence of the protein grafting oligonucleotides 1502. The protein grafting oligonucleotides 1502 can be treated with one or more cell adherent materials (e.g., protein or peptide) 160. The cell adhesive material (e.g., protein or peptide) can couple to the protein grafting oligonucleotides 1502. The cell adhesive material (e.g., protein or peptide) can promoteadhesion of the cell 110 to the oligonucleotides or surface bottom surface 101. The cell adhesive material can be linked with a protein or peptide derivatized with a biotin functional group configured to bind to the streptavidin portion of the complementary binding sequence- streptavidin 1504.

[0107] In some cases, oligonucleotides can have multiple categories of binding sequences. For example, the one or more oligonucleotides can have a first binding sequence (poly T) that couples to biomolecule (including mRNA released from a cell). The protein grafting oligonucleotides 1502 can couple to complementary binding sequence-streptavidin 1504 and the complementary binding sequence-streptavidin 1504 can couple to biotin derivatized cell adherent material 160. Since the cell adherent material 160 and the complementary binding sequence-streptavidin 1504 are both not configured to bind to the one or more oligonucleotides 105, both cell adherent material 160 and complementary binding sequence-streptavidin 1504 do not interfere with the binding of mRNA released from a cell to the first binding sequence of the one or more oligonucleotides. Referring back to FIG. 2, under certain circumstances, poly A streptavidin and mRNA can compete with the binding sequence of the one or more oligonucleotides.

[0108] The cell adhesive material can be biotinylated. In some cases, the cell adhesive material comprises laminin, fibronectin, poly-l-lysine (PLL), poly-d-lysine (PDL), vitronectin, collagen, gelatin, RGD peptide (arginylglycylaspartic acid), cell adhesive peptides such as, for example, DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13), or any combination thereof.

[0109] A hydrogel chamber 108 can be formed around the cell 110 that is coupled to the oligonucleotides treated with cell adhesive proteins. The cell 110 can be lysed such that cellular components are released from the cell into the hydrogel chamber. Cellular components can include mRNA. Once released from the cell, cellular components (including mRNA) can settle through the channel 100 and couple to the oligonucleotides 105.

[0110] FIG. 3 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface can be co-spotted with oligonucleotides 105 and cell adhesive biomolecules (proteins) 160. The cell adhesive biomolecules 160 can comprise poly-l-lysine (PLL), poly-d- lysine (PDL), laminin, fibronectin, vitronectin, collagen, gelatin, streptavidin, RGD peptide(arginylglycylaspartic acid), cell adhesive peptides (e.g., DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13)), biotinylated fibronectin, or biotinylated laminin, or any combination thereof. The cell adhesive biomolecule can promote adhesion of the cell 110 to the oligonucleotides or surface bottom surface 101.

[0111] In some cases, a density of the cell adherent biomolecules on the surface is about 200 biomolecules / pm2to about 5xl07biomolecules / pm2. In some cases, a density of the cell adherent biomolecules on the surface is about 200 biomolecules / pm2to about 1,000 biomolecules / pm2, about 200 biomolecules / pm2to about 10,000 biomolecules / pm2, about 200 biomolecules / pm2to about 1,000,000 biomolecules / pm2, about 200 biomolecules / pm2to about 50,000,000 biomolecules / pm2, about 1,000 biomolecules / pm2to about 10,000 biomolecules / pm2, about 1,000 biomolecules / pm2to about 1,000,000 biomolecules / pm2, about 1,000 biomolecules / pm2to about 50,000,000 biomolecules / pm2, about 10,000 biomolecules / pm2to about 1,000,000 biomolecules / pm2, about 10,000 biomolecules / pm2to about 50,000,000 biomolecules / pm2, or about 1,000,000 biomolecules / pm2to about 50,000,000 biomolecules / pm2. In some cases, a density of the cell adherent biomolecules on the surface is about 200 biomolecules / pm2, about 1,000 biomolecules / pm2, about 10,000 biomolecules / pm2, about 1,000,000 biomolecules / pm2, or about 50,000,000 biomolecules / pm2. In some cases, a density of the cell adherent biomolecules on the surface is at least about 200 biomolecules / pm2, about 1,000 biomolecules / pm2, about 10,000 biomolecules / pm2, or about 1,000,000 biomolecules / pm2. In some cases, a density of the cell adherent biomolecules on the surface is at most about 1,000 biomolecules / pm2, about 10,000 biomolecules / pm2, about 1,000,000 biomolecules / pm2, or about 50,000,000 biomolecules / pm2.

[0112] A hydrogel chamber can be formed around the cell 110. The cell 110 can be lysed such that cellular components are released from the cell into the hydrogel chamber. Cellular components can include mRNA. Once released from the cell, cellular components (including mRNA) can settle through the channel 100 and couple to the oligonucleotides 105.

[0113] FIG. 4 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface can be co-spotted with oligonucleotides 105. One or more cell adherent particles 170 can be disposed on the oligonucleotides 105. The particles may be a polymer particle at least partially coated with a cell adherent coating. The particles (or cell adherentcoating thereon) can comprise poly-l-lysine (PLL), poly-d-lysine (PDL), laminin, fibronectin, vitronectin, collagen, gelatin, RGD peptide (arginylglycylaspartic acid), cell adhesive peptides (e g., DGEA (SEQ ID NO: 1), IKVAV (SEQ ID NO: 2), YIGSR (SEQ ID NO: 3), KQAGDV (SEQ ID NO: 4), CKKQRFRHRNRKG (SEQ ID NO: 5), LIGRKK (SEQ ID NO: 6), SPPRRARV (SEQ ID NO: 7), NGRAHA (SEQ ID NO: 8), ID APS (SEQ ID NO: 9), REDV (SEQ ID NO: 10), KRSR (SEQ ID NO: 11), VGVAPG (SEQ ID NO: 12), CNYYSNS (SEQ ID NO: 13)), streptavidin, biotinylated fibronectin, or biotinylated laminin, or any combination thereof.

[0114] A cell 110 can couple to the cell adherent particle 170. A hydrogel chamber 108 can be formed around the cell 110 that is coupled to the cell adherent particle. The cell 110 can be lysed such that cellular components are released from the cell into the hydrogel chamber. Cellular components can include mRNA. Once released from the cell, cellular components (including mRNA) can settle through the channel 100 and couple to the oligonucleotides 105.

[0115] In some cases, a density of the cell adherent particles on the surface is 10 particles / mm2to 2,500 particles / mm2. In some cases, a density of the cell adherent particles on the surface is 10 particles / mm2to 50 particles / mm2, 10 particles / mm2to 100 particles / mm2, 10 particles / mm2to 500 particles / mm2, 10 particles / mm2to 1,000 particles / mm2, 10 particles / mm2to 2,500 particles / mm2, 50 particles / mm2to 100 particles / mm2, 50 particles / mm2to 500 particles / mm2, 50 particles / mm2to 1,000 particles / mm2, 50 particles / mm2to 2,500 particles / mm2, 100 particles / mm2to 500 particles / mm2, 100 particles / mm2to 1,000 particles / mm2, 100 particles / mm2to 2,500 particles / mm2, 500 particles / mm2to 1,000 particles / mm2, 500 particles / mm2to 2,500 particles / mm2, or 1,000 particles / mm2to 2,500 particles / mm2. In some cases, a density of the cell adherent particles on the surface is 10 particles / mm2, 50 particles / mm2, 100 particles / mm2, 500 particles / mm2, 1,000 particles / mm2, or 2,500 particles / mm2. In some cases, a density of the cell adherent particles on the surface is at least 10 particles / mm2, 50 particles / mm2, 100 particles / mm2, 500 particles / mm2, or 1,000 particles / mm2. In some cases, a density of the cell adherent particles on the surface is at most 50 particles / mm2, 100 particles / mm2, 500 particles / mm2, 1,000 particles / mm2, or 2,500 particles / mm2.

[0116] FIG. 5 shows a cross-sectional side view illustration of a portion of a channel 100 of a fluidic device provided herein. The fluidic device may comprise a channel 100. The channel 100 may comprise a top surface 102. Further, the channel 100 may comprise a bottom surface 101. The bottom surface may comprise spots with one or more oligonucleotides 105. In some cases, the bottom surface is coated with a cell adherent support or coating 130. In some embodiments, the top surface 102 and the bottom surface 101 are disposed, placed, or positioned opposite of one another. The channel 100 may receive a cell 110. The channel 100 can also receive one ormore polymer precursors. In some embodiments, the top surface 102 and / or the bottom surface 101 can be optically transmissive so that visible and / or UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the fluidic device, and measurement of the cell or components thereof. A hydrogel chamber 108 can be formed around a cell 110. In some cases, the cell 110 is encapsulated within the hydrogel chamber 108. The cell adherent support or coating 130 can promote adhesion of the cell to the bottom surface 101. The cell 110 can be lysed such that cellular components are released from the cell into the hydrogel chamber. Cellular components can include mRNA. Once released from the cell, cellular components (including mRNA) can settle through the channel 100 and couple to the oligonucleotides 105 on the bottom surface 101.

[0117] The cell adherent coating can include one material or layers of one or more materials. The cell adherent coating can include fibronectin, poly-l-ornithine, or laminin, or any combination thereof.Cage Enclosure of Adherent Cells on a Surface

[0118] Hydrogel chambers can be formed to encapsulate cells as described elsewhere herein. The cells can be adherent cells. In some cases, cells are encapsulated in hydrogel chambers prior to incubation, adhesion, and spreading. FIGs. 6 and 7B show a top view of a fluidic device with one or more cells disposed thereon. A portion of the cells can be encapsulated in hydrogel chambers. After cage enclosure, the cells may adhere to a surface of the fluidic device.Following cage enclosure, the adherent cells can be incubated for a period of time. One or more live assays can be performed on the cells encapsulated in the hydrogel chambers. The assays may comprise detecting or measuring a characteristic or property of the cell. The adherent cells can be fixed and stained. The cells can be lysed, releasing mRNA into the hydrogel chambers. As discussed herein, mRNA released from the cells can diffuse to one or more oligonucleotides on a surface of the fluidic device. An image of induced (top) versus uninduced cells (bottom) is shown in FIG. 7A where IMR90 cells were caged, fixed, and stained on a fibronectin (50pg / mL) coated (30 mins, 23 °C) fluidic device. IMR90 (fibroblast) cells were treated with etoposide (cancer treatment drug) or cell media for 5 days to yield induced (senescent) and uninduced (healthy) cells respectively. The fibroblasts were encapsulated in hydrogel chambers and allowed to adhere to the fluidic device's fibronectin-coated surface. The IMR90 cells were then fixed with 4% paraformaldehyde (PF A), permeabilized with Triton X-100, and stained with pl6 and p21 conjugated antibodies. Brightfield and fluorescent images were acquired using lOx objective and overlay ed in Imaged. For induced cells, some of the cages show increased expression of P16 and P21 indicating the onset of the senescent state. For the uninduced cells, all of the cages show a significantly lower expression of P16 and P21 and did not show anindicating of transitioning to the senescent state. It is worthwhile to note that for uninduced and induced cells, the fibronectin coating caused the cells to adhere to the fibronectin treated surface.

[0119] Hydrogel chambers can be formed to encapsulate cells as described elsewhere herein. The cells can be adherent cells. In some cases, cells are encapsulated in hydrogel chambers after adhesion and spreading. FIG. 8 shows a top view of a fluidic device with one or more cells disposed thereon. The cells can be incubated on the surface for a period of time, allowing the cells to adhere and spread on the surface. A portion of the adhered cells can be encapsulated in hydrogel chambers. Following cage enclosure, the adherent cells can be fixed and stained. The cells can be lysed, releasing mRNA into the hydrogel chambers. As discussed herein, mRNA released from the cells can diffuse to one or more oligonucleotides on a surface of the fluidic device. FIG. 9A shows an image of adherent neurons caged post-incubation. The surface of the fluidic device was coated with 0.1 mg / ml poly-l-omithine (2h, 37C) followed by 20 ug / ml laminin (16h, 4C). After coating, cortical neurons derived from induced pluripotent stem cells (iXCells Biotechnologies #40HU-009 - IM) were thawed, resuspended in fresh medium and seeded into the fludic device. Neurons were cultured for 5 days in the fluidic device, and then encapsulated in hydrogel chambers and imaged at 10X in the brightfield channel. FIG. 9B shows a top view of a fluidic device with one or more neurons that are incubated and adhered to a surface prior to cage enclosure.Enclosure of Suspension Cells Within Cages

[0120] Hydrogel chambers can be formed to encapsulate cells as described elsewhere herein. The cells can include suspension cells, which may be cells that are not adhered to a surface. Under quiescent conditions (e.g., in between media refreshment steps), a suspension cell may diffuse throughout the interior of a hydrogel chamber or may settle onto a bottom fluidic device surface.

[0121] A suspension cell input into a fluidic device may transition to an adherent state. In some cases, such a cell may adhere to a surface of the fluidic device prior to hydrogel chamber formation. In other cases, a cell may adhere to a surface of the fluidic device after it is enclosed in a hydrogel chamberProtein Capture Antibody Layer

[0122] In an aspect, a surface of a fluidic device as described herein has an antibody layer to capture one or more proteins. The surface may be a bottom surface of a fluidic device. The antibody layer can be located on a same surface as one or more oligonucleotides. The antibody layer can be located on a same surface as a cell adherent support. The antibody layer can be usedto capture proteins secreted from a cell. The antibody layer can be used to capture surface proteins from a cell.

[0123] While antibodies are disclosed as an example capture element that may be coupled to a surface of a fluidic device to promote analyte capture, a surface may be functionalized with other capture elements such as antibody fragments (e.g., nanobodies, minibodies, scFvs, monobodies, diabodies, triabodies, Fab fragments, and the like), aptamers, affimers, DARPins, knottins, and combinations thereof. A surface may include a single capture element targeted to a single analyte, or may include a plurality of capture elements targeted to separate analytes.

[0124] A capture element (e.g., an antibody, an aptamer, or another capture element disclosed herein) may be coupled to a surface of a fluidic device prior to or during a cellular assay. In such a method, the capture element may be input into the fluidic device to couple to an adherent support on a surface of the fluidic device. An outline of such a method is provided in EXAMPLE 3, which covers antibody layer formation on poly-l-lysine (PLL) adherent supports on a surface of a fluidic device. Generalizing from this example, in some methods, a capture element is input into a fluidic device, wherein the antibody or other capture element couples to an adherent support on a surface of the fluidic device. In some cases, the adherent support is coupled to a top surface of the fluidic device. In some cases, the adherent support is coupled to a bottom surface of the fluidic device. In some cases, the adherent support is coupled to a top surface and a bottom surface of the fluidic device. In some cases, the antibody or other capture element is input into the fluidic device in a buffer that contains about 1 to about 250 pg / mL, about 5 to about 100 pg / mL, or about 15 to about 50 pg / mL of the antibody or the capture surface. The buffer may then be incubated in the fluidic device for about 1 to about 300 minutes, about 5 to about 60 minutes, or about 10 to about 30 minutes. The buffer may then be removed from the fluidic device through one or more wash steps to remove unbound capture elements from the fluidic device.

[0125] As a further example, a capture element may be coupled to a functional group on the surface of a fluidic device. In such methods, the capture element may be input into the fluidic device, wherein the capture element couples to a functional group on a surface of the fluidic device. Examples of functional groups consistent with the present disclosure include biotin, streptavidin, strep-tactin a strep tag, digoxigenin, tactin, and neutravidin. The capture element may include a binding element that couples to the functional group. For example, when the surface includes streptavidin, the capture element may include biotin or a strep tag that couples to the streptavidin. After a portion of the capture elements couple to functional groups on the surface of the fluidic device, unbound capture elements can then be washed out of the fluidic device.Cellular Measurement Methods

[0126] A disclosed method can include one or more forms of cellular analysis. Analysis may be performed on a single cell or collection of cells that are enclosed within a chamber within a fluidic device. Accordingly, cells within an individual chamber can be tracked independently of other cells that are present in the fluidic device. When two or more forms of analysis are performed on a cell, the two or more forms of analysis can be performed sequentially or in tandem.

[0127] Examples of cellular characteristics that can be measured in a disclosed method include cytotoxicity, proliferation rate, activation status, cellular identity, purity, gene expression profile, transcriptome, surface marker expression, gRNA expression, soluble factor secretion, activation status, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), morphology, subcellular localization, intracellular protein expression, or a combination thereof. A cell may be subjected to one or more forms of analysis at multiple time points. For example, the morphology, surface marker expression, activation status, soluble factor secretion profile, and / or one or more other measurements may be made on a cell as it transitions from a suspended state to an adhered state as the cell adheres to an adherent support disclosed herein.

[0128] A method disclosed herein can include detecting a guide ribonucleic acid (gRNA) associated with a genetic modification of a cell. For example, after determining whether a cell adheres to an adherent support, a genetic modification of the cell can be detected by detecting gRNA associated with the genetic modification, and the genetic modification can be associated with the adherence or non-adherence of the cell to the cell adherent support. The cell can be transiently or stably transfected with a nucleic acid encoding a gRNA specific for a particular genomic sequence. The guide RNA can be coupled to a barcode, an exogenous messenger RNA (e.g., a selection marker), a capture sequence (e.g., a polyA tail), or a combination thereof. The cell can express a Cas protein that can utilize the gRNA. Alternatively, a Cas protein can be delivered to the cell, for example in a chitosan particle or liposome that is configured for uptake by the cell. Cell growth, movement, or other characteristic or characteristics can then be correlated with a genomic edit imparted by a particular gRNA sequence. In one such method, the cells can be lysed to release guide RNA, the guide RNA can optionally be captured on a nucleic acid barcode, and then be used as a template for generating a cDNA molecule comprising a complement of the guide RNA sequence, and optionally additional sequences coupled to the guide RNA such as the exogenous mRNA, the barcode, or a combination thereof. The cDNA molecule can be coupled to a spatial location tag corresponding to a unique location within the channel of the fluidic device, such that the spatial location tag can be associated with a particular cell or chamber in the fluidic device.

[0129] In some aspects, a method includes determining a proliferation rate of a cell. It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. Proliferation rate can be determined by counting cells at least partially enclosed by one or more chambers generated during an assay. For example, one or more cells can be counted periodically (e.g., with fluorescence or brightfield imaging) following at least partial enclosure within one or more chambers to determine a rate of change in the number of cells. Separate proliferation rates can be determined for each cell or collection of cells enclosed by a unique chamber or collection of chambers. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Intracellular dyes for dye dilution can include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. After counts are recorded for each chamber, further assays may be conducted on the clonal populations within the chambers to identify the cell types, for example, by an assessment of cell surface proteins, cell protein secretions, transcriptome, or the like.

[0130] In some aspects, a method includes detecting a soluble factor secreted by a cell. Soluble factor analysis can include disposing a capture surface (e.g., a bead) near the cell, wherein the capture surface includes an affinity reagent (e.g., an aptamer or an antibody) that binds the soluble factor, and detecting the soluble factor bound to the capture surface. Disposing the capture surface near to the cell can include enclosing or at least partially enclosing the capture surface with the cell within one or more chambers, and optionally removing non-enclosed capture surfaces from the fluidic device that contains the cell. In an embodiment, the capture surface is a bead. As used herein, the term “bead” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide-based 30 to 10000 pm particle. However, further capture surfaces, such as nanotubes, nucleic acid nanostructures, and antibody Fc domains may be used. The affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof.

[0131] Soluble factor detection may also be performed with a bispecific binding agent capable of simultaneously binding to a cell and to a soluble factor secreted by the cell. The bispecific binding agent can be coupled to a target cell of interest and then used to capture soluble factors secreted by the cell. In this way, the bispecific binding agent may couple the soluble factor to the surface of the cell. The soluble factor may then be detected, for example by coupling a detectable binding agent such as a fluorescent antibody to the soluble factor coupled to the surface of the cell and measuring the detectable binding agent.

[0132] Examples of soluble factors include a cytokine, an immune active molecule, an interleukin, an interferon, a colony stimulating factor, a tumor necrosis factor, or a granzyme. Thecytokine may be interferon-y (IFN-y) and interferon-a (IFN-a), an interleukins such as interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin- 10 (IL-10), interleukin- 13 (IL-13), interleukin- 15 (IL-15), interleukin-21 (IL-21), or interleukin-23 (IL-23), a colony stimulating factor (CSFs) such as granulocyte-macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or a tumor necrosis factors (TNF) such as TNF-a or TNF-p. In another embodiment, the secreted factor comprises an effector molecule such as a granzyme.

[0133] A soluble factor bound to a binding agent can be detected by contacting the soluble factor bound to said capture surface with a labeled ‘detection’ antibody configured to bind to the soluble factor, and detecting the labeled antibody. Multiple soluble factors can be detected in a single assay by providing a capture surface or plurality of capture surfaces that comprise a plurality of affinity reagents configured to bind the plurality of soluble factors, contacting the plurality of soluble factors bound to the capture surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of soluble factors, and detecting a plurality of labels coupled to the plurality of antibodies.

[0134] In some aspects, a method includes sequencing at least a portion of a transcriptome of a cell. A chamber may co-enclose a cell with oligonucleotide barcodes within a fluidic device. The oligonucleotide barcodes may be coupled to one or more surfaces of the fluidic device. mRNA may be released from the one or more cells (e.g., by lysing the one or more cells) and captured on the oligonucleotide barcodes. The oligonucleotide barcodes may be extended using at least a portion of the captured mRNA as templates. Similarly, mRNA may be extended using at least a portion of an oligonucleotide barcode as a template. Extended nucleic acid barcodes, extended mRNA molecules, or an additional nucleic acid extended using at least a portion of an extended nucleic acid barcode or extended mRNA molecule as a template may be eluted from the fluidic device and sequenced. Examples of sequencing methods consistent with the present disclosure include nanopore sequencing, pyrosequencing, sequencing-by-hybridization, sequencing-by- ligation, sequencing-by-synthesis, single-molecule sequencing, digital gene expression, next generation sequencing, shotgun sequencing, Sanger sequencing, ion torrent sequencing, as well as other next-generation-sequencing methods known in the art.

[0135] The nucleic acid barcodes may contain spatial barcode sequences that are uniquely associated with the cell and / or the one or more chambers. Accordingly, extended nucleic acid barcodes (which contain the spatial barcodes) and mRNA extended using the nucleic acid barcodes as templates (which contain complements of the spatial barcodes) may be associated with a particular cell or a chamber. The nucleic acid barcodes may also contain unique molecular identifier sequences to facilitate mRNA quantitation by normalizing sequencing counts ofextended nucleic acid barcodes and / or mRNA extended using the nucleic acid barcodes as templates. For example, during sequencing, the number of instances of each mRNA sequence may be determined based on the number of unique molecular identifier sequences associated with that mRNA sequence.

[0136] In some cases, mRNA and / or nucleic acid barcode extension involves reverse transcription. Reverse transcription reagents may comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murine leukemia virus (MMLV)), dNTPs, optional RNase inhibitor, buffer.

[0137] In some aspects, a method includes measuring a surface marker expressed by a cell. Such a method may include combining a cell with a detection antibody (e.g., inputting the detection antibody into a fluidic device that contains the cell), wherein the detection antibody couples to the surface marker on the surface of the cell, and detecting a detectable moiety coupled to the detection antibody. In certain aspects, the detectable moiety is an optically detectable moiety such as a fluorophore or a dye. Surface markers that may be measured in a disclosed method comprise CD3, CD4, CD8, CD 19, CD25, CD45, or CD56, or any combination thereof

[0138] In some aspects, a method includes measuring activation of one or more cells. Cellular activation can be detected using numerous assays disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation or changes in proliferation, changes in morphology, change in cytotoxicity, or a combination thereof.Hydrogel Compositions

[0139] In some embodiments, a channel of a fluidic device of a system of the invention comprises one or more polymer precursors for forming chambers. In some embodiments, the one or more polymer precursors comprise hydrogel precursors. 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-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA), poly(lactic-co- gly colic acid) (PLGA), poly caprolactone (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. Insome 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. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises an optically cleavable hydrogel. In some embodiments, the degrading comprises exposing the hydrogel polymer wall to UV light. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are made of different materials. In some embodiments, the degrading in (b) does not degrade the hydrogel chamber. In some embodiments, the method further comprises degrading the hydrogel chamber. In some embodiments, the method further comprises imaging the analyte, the first biological material, the hydrogel chamber, the fluidic device, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a cPEG monomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (I):

[0140] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (II):(II).

[0141] In some embodiments, n is between about 0 to about 100, or optionally n is between about5 to about 50. In some embodiments, x is between about 1 to about 10, or optionally x is between about 2 to about 6. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (III):(III).

[0142] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IV):

[0143] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (V):

[0144] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VI):

[0145] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VII):(VII).

[0146] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (VIII):

[0147] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (IX):

[0148] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (X):(X).

[0149] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XI):

[0150] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XII):

[0151] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer including a structure (XIII):(XIII).

[0152] In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a monomer, the monomer comprising: an oligomeric domain comprising three or more arms, wherein each arm of said oligomeric domain comprises a degradable unit and a crosslinkable unit, wherein the crosslinkable unit of an arm of the three or more arms is configured to crosslink with another crosslinkable unit of another polymer precursor in response to a first stimulus, thereby obtaining the polymerized form of the monomer, and wherein the degradable unit is configured to be cleaved in response to a second stimulus, thereby solubilizing the polymerized form of the monomer. In some embodiments, the oligomeric domain comprises four or more arms. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a degradable functional group. In some embodiments, said degradable function group comprises disulfide, Beta-thioether ester, Amidomethylol and vicinal diol, Vicinal diol, Alginate backbone, Dextran backbone, Chitosan backbone, Hyaluronic acid backbone, Chondroitin sulfate backbone, or Carboxy methyl cellulose backbone, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a polymerized form of a hydrogel macromonomer. In some embodiments, the hydrogel macromonomer comprisescPEG, cSEL-BTEEC, cSEL-DHEBA, cSEL-diol, cSEL-alginate, cSEL-dextran, cSEL-chitosan, cSEL-hyaluronic acid, cSEL-chondroitin sulfate, or cSEL-cellulose, or a combination thereof. In some embodiments, the degradation unit is degraded by inputting a degradation reagent into the fluidic device. In some embodiments, the degradation reagent comprises DTT, TCEP, BME, GSH, DMEM, RPMI, PBS buffer, DMEM, RPMI, PBS buffer, sodium (meta)periodate, Alginate lyase (enzyme), Dextranase, Lysozyme and chitinase, Hyaluronidase, Chondroitinase, or Cellulases, or a combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises at least one beta-thioether ester. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a PEG-macromonom er containing beta-thioether esters. In some embodiments, the beta-thioether ester is formed by reacting an acrylate with a thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Michael donor. In some embodiments, the Michael donor is PEG-thiol. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a cSEL beta-thioether ester with one beta-thioether ester per arm. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises are formed from any material that comprises a PEG with a Michael acceptor chain. In some embodiments, the Michael acceptor chain comprises PEG-acrylamide, PEG- vinyl sulfone, PEG- maleimide, or PEG-carbonyl acrylic, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by cleavage of disulfide bonds. In some embodiments, the disulfide bonds are cleavable by one or more reducing agents. In some embodiments, the one or more reducing agents comprise DTT, TCEP, BME, or GSH, or any combination thereof. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more arms each comprising one or more amides.

[0153] In some embodiments, the hydrogel chamber or the hydrogel polymer wall is degradable by oxidative cleavage of vicinal diol by sodium (meta)periodate. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photocleavable 4-arm PEG- macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall is photodegradable via an ortho-nitrobenzyl moiety. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a Coumarin-based photodegradable macromonomer. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a 4-arm PEG- acrylamide comprising one or more disulfides. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises one or more cage disulfide bonds in a hydrogel cage formation. In some embodiment, the hydrogel cages degrade using light and a photoinitiator. In some embodiments, the hydrogel chamber or the hydrogel polymer wall enables hydrogel formation. In some embodiments, the hydrogel enables spatiotemporal control of hydrogel cage degradation, therefore enabling selective retention of cells with a single hydrogel formulation. Insome embodiments, upon exposure to light, photogenerated radicals’ initial multiple fragmentation and disulfide exchange reactions, thereby permitting and promoting photodeformation, photowelding and photodegradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, one or more polymer precursors enable formation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the hydrogel exhibits a chemical or physical change in response to an external stimulus. In some embodiments, the hydrogel chamber or the hydrogel polymer wall comprises a photolabile nitrobenxyl ester which lyses upon photon absorption, thereby allowing a user to exogenously control degradation of the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the method further comprises controlling a network degradation of the hydrogel chamber or the hydrogel polymer wall by concentration of a photoinitaitor infused into the hydrogel chamber or the hydrogel polymer wall. In some embodiments, the first hydrogel polymer wall comprises a shape configured to contain the first biological material. In some embodiments, the fluidic device comprises a top layer, a bottom layer, and a spacer layer.

[0154] In some embodiments, the spacer layer includes a cut-out region, where the spacer layer is sandwiched in between the bottom layer and the top layer to form a channel in the cut-out region. In some embodiments, the hydrogel chamber is at least partly formed by the top layer and the bottom layer. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are the same material. In some embodiments, the hydrogel chamber and the hydrogel polymer wall are different materials.

[0155] In some embodiments, hydrogel chambers of the invention are degradable or depolymerizable either generally within a channel or “on demand” within a channel. Hydrogel chambers that are generally degradable are degraded by treatment with a degradation agent, or equivalently, a depolymerization agent that is exposed to all chambers within channel. Depolymerization agents can include, but are not limited to, heat, light, and / or chemical depolymerization reagents (also sometimes referred to a cleaving reagents or degradation reagents). In some embodiments, on demand degradation may be implemented using polymer precursors that permit photo-crosslinking and photo-degradation, for example, using different wavelengths for crosslinking and for degradation. For example, Eosin Y may be used for radical polymerization at defined regions using 500 nm wavelength, after which illumination at 380 nm can be used to cleave the cross linker. In other embodiments, photo-caged hydrogel cleaving reagents may be included in the formation of polymer matrix walls. For example, acid labile crosslinkers (such as esters, or the like) can be used to create the hydrogel and then UV light can be used to generate local acidic conditions which, in turn, degrades the hydrogel. In some embodiments, the at least one polymer matrix is degradable by at least one of: (i) contacting theat least one polymer matrix with a cleaving reagent; (ii) heating the at least one polymer matrix to at least 90 °C; or (iii) exposing the at least one polymer matrix to a wavelength of light that cleaves a photo-cleavable cross linker that cross links the polymer of the at least one polymer matrix. In some embodiments, the at least one polymer matrix comprises a hydrogel. In some embodiments, the cleaving reagent degrades the hydrogel. In some embodiments, the cleaving reagent comprises a reducing agent, an oxidative agent, an enzyme, a pH based cleaving reagent, or a combination thereof. In some embodiments, the cleaving reagent comprises dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THP), or a combination thereof. In some embodiments, the surface of the polymer matrix or hydro gel may be functionalized by coupling a functional group to the polymer matrix or hydrogel. Some nonlimiting 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 polymer matrix may be used to capture biomolecules inside a polymer matrix compartment formed adjacent to (e.g., around or on) the biological component. 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).

[0156] In some embodiments, existing polymer matrix walls may be partially degraded, e.g. to change porosity. In some embodiments, polymer precursors may include degradable beads that form part of, and are embedded in, the polymer matrix walls when synthesized, after which either on-demand or generally, may be degraded, thereby creating an increase in porosity.

[0157] In some embodiments, the hydrogel chamber is made of a first material that degrades upon exposure to a first stimulus. In some cases, a hydrogel polymer wall is made of a second material that degrades upon exposure to a second stimulus. The first stimulus and second stimulus can be different. In some cases, the first stimulus comprises light, and the second stimulus comprises a degradation reagent. In some cases, the first stimulus comprises a degradation reagent, and the second stimulus comprises light. In some cases, the first stimulus comprises a first degradation reagent, and the second stimulus comprises a second degradation reagent different from the first degradation reagent. In some cases, the first stimulus comprises light in a first wavelength range, and the second stimulus comprises a light in a second wavelength range different from the first wavelength range.

[0158] The pore size in the polymer matrix may be modulated using a chemical reagent, or by applying heat, electrical field, light, or another suitable stimulus. In other words, the polymer matrix may comprise tunable properties (e.g., the pore size) In some cases, the polymer matrix may comprise 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.Computer Systems

[0159] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 14 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0160] 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, storageunit 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.

[0161] 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.

[0162] 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).

[0163] 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.

[0164] 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.

[0165] 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 executableor 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.

[0166] 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.

[0167] Aspects of the systems and methods provided herein, such as the computer system 1501, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that may be carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0168] 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 coaxialcables; 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.

[0169] The computer system 1501 can include or be in communication with an electronic display 1535 that comprises a user interface (UI) 1540 for providing, for example, an image of a biological component, a barcode, a signal or measurement of a local parameter. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0170] 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.EXAMPLESExample 1Fluidic Device With Adherent Support and Nucleic Acid Barcodes Co-Spotted on Bottom Surface

[0171] This example covers the fabrication and use of a fluidic device with a bare glass top surface and oligonucleotide barcodes and an adherent support coupled to a bottom surface. The fluidic device was formed by bonding top and bottom glass slides to a double-sided adhesive spacer layer with cut-out regions defining fluidic device channels. Prior to fabrication of the fluidic device, the top surface of the bottom glass slide (corresponding to the bottom surface of the interior of the fluidic device) was coated with a polymer functionalized with NHS-ester groups. Then, oligonucleotide barcode precursors, gelatin, and a dye were co-spotted in arrays of discrete circular sites on the top surface of the bottom glass slide. The oligonucleotide barcode precursors contained 5 ’-end amine groups that reacted with the NHS-ester groups on the polymer coating to couple the oligonucleotide barcode precursors to the bottom glass slide. Thegelatin noncovalently adhered to the bottom glass slide. After co-spotting, the remaining NHS ester groups were quenched with an ethanolamine solution. The presence of the dye in the discrete circular sites was later confirmed by imaging. The oligonucleotides were ligated to the 3’ ends of the oligonucleotide barcode precursors to form oligonucleotide barcodes. The bottom glass slide was then incubated at 30°C and 70% relative humidity for 20 hours. The fluidic device was then formed by bonding the bottom glass slide and the top glass slide to the doublesided adhesive spacer layer.

[0172] The fluidic device was then used for a cell incubation. IMR-90 fibroblast cells were input into the fluidic device and incubated overnight at 37°C. The cells were then subjected to brightfield imaging at 4x magnification. A representative image of the cells is shown in FIG. 16. In this image, a discrete circular site is indicated by the label 1601 and a cell is indicated by the label 1602. The majority of cells (e.g., 1602) in this image are localized to discrete circular sites (e.g., 1601), and exhibit morphologies that are consistent with surface adherence. One cell (labeled 1604) simultaneously adhered to two discrete circular sites (labeled 1603 A and 1063B). In this example, adhered cells tended to link to the discrete circular sites and not between the discrete circular sites. For this reason, under certain circumstances, forming enclosures around the cells can be simpler since all of the discrete circular sites can be used as location for forming the polymer enclosure.Example 2Fluidic Device With a Cell Adherent Support Coupled to Oligonucleotide Barcodes

[0173] This example covers the use of a fluidic device with an adherent support coupled to barcodes on a bottom surface of the fluidic device. The fluidic device was prepared by loading 25 pg / mL of a streptavidin-oligonucleotide fusion into the fluidic device. The streptavidinoligonucleotide fusion hybridized to oligonucleotide barcodes coupled to its bottom surface in an array of discrete sites. Biotinylated fibronectin was then added to the fluidic device to couple to the streptavidin-oligonucleotide fusion on the oligonucleotide barcodes. The fluidic device was then washed with phosphate buffered saline to remove unbound fibronectin from the fluidic device.

[0174] IMR-90 cells were then input into the fluidic device. The cells were incubated to allow the cells to adhere to the fibronectin FIG. 17 provides a representative image of cells in the fluidic device. Within this image, a representative discrete site is labeled as 1701, and a representative cell is labeled as 1702. As shown in this image, cells localized to the oligonucleotide barcode-containing discrete sites along the bottom surface of the fluidic device.Example 3Secreted Protein Capture on Fluidic Device Surfaces

[0175] This example covers a process for functionalizing top and bottom fluidic device surfaces with capture antibodies. Three fluidic devices were prepared and then used to measure cytokine secretion from cells: (1) a first fluidic device with a bare glass bottom surface and an antibody-coated top surface, (2) a second fluidic device with an antibody-coated bottom surface and a bare glass top surface, and (3) a third fluidic device with an antibody-coated bottom surface and a bare glass top surface. The first, second, and third fluidic devices were constructed by attaching top and bottom glass layers to a spacer layer formed from a double-sided adhesive. The spacer layer contained polygonal cut-out regions that defined channels of the fluidic devices.

[0176] Before the glass layers were attached to the spacer, the top glass layer of the first fluidic device and the bottom glass layers of the second and third fluidic devices were subjected to ten minutes of plasma treatment. These glass layers were then covered with 0.01% poly-L-lysine (PLL) solution and incubated for 1 hour at room temperature, during which time PLL deposited on these surfaces. The glass layers were then washed with deionized water, dried with nitrogen gas, vacuum sealed, and then bonded to spacer layers for fluidic device fabrication.

[0177] Following fabrication, antibodies were coupled to the PLL-functionalized surfaces of the first, second, and third fluidic devices. Briefly, the fluidic devices were filled with pH 9.5 carbonate buffer containing 20 pg / mL anti-IFN-y antibodies. The buffer was incubated in the fluidic devices for 2.5 hours to allow the antibodies to adhere to the PLL-functionalized surfaces in the fluidic devices. The fluidic devices were then washed 3 -times with ELISA wash buffer, filled with 3% bovine serum albumin solution, and incubated for another 2 hours at room temperature.

[0178] NK-92 cells were input into each fluidic device. Phorbol 12-myristate 13-acetate (PMA) and ionomycin were added to the first and second fluidic devices to induce IFN-y secretion by the NK-92 cells. PMA and ionomycin were not added to the third fluidic device. Individual NK-92 cells were enclosed in hydrogel chambers. Next, fluorescently labeled anti- IFN-y detection antibodies were added to each fluidic device. The first, second, and third fluidic devices were then subjected to fluorescence and brightfield imaging.

[0179] Images of the fluidic devices are shown in FIGS. 18A-F. FIGS. 18A-C are fluorescence images of the first, second, and third fluidic devices, respectively. FIGS. 18D-F are combined fluorescence and brightfield images of the first, second, and third fluidic devices, respectively. In FIGS. 18A and 18D (for the first fluidic device) and in FIGS. 18B and 18E (for the second fluidic device), fluorescence intensity is seen primarily within chambers. These results suggest that IFN-y secreted by the NK-92 cells coupled to surface-bound antibodies inthe first and second fluidic devices. Fluorescence intensity was primarily localized inside of chambers, indicating that chamber polymer walls limited IFN-y diffusion out of chambers. Negligible fluorescence intensity was observed in FIGS. 18C and 18F.Example Embodiments

[0180] The embodiments listed below are example embodiments of the systems and methods described herein, and do not limit the description above:

[0181] Embodiment 1. A fluidic device for processing a biological component, comprising: a surface, wherein the surface comprises (i) one or more oligonucleotides configured to associate with a biomolecule, and (ii) a cell adherent support, wherein the cell adherent support is coupled to at least a portion of the one or more oligonucleotides.

[0182] Embodiment 2. The fluidic device of embodiment 1, wherein the fluidic device further comprises a hydrogel chamber.

[0183] Embodiment 3. The fluidic device of embodiment 1, wherein the one or more oligonucleotides each comprise a barcode sequence.

[0184] Embodiment 4. The fluidic device of embodiment 1, wherein the one or more oligonucleotides each comprise a spatial location tag corresponding to a unique location of the oligonucleotide on the fluidic device.

[0185] Embodiment 5. The fluidic device of any one of embodiments 1-4, wherein the one or more oligonucleotides comprise a binding sequence configured to couple to the biomolecule.

[0186] Embodiment 6. The fluidic device of embodiment 5, wherein the binding sequence comprises a polyT sequence, and wherein the biomolecule comprises a messenger ribonucleic acid (mRNA) comprising a polyadenylated (poly A) tail.

[0187] Embodiment 7. The fluidic device of any one of embodiments 1-6, wherein the one or more oligonucleotides are distributed amongst one or more reaction sites on the surface, wherein each reaction site has a density of the one or more oligonucleotides from about 2 x 102 to about 5 x 107 oligonucleotides / pm2.

[0188] Embodiment 8. The fluidic device of any one of embodiments 1-7, wherein the one or more oligonucleotides are treated with one or more cell adhesive proteins.

[0189] Embodiment 9. The fluidic device of any one of embodiments 1-8, wherein the one or more oligonucleotides comprise a binding sequence, wherein a poly A-streptavidin is coupled to the binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A- streptavidin.

[0190] Embodiment 10. The fluidic device of any one of embodiments 1-9, wherein the one or more oligonucleotides comprise: (i) a first binding sequence, wherein a poly A-streptavidin is coupled to the first binding sequence, and wherein a cell adhesive protein is configured tocouple to the poly A-streptavidin; and (ii) a second binding sequence, wherein the second binding sequence is configured to associate with the biomolecule.

[0191] Embodiment 11 The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein is biotinylated.

[0192] Embodiment 12. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises laminin.

[0193] Embodiment 13. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises fibronectin.

[0194] Embodiment 14. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises poly-l-lysine (PLL).

[0195] Embodiment 15. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises poly-d-lysine (PDL).

[0196] Embodiment 16. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises vitronectin.

[0197] Embodiment 17. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises collagen.

[0198] Embodiment 18. The fluidic device of any one of embodiments 8-10, wherein the cell adhesive protein comprises gelatin.

[0199] Embodiment 19. The fluidic device of any one of embodiments 1-18, wherein the cell adherent support comprises one or more cell adherent biomolecules.

[0200] Embodiment 20. The fluidic device of embodiment 19, wherein the one or more cell adherent biomolecules comprise a protein.

[0201] Embodiment 21. The fluidic device of embodiment 20, wherein the protein comprises poly-l-lysine (PLL).

[0202] Embodiment 22. The fluidic device of embodiment 20, wherein the protein comprises poly-d-lysine.

[0203] Embodiment 23. The fluidic device of embodiment 20, wherein the protein comprises laminin.

[0204] Embodiment 24. The fluidic device of embodiment 23, wherein the cell adherent support further comprises poly-l-ornithine (PLO).

[0205] Embodiment 25. The fluidic device of embodiment 20, wherein the protein comprises fibronectin.

[0206] Embodiment 26. The fluidic device of embodiment 20, wherein the protein comprises vitronectin.

[0207] Embodiment 27. The fluidic device of embodiment 20, wherein the protein comprises collagen.

[0208] Embodiment 28. The fluidic device of embodiment 20, wherein the protein comprises gelatin.

[0209] Embodiment 29. The fluidic device of embodiment 19, wherein the one or more cell adherent biomolecules comprise streptavidin.

[0210] Embodiment 30. The fluidic device of embodiment 19, wherein the one or more cell adherent biomolecules comprise biotinylated fibronectin.

[0211] Embodiment 31. The fluidic device of embodiment 19, wherein the one or more cell adherent biomolecules comprise biotinylated laminin.

[0212] Embodiment 32. The fluidic device of any one of embodiments 19-31, wherein a density of the one or more cell adherent biomolecules on the cell adherent support is 200 to about 5 x 107cell adherent biomolecules / pm2.

[0213] Embodiment 33. The fluidic device of any one of embodiments 1-32, wherein the cell adherent support comprises one or more cell adherent particles.

[0214] Embodiment 34. The fluidic device of embodiment 33, wherein the one or more cell adherent particles comprise a polymer particle at least partially coated with a particle cell adherent coating.

[0215] Embodiment 35. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises a fibronectin coating.

[0216] Embodiment 36. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises poly-l-ornithine.

[0217] Embodiment 37. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises a laminin coating.

[0218] Embodiment 38. The fluidic device of embodiment 34, wherein the particle cell adherent coating further comprises poly-l-omithine.

[0219] Embodiment 39. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises poly-l-lysine.

[0220] Embodiment 40. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises poly-d-lysine.

[0221] Embodiment 41. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises vitronectin.

[0222] Embodiment 42. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises collagen.

[0223] Embodiment 43. The fluidic device of embodiment 34, wherein the particle cell adherent coating comprises gelatin.

[0224] Embodiment 44. The fluidic device of any one of embodiments 33-43, wherein a density of the one or more cell adherent particles on the surface is from about 10 to about 2500 particles / mm2.

[0225] Embodiment 45. The fluidic device of any one of embodiments 1-44, wherein the surface comprises one or more flow channels, wherein a flow channel of the one or more flow channels comprises one or more arrays, wherein an array of the one or more arrays comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises an oligonucleotide comprising: (a) a first sequence unique to a spatial location of the reaction site within the array; and (b) a second sequence unique to a spatial location of the array of the one or more arrays.

[0226] Embodiment 46. The fluidic device of embodiment 45, wherein each array comprises greater than 500 reaction sites.

[0227] Embodiment 47. The fluidic device of embodiment 45 or 46, wherein each array comprises greater than 1000 reaction sites.

[0228] Embodiment 48. The fluidic device of any one of embodiments 45-47, wherein each array comprises greater than 1500 reaction sites.

[0229] Embodiment 49. The fluidic device of any one of embodiments 45-48, wherein the surface comprises at least 5 flow channels.

[0230] Embodiment 50. The fluidic device of any one of embodiments 45-49, wherein the surface comprises at least 10 flow channels.

[0231] Embodiment 51. The fluidic device of any one of embodiments 45-50, wherein a flow channel of the one or more flow channels comprises at least 5 arrays.

[0232] Embodiment 52. The fluidic device of any one of embodiments 45-51, wherein a flow channel of the one or more flow channels comprises at least 10 arrays.

[0233] Embodiment 53. The fluidic device of any one of embodiments 45-52, wherein a flow channel of the one or more flow channels comprises at least 15 arrays.

[0234] Embodiment 54. The fluidic device of any one of embodiments 45-53, wherein the one or more reaction sites are separated by a pitch distance of about 100 to 150 pm.

[0235] Embodiment 55. The fluidic device of any one of embodiments 45-54, wherein each reaction site has a diameter of about 50 to 100 pm.

[0236] Embodiment 56. The fluidic device of embodiment 2, wherein the hydrogel chamber is formed upon polymerization of one or more polymer precursors.

[0237] Embodiment 57. The fluidic device of embodiment 56, wherein the fluidic device is in optical communication with a spatial energy modulation element configured to selectively direct energy to the fluidic device.

[0238] Embodiment 58. The fluidic device of any one of embodiments 1-57, further comprising a detector in optical communication with the fluidic device.

[0239] Embodiment 59. The fluidic device of any one of embodiments 1-58, wherein the biomolecule comprises genetic material.

[0240] Embodiment 60. The fluidic device of embodiment 59, wherein the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0241] Embodiment 61. The fluidic device of embodiment 59, wherein the genetic material comprises messenger RNA (mRNA).

[0242] Embodiment 62. The fluidic device of any one of embodiments 1-61, wherein the fluidic device further comprises a cell.

[0243] Embodiment 63. The fluidic device of embodiment 62, wherein the biomolecule comprises a component of the cell.

[0244] Embodiment 64. The fluidic device of embodiment 63, wherein the component of the cell comprises messenger ribonucleic acid (mRNA).

[0245] Embodiment 65. The fluidic device of any one of embodiments 62-64, wherein the cell comprises an adherent cell.

[0246] Embodiment 66. The fluidic device of embodiment 65, wherein the cell adherent support is configured to couple to said adherent cell.

[0247] Embodiment 67. The fluidic device of any one of embodiments 1-66, wherein the cell adherent support is functionalized for association with an additional biomolecule.

[0248] Embodiment 68. The fluidic device of embodiment 67, wherein the additional biomolecule comprises a secreted protein.

[0249] Embodiment 69. The fluidic device of embodiment 67, wherein the additional biomolecule comprises a surface protein.

[0250] Embodiment 70. The fluidic device of any one of embodiments 1-69, wherein the cell adherent support is configured to enable adhesion and spreading of adherent cells.

[0251] Embodiment 71. The fluidic device of any one of embodiments 1-70, wherein the surface is a bottom surface of the fluidic device.

[0252] Embodiment 72. The fluidic device of embodiment 71, wherein the bottom surface is optically transparent, allowing for imaging of the fluidic device.

[0253] Embodiment 73. The fluidic device of embodiment 70 or 71, wherein a top surface of the fluidic device is optically transparent, allowing for imaging of the fluidic device.

[0254] Embodiment 74. The fluidic device of any one of embodiments 1-73, wherein the surface further comprises an antibody layer configured to capture one or more proteins.

[0255] Embodiment 75. The fluidic device of embodiment 74, wherein the antibody layer is configured to bind one or more cytokines.

[0256] Embodiment 76. The fluidic device of any one of embodiments 1-75, wherein the one or more oligonucleotides comprise a binding sequence, and wherein a complementary binding sequence-fibronectin conjugate is coupled to the binding sequence.

[0257] Embodiment 77. The fluidic device of any one of embodiments 1-76, wherein the one or more oligonucleotides comprise a binding sequence, and wherein a complementary binding sequence-laminin conjugate is coupled to the binding sequence.

[0258] Embodiment 78. A method for processing one or more adherent cells, comprising: (a) introducing a sample comprising the one or more adherent cells to a fluidic device, wherein the fluidic device comprises a surface, wherein the surface (i) is functionalized for association with a biomolecule and (ii) comprises a cell adherent support; (b) encapsulating an adherent cell of the one or more adherent cells in a hydrogel chamber; and (c) releasing the biomolecule from the adherent cell such that the biomolecule associates with the surface.

[0259] Embodiment 79. The method of embodiment 78, wherein the surface is a bottom surface of the fluidic device.

[0260] Embodiment 80. The method of embodiment 79, wherein the bottom surface is optically transparent, allowing for imaging of the fluidic device.

[0261] Embodiment 81. The method of embodiment 78 or 79, wherein a top surface of the fluidic device is optically transparent, allowing for imaging of the fluidic device.

[0262] Embodiment 82. The method of any one of embodiments 78-81, wherein the adherent cell is coupled to the cell adherent support.

[0263] Embodiment 83. The method of embodiment 82, wherein the adherent cell is coupled to the cell adherent support prior to (b).

[0264] Embodiment 84. The method of embodiment 82, wherein the adherent cell is coupled to the cell adherent support subsequent to (b).

[0265] Embodiment 85. The method of any one of embodiments 78-84, wherein the releasing in (c) comprises lysing the adherent cell to release the biomolecule.

[0266] Embodiment 86. The method of any one of embodiments 78-85, wherein the surface is functionalized with one or more oligonucleotides.

[0267] Embodiment 87. The method of embodiment 86, wherein the one or more oligonucleotides each comprise a barcode sequence.

[0268] Embodiment 88. The method of embodiment 86, wherein the one or more oligonucleotides each comprise a spatial location tag corresponding to a unique location of the oligonucleotide on the surface.

[0269] Embodiment 89. The method any one of embodiments 86-88, wherein the one or more oligonucleotides comprise a binding sequence configured to couple to the biomolecule.

[0270] Embodiment 90. The method of embodiment 86, wherein the binding sequence comprises a polyT sequence, and wherein the biomolecule comprises a messenger ribonucleic acid (mRNA) comprising a polyadenylated (poly A) tail.

[0271] Embodiment 91. The method of any one of embodiments 86-90, wherein the one or more oligonucleotides are distributed amongst one or more reaction sites on the surface, wherein each reaction site has a density of the one or more oligonucleotides from about 2 x 102to about 5 x 107oligonucleotides / pm2.

[0272] Embodiment 92. The method of any one of embodiments 86-91, wherein the one or more oligonucleotides are treated with one or more cell adhesive proteins.

[0273] Embodiment 93. The method of any one of embodiments 86-92, wherein the one or more oligonucleotides comprise a binding sequence, wherein a poly A-streptavidin is coupled to the binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A- streptavidin.

[0274] Embodiment 94. The method of any one of embodiments 86-92, wherein the one or more oligonucleotides comprise: (i) a first binding sequence, wherein a poly A-streptavidin is coupled to the first binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin; and (ii) a second binding sequence, wherein the second binding sequence is configured to associate with the biomolecule.

[0275] Embodiment 95. The method of any one of embodiments 92-94, wherein the cell adhesive protein is biotinylated.

[0276] Embodiment 96. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises laminin.

[0277] Embodiment 97. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises fibronectin.

[0278] Embodiment 98. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises poly-l-lysine (PLL).

[0279] Embodiment 99. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises poly-d-lysine (PDL).

[0280] Embodiment 100. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises vitronectin.

[0281] Embodiment 101. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises collagen.

[0282] Embodiment 102. The method of any one of embodiments 92-94, wherein the cell adhesive protein comprises gelatin.

[0283] Embodiment 103. The method of any one of embodiments 78-102, wherein the cell adherent support comprises one or more cell adherent biomolecules.

[0284] Embodiment 104. The method of embodiment 103, wherein the one or more cell adherent biomolecules comprise a protein.

[0285] Embodiment 105. The method of embodiment 104, wherein the protein comprises poly- 1-lysine (PLL).

[0286] Embodiment 106. The method of embodiment 104, wherein the protein comprises poly- d-lysine (PDL).

[0287] Embodiment 107. The method of embodiment 104, wherein the protein comprises laminin.

[0288] Embodiment 108. The method of embodiment 107, wherein the cell adherent support further comprises poly-l-omithine (PLO).

[0289] Embodiment 109. The method of embodiment 104, wherein the protein comprises fibronectin.

[0290] Embodiment 110. The method of embodiment 104, wherein the protein comprises vitronectin.

[0291] Embodiment 111. The method of embodiment 104, wherein the protein comprises collagen.

[0292] Embodiment 112. The method of embodiment 104, wherein the protein comprises gelatin.

[0293] Embodiment 113. The method of embodiment 104, wherein the one or more cell adherent biomolecules comprise streptavidin.

[0294] Embodiment 114. The method of embodiment 103, wherein the one or more cell adherent biomolecules comprise biotinylated fibronectin.

[0295] Embodiment 115. The method of embodiment 103, wherein the one or more cell adherent biomolecules comprise biotinylated laminin.

[0296] Embodiment 116. The method of any one of embodiments 103-115, wherein a density of the one or more cell adherent biomolecules on the cell adherent support is about 200 to about 5 x 107cell adherent biomolecules / pm2.

[0297] Embodiment 117. The method of any one of embodiments 78-116, wherein the cell adherent support comprises one or more cell adherent particles.

[0298] Embodiment 118. The method of embodiment 117, wherein the one or more cell adherent particles comprise a polymer particle at least partially coated with a particle cell adherent coating.

[0299] Embodiment 119. The method of embodiment 117, wherein the particle cell adherent coating comprises fibronectin.

[0300] Embodiment 120. The method of embodiment 117, wherein the particle cell adherent coating comprises poly-l-ornithine.

[0301] Embodiment 121. The method of embodiment 117, wherein the particle cell adherent coating comprises a laminin coating.

[0302] Embodiment 122. The method of embodiment 121, wherein the particle cell adherent coating further comprises poly-l-ornithine.

[0303] Embodiment 123. The method of embodiment 117, wherein the particle cell adherent coating comprises poly-l-lysine.

[0304] Embodiment 124. The method of embodiment 117, wherein the particle cell adherent coating comprises poly-d-lysine.

[0305] Embodiment 125. The method of embodiment 117, wherein the particle cell adherent coating comprises vitronectin.

[0306] Embodiment 126. The method of embodiment 117, wherein the particle cell adherent coating comprises collagen.

[0307] Embodiment 127. The method of embodiment 117, wherein the particle cell adherent coating comprises gelatin.

[0308] Embodiment 128. The method of any one of embodiments 117-127, wherein a density of the one or more cell adherent particles on the surface is from about 1 to about 2500 particles / mm2.

[0309] Embodiment 129. The method of any one of embodiments 117-128, wherein the surface is functionalized with one or more oligonucleotides, and wherein the one or more cell adherent particles rest on top of the one or more oligonucleotides.

[0310] Embodiment 130. The method of any one of embodiments 78-129, wherein the surface comprises one or more flow channels, wherein a flow channel of the one or more flow channels comprises one or more arrays, wherein an array of the one or more arrays comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises an oligonucleotide comprising: (a) a first sequence unique to a spatial location of the reaction site within the array; and (b) a second sequence unique to a spatial location of the array of the one or more arrays.

[0311] Embodiment 131. The method of embodiment 130, wherein each array comprises greater than 500 reaction sites.

[0312] Embodiment 132. The method of embodiment 130 or 131, wherein each array comprises greater than 1000 reaction sites.

[0313] Embodiment 133. The method of any one of embodiments 130-132, wherein each array comprises greater than 1500 reaction sites.

[0314] Embodiment 134. The method of any one of embodiments 130-133, wherein the surface comprises at least 5 flow channels.

[0315] Embodiment 135. The method of any one of embodiments 130-134, wherein the surface comprises at least 10 flow channels.

[0316] Embodiment 136. The method of any one of embodiments 130-135, wherein the surface comprises at least 15 flow channels.

[0317] Embodiment 137. The method of any one of embodiments 130-136, wherein the surface comprises at least 30 arrays.

[0318] Embodiment 138. The method of any one of embodiments 130-137, wherein a flow channel of the one or more flow channels comprises at least 5 arrays.

[0319] Embodiment 139. The method of any one of embodiments 130-138, wherein a flow channel of the one or more flow channels comprises at least 10 arrays.

[0320] Embodiment 140. The method of any one of embodiments 130-139, wherein the one or more reaction sites are separated by a pitch distance of about 100 to 150 pm.

[0321] Embodiment 141. The method of any one of embodiments 130-140, wherein each reaction site has a diameter of about 50 to 100 pm.

[0322] Embodiment 142. The method of embodiment 78, wherein the hydrogel chamber is formed upon polymerization of one or more polymer precursors.

[0323] Embodiment 143. The method of embodiment 142, wherein the fluidic device is in optical communication with a spatial energy modulation element configured to selectively direct energy to the fluidic device.

[0324] Embodiment 144. The method of any one of embodiments 78-143, further comprising a detector in optical communication with the fluidic device.

[0325] Embodiment 145. The method of any one of embodiments 78-144, wherein the biomolecule comprises genetic material.

[0326] Embodiment 146. The method of embodiment 145, wherein the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0327] Embodiment 147. The method of embodiment 145, wherein the genetic material comprises messenger RNA (mRNA).

[0328] Embodiment 148. The method of any one of embodiments 78-147, wherein the fluidic device further comprises a cell.

[0329] Embodiment 149. The method of embodiment 148, wherein the biomolecule comprises a component of the cell.

[0330] Embodiment 150. The method of embodiment 149, wherein the component of the cell comprises messenger ribonucleic acid (mRNA).

[0331] Embodiment 151. The method of any one of embodiments 148-150, wherein the cell is an adherent cell.

[0332] Embodiment 152. The method of embodiment 151, wherein the cell adherent support is configured to couple to said adherent cell.

[0333] Embodiment 153. The method of any one of embodiments 78-152, wherein the cell adherent support is functionalized for association with an additional biomolecule.

[0334] Embodiment 154. The method of embodiment 153, wherein the additional biomolecule comprises a secreted protein.

[0335] Embodiment 155. The method of embodiment 153, wherein the additional biomolecule comprises a surface protein.

[0336] Embodiment 156. The method of any one of embodiments 78-155, wherein the surface further comprises an antibody layer configured to capture one or more proteins.

[0337] Embodiment 157. The method of embodiment 156, wherein the one or more proteins comprise cytokines.

[0338] Embodiment 158. The method any one of embodiments 78-157, further comprising, prior to (c), performing one or more assays on the one or more adherent cells.

[0339] Embodiment 159. The method of embodiment 158, wherein the one or more assays comprises a fluorescence assay.

[0340] Embodiment 160. The method of embodiment 158, wherein the one or more assays comprise a secretion assay.

[0341] Embodiment 161. The method of embodiment 160, wherein the adherent cell secretes one or more cytokines.

[0342] Embodiment 162. The method of embodiment 158, wherein the one or more assays comprise measuring one or more surface markers of the adherent cell.

[0343] Embodiment 163. The method of embodiment 162, wherein the one or more surface markers comprise CD3, CD4, CD8, CD19, CD25, CD45, or CD56, or any combination thereof.

[0344] Embodiment 164. The method of embodiment 158, wherein the one or more assays comprises a morphological assay.

[0345] Embodiment 165. A method for processing a cell, comprising: (a) introducing a sample comprising a plurality of cells to a fluidic device, wherein the one or more cells settle on a surface of the fluidic device, wherein the surface comprises one or more reaction zones, and wherein a reaction zone of the one or more reaction zones: (i) is functionalized for association with a biomolecule, and (ii) comprises a cell adherent support, wherein the plurality of cells settle on the surface; (b) incubating the one or more cells on the surface such that the one or more cells transform to an adherent state; (c) encapsulating a cell of the one or more cells and at least a portion of the reaction zone in a hydrogel chamber; and (d) lysing the cell to release messenger ribonucleic acid (mRNA) molecules, wherein the mRNA molecules settle to the surface.

[0346] Embodiment 166. The method of embodiment 165, wherein the surface is further functionalized with one or more protein capture antibodies.

[0347] Embodiment 167. The method of embodiment 166, wherein prior to (d), one or more proteins are secreted from the cell.

[0348] Embodiment 168. The method of embodiment 167, wherein the method further comprises capturing the secreted proteins with the protein capture antibodies.

[0349] Embodiment 169. The method of any one of embodiments 165-168, wherein the cell comprises an adherent cell.

[0350] Embodiment 170. The method of embodiment 169, wherein the cell adherent support is configured to couple to said adherent cell.

[0351] Embodiment 171. The method of any one of embodiments 165-170, wherein (b) occurs prior to (c).

[0352] Embodiment 172. The method of any one of embodiments 165-170, wherein (c) occurs prior to (b).

[0353] Embodiment 173. A fluidic device for processing a biological component, comprising: a surface, wherein the surface: (i) is functionalized for association with a biomolecule, and (ii) comprises a cell adherent support; and a hydrogel chamber.

[0354] While the present invention has been described with reference to several particular example embodiments, those skilled in the art will recognize that many changes may be made thereto without departing from the spirit and scope of the present invention. The present invention is applicable to a variety of sensor implementations and other subject matter, in addition to those discussed above.

Claims

CLAIMSWhat is claimed is:

1. A method for processing one or more adherent cells, comprising:(a) introducing a sample comprising the one or more adherent cells to a fluidic device, wherein the fluidic device comprises a surface, wherein the surface (i) is functionalized for association with a biomolecule and (ii) comprises a cell adherent support;(b) encapsulating an adherent cell of the one or more adherent cells in a hydrogel chamber; and(c) releasing the biomolecule from the adherent cell such that the biomolecule associates with the surface.

2. The method of claim 1, wherein the surface is a bottom surface of the fluidic device.

3. The method of claim 2, wherein the bottom surface is optically transparent, allowing for imaging of the fluidic device.

4. The method of claim 1 or 2, wherein a top surface of the fluidic device is optically transparent, allowing for imaging of the fluidic device.

5. The method of claim 1, wherein the adherent cell is coupled to the cell adherent support.

6. The method of claim 5, wherein the adherent cell is coupled to the cell adherent support prior to (b).

7. The method of claim 5, wherein the adherent cell is coupled to the cell adherent support subsequent to (b).

8. The method of claim 1, wherein the releasing in (c) comprises lysing the adherent cell to release the biomolecule.

9. The method of claim 1, wherein the surface is functionalized with one or more oligonucleotides.

10. The method of claim 9, wherein the one or more oligonucleotides each comprise a barcode sequence.

11. The method of claim 9, wherein the one or more oligonucleotides each comprise a spatial location tag corresponding to a unique location of the oligonucleotide on the surface.

12. The method of claim 9, wherein the one or more oligonucleotides comprise a binding sequence configured to couple to the biomolecule.

13. The method of claim 9, wherein the binding sequence comprises a polyT sequence, and wherein the biomolecule comprises a messenger ribonucleic acid (mRNA) comprising a polyadenylated (poly A) tail.

14. The method of claim 9, wherein the one or more oligonucleotides are distributed amongst one or more reaction sites on the surface, wherein each reaction site has a density of the one or more oligonucleotides from about 2 x 102to about 5 x 107oligonucleotides / pm2.

15. The method of claim 9, wherein the one or more oligonucleotides are treated with one or more cell adhesive proteins.

16. The method of claim 9, wherein the one or more oligonucleotides comprise a binding sequence, wherein a poly A-streptavidin is coupled to the binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin.

17. The method of claim 9, wherein the one or more oligonucleotides comprise: (i) a first binding sequence, wherein a poly A-streptavidin is coupled to the first binding sequence, and wherein a cell adhesive protein is configured to couple to the poly A-streptavidin; and (ii) a second binding sequence, wherein the second binding sequence is configured to associate with the biomolecule.

18. The method of claim 15, wherein the cell adhesive protein is biotinylated.

19. The method of claim 15, wherein the cell adhesive protein comprises laminin.

20. The method of claim 15, wherein the cell adhesive protein comprises fibronectin.

21. The method of claim 15, wherein the cell adhesive protein comprises poly-1 -lysine(PLL).

22. The method of claim 15, wherein the cell adhesive protein comprises poly-d-lysine (PDL).

23. The method of claim 15, wherein the cell adhesive protein comprises vitronectin.

24. The method of claim 15, wherein the cell adhesive protein comprises collagen.

25. The method of claim 15, wherein the cell adhesive protein comprises gelatin.

26. The method of claim 1, wherein the cell adherent support comprises one or more cell adherent biomolecules.

27. The method of claim 26, wherein the one or more cell adherent biomolecules comprise a protein.

28. The method of claim 27, wherein the protein comprises poly-l-lysine (PLL).

29. The method of claim 27, wherein the protein comprises poly-d-lysine (PDL).

30. The method of claim 27, wherein the protein comprises laminin.

31. The method of claim 30, wherein the cell adherent support further comprises poly-1- omi thine (PLO).

32. The method of claim 27, wherein the protein comprises fibronectin.

33. The method of claim 27, wherein the protein comprises vitronectin.

34. The method of claim 27, wherein the protein comprises collagen.

35. The method of claim 27, wherein the protein comprises gelatin.

36. The method of claim 27, wherein the one or more cell adherent biomolecules comprise streptavidin.

37. The method of claim 26, wherein the one or more cell adherent biomolecules comprise biotinylated fibronectin.

38. The method of claim 26, wherein the one or more cell adherent biomolecules comprise biotinylated laminin.

39. The method of claim 26, wherein a density of the one or more cell adherent biomolecules on the cell adherent support is about 200 to about 5 x 107cell adherent biomolecules / pm2.

40. The method of claim 1, wherein the cell adherent support comprises one or more cell adherent particles.

41. The method of claim 40, wherein the one or more cell adherent particles comprise a polymer particle at least partially coated with a particle cell adherent coating.

42. The method of claim 40, wherein the particle cell adherent coating comprises fibronectin.

43. The method of claim 40, wherein the particle cell adherent coating comprises poly-1- ornithine.

44. The method of claim 40, wherein the particle cell adherent coating comprises a laminin coating.

45. The method of claim 44, wherein the particle cell adherent coating further comprises poly-l-ornithine.

46. The method of claim 40, wherein the particle cell adherent coating comprises poly-1- lysine.

47. The method of claim 40, wherein the particle cell adherent coating comprises poly-d- lysine.

48. The method of claim 40, wherein the particle cell adherent coating comprises vitronectin.

49. The method of claim 40, wherein the particle cell adherent coating comprises collagen.

50. The method of claim 40, wherein the particle cell adherent coating comprises gelatin.

51. The method of claim 40, wherein a density of the one or more cell adherent particles on the surface is from about 1 to about 2500 particles / mm2.

52. The method of claim 40, wherein the surface is functionalized with one or more oligonucleotides, and wherein the one or more cell adherent particles rest on top of the one or more oligonucleotides.

53. The method of claim 1, wherein the fluidic device comprises one or more flow channels; wherein a portion of the surface of the fluidic device comprises one or more arrays; wherein an array of the one or more arrays comprises one or more reaction sites, and wherein a reaction site of the one or more reaction sites comprises an oligonucleotide comprising: (a) a first sequence uniqueto a spatial location of the reaction site within the array; and (b) a second sequence unique to a spatial location of the array of the one or more arrays.

54. The method of claim 53, wherein each array comprises greater than 500 reaction sites.

55. The method of claim 53, wherein each array comprises greater than 1000 reaction sites.

56. The method of claim 53, wherein each array comprises greater than 1500 reaction sites.

57. The method of claim 53, wherein the surface comprises at least 5 flow channels.

58. The method of claim 53, wherein the surface comprises at least 10 flow channels.

59. The method of claim 53, wherein the surface comprises at least 15 flow channels.

60. The method of claim 53, wherein the surface comprises at least 30 arrays.

61. The method of claim 53, wherein a flow channel of the one or more flow channels comprises at least 5 arrays.

62. The method of claim 53, wherein a flow channel of the one or more flow channels comprises at least 10 arrays.

63. The method of claim 53, wherein the one or more reaction sites are separated by a pitch distance of about 100 to 150 pm.

64. The method of claim 53, wherein each reaction site has a diameter of about 50 to 100 pm.

65. The method of claim 1, wherein the hydrogel chamber is formed upon polymerization of one or more polymer precursors.

66. The method of claim 65, wherein the fluidic device is in optical communication with a spatial energy modulation element configured to selectively direct energy to the fluidic device.

67. The method of claim 1, further comprising a detector in optical communication with the fluidic device.

68. The method of claim 1, wherein the biomolecule comprises genetic material.

69. The method of claim 68, wherein the genetic material comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

70. The method of claim 68, wherein the genetic material comprises messenger RNA (mRNA).

71. The method of claim 1, wherein the fluidic device further comprises a cell.

72. The method of claim 71, wherein the biomolecule comprises a component of the cell.

73. The method of claim 72, wherein the component of the cell comprises messenger ribonucleic acid (mRNA).

74. The method of claim 71, wherein the cell is an adherent cell.

75. The method of claim 74, wherein the cell adherent support is configured to couple to said adherent cell.

76. The method of claim 1, wherein the cell adherent support is functionalized for association with an additional biomolecule.

77. The method of claim 76, wherein the additional biomolecule comprises a secreted protein.

78. The method of claim 76, wherein the additional biomolecule comprises a surface protein.

79. The method of claim 1, wherein the surface further comprises an antibody layer configured to capture one or more proteins.

80. The method of claim 79, wherein the one or more proteins comprise cytokines.

81. The method of claim 1, further comprising, prior to (c), performing one or more assays on the one or more adherent cells.

82. The method of claim 81, wherein the one or more assays comprises a fluorescence assay.

83. The method of claim 81, wherein the one or more assays comprise a secretion assay.

84. The method of claim 83, wherein the adherent cell secretes one or more cytokines.

85. The method of claim 81, wherein the one or more assays comprise measuring one or more surface markers of the adherent cell.

86. The method of claim 85, wherein the one or more surface markers comprise CD3, CD4, CD8, CD 19, CD25, CD45, or CD56, or any combination thereof.

87. The method of claim 81, wherein the one or more assays comprises a morphological assay.

88. A method for processing a cell, comprising:(a) introducing a sample comprising a plurality of cells to a fluidic device, wherein the one or more cells settle on a surface of the fluidic device, wherein the surface comprises one or more reaction zones, and wherein a reaction zone of the one or more reaction zones: (i) is functionalized for association with a biomolecule, and (ii) comprises a cell adherent support, wherein the plurality of cells settle on the surface;(b) incubating the one or more cells on the surface such that the one or more cells transform to an adherent state;(c) encapsulating a cell of the one or more cells and at least a portion of the reaction zone in a hydrogel chamber; and(d) lysing the cell to release messenger ribonucleic acid (mRNA) molecules, wherein the mRNA molecules settle to the surface.

89. The method of claim 88, wherein the surface is further functionalized with one or more protein capture antibodies.

90. The method of claim 89, wherein prior to (d), one or more proteins are secreted from the cell.

91. The method of claim 90, wherein the method further comprises capturing the secreted proteins with the protein capture antibodies.

92. The method of claim 88, wherein the cell comprises an adherent cell.

93. The method of claim 92, wherein the cell adherent support is configured to couple to said adherent cell.

94. The method of claim 88, wherein (b) occurs prior to (c).

95. The method of claim 88, wherein (c) occurs prior to (b).

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