Custom printed structures for cell growth and motility assays

Abstract

Disclosed herein are systems and methods for measuring cell growth and movement. The methods can include synthesizing custom obstacle courses around individual cells or cell aggregates, and measuring their movement and / or growth through the obstacle course. The cells and cell aggregates can be subjected to further analyses to correlate phenotypic and genotypic traits with the movement and / or growth.

Description

Attorney Docket No. 59528-732601CUSTOM PRINTED STRUCTURES FOR CELL GROWTH AND MOTILITY ASSAYSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 703,115 filed October 3, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Cell growth and motility are highly regulated activities that are central to numerous physiological and morphogenesis processes and diseases. More broadly, directing movement and new growths in response to stimuli is an inherent characteristic of cells that is essential for survival and development. For example, during wound healing, cell aggregates are mechanically guided by extracellular matrix protrusions and chemotactic signals to reform damaged tissue (Friedl & Gilmour, Nature Reviews Molecular Cell biology 10, 445-457 (2009)). In brain development, cell positioning and neurite extension and branching are directed by a complex system of mechanical and chemical cues (Kollins & Davenport. Branching Morphogenesis in Vertebrate Neurons. In: Madame Curie Bioscience Database [Internet], Austin (TX): Landes Bioscience; 2000-2013).

[0003] It is unsurprising, then, that uncontrolled and disregulated growth and movement are common features of developmental conditions and diseases. In cancer, tumor cell motility is often determinative of severity and outcome. Often by way of mutation, tumor cells can adopt diverse modes of movement to escape their native environments and metastasize in distant tissues. Two cancers that exhibit similar growth, proliferation, and immunomodulation can nonetheless exhibit different motilities that underlie drastically different morbidities and mortality rates. In Alzheimer’s disease, neurite outgrowth dysregulation correlates with neurodegeneration and cell death (Saad et al., J. Alzheimers Dis. 43(3), 993-1006 (2015)), while Alzheimer’s disease severity appears to affect astrocyte density and morphology. Monterey et al., Front Neurol. 12, 619626 (2021)).

[0004] Despite their importance in health and disease, cell growth and motility have remained challenging to study. In cancer, the mechanistic and oncogenic underpinnings of tumor cell motility remain poorly understood relative to proliferative capacity and other oncogenic characteristics (see, e.g., Meehan et al., J. Biol. Chem. 279, 1562-1569 (2004); Sahni et al., J. Biol. Chem. 289, 9692-9709 (2014); Stuelten et al., Nat. Rev. Cancer 18(5), 296-312 (2019)). In Alzheimer’s disease, the relationship between neuron and astrocyteAttorney Docket No. 59528-732601 morphology and disease progression have not been fully elucidated (Preman et al., Cells 10(3), 540 (2021)).

[0005] Research on cell growth and motility is limited by inability to correlate cell behavior and morphology with other traits. Conventional cell motility and growth assays such as scratch-wound, fixation, and staining assays measure aggregate movement or morphological changes within populations of cells. These assays are also limited in their ability to analyze individual cells for additional characteristics that may relate to their growth or movement. Accordingly, the ability to correlate cell growth and movement to phenotypic and genotypic traits is limited.SUMMARY

[0006] Pursuant to the need for rapid, highly multiplexed, and parallelizable assays for studying the growth and movement of cells, the present disclosure provides systems and methods for simultaneously measuring growth, movement, and additional phenotypic and genotypic features of individual cells and cell aggregates in custom-printed obstacle courses and chambers.

[0007] In an aspect, provided herein is a method for measuring cellular growth, comprising: (a) inputting a cell or an aggregate of cells into a fluidic device; (b) inputting a polymer precursor into the fluidic device; (c) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells; (d) synthesizing a pathway adjacent to the chamber; and (e) measuring growth of the cell or the aggregate of cells through at least a portion of the pathway.

[0008] In some aspects, the synthesizing of the pathway in (d) uses the polymer precursor. In some aspects, the synthesizing of the pathway in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

[0009] In some aspects, the method further comprises synthesizing an additional chamber, wherein the additional chamber is fluidically coupled to the pathway. In some aspects, the additional chamber is synthesized using the polymer precursor. In some aspects, the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor. In some aspects, the growth of the cell or the aggregate of cells is towards the additional chamber. In some aspects, an additional cell, an aggregate of additional cells, a bead, or a combination thereof are disposed within the additional chamber. In some aspects, the growth of the cell or theAttorney Docket No. 59528-732601 aggregate of cells is towards the additional cell, the aggregate of additional cells, the bead, or the combination thereof.

[0010] In another aspect, provided herein is a method for measuring cellular growth, comprising: (a) inputting a cell or an aggregate of cells, an additional cell or an aggregate of additional cells, and a polymer precursor into a fluidic device; (b) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells, and wherein the additional cell or the aggregate of additional cells are outside of the chamber; and (c) measuring growth of the cell or the aggregate of cells from the chamber towards the additional cell or the aggregate of additional cells.

[0011] In some aspects, the method further comprises, prior to the measuring the growth of the cell or the aggregate of cells in (c), incubating the additional cell or the aggregate of additional cells at least until the additional cell or the aggregate of additional cells adhere to a position on a surface of the fluidic device. In some aspects, the method further comprises synthesizing an additional chamber, wherein the additional chamber and at least partially encloses the additional cell or the aggregate of additional cells. In some aspects, the additional chamber is synthesized using the polymer precursor. In some aspects, the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

[0012] In another aspect, provided herein is a method for measuring cellular growth, comprising: (a) inputting a cell or an aggregate of cells into a fluidic device; (b) inputting a polymer precursor into the fluidic device; (c) synthesizing a first chamber using the polymer precursor within the fluidic device, wherein the first chamber at least partially encloses the cell or the aggregate of cells; (d) synthesizing a second chamber; and (e) measuring growth of the cell or the aggregate of cells from the first chamber towards the second chamber.

[0013] In some aspects, the synthesizing of the second chamber in (d) uses the polymer precursor. In some aspects, the synthesizing of the second chamber in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor. In some aspects, an additional cell, an aggregate of additional cells, a bead, or a combination thereof are disposed within the second chamber.

[0014] In some aspects, the growth comprises axonal growth, dendritic growth, neurite growth, pseudopodium growth, filopodium growth, lamellipodium growth, a growth cone, acrosome growth, comet tail growth, or a combination thereof. In some aspects, the measuring comprises determining a rate or a distance of the growth of the first cell or theAttorney Docket No. 59528-732601 aggregate of the first cells. In some aspects, the method further comprises determining whether a direction of the growth of the first cells or the aggregate of the first cells is random or directed. In some aspects, the method further comprises determining whether the additional cell, the aggregate of additional cells, the bead, or the combination thereof increases the growth, decreases the growth, randomizes a direction of the growth, or directs the direction of the growth of the first cell or the aggregate of the first cells. In some aspects, the additional cell or the aggregate of additional cells grows towards the cell or the aggregate of cells. In some aspects, the cell or the aggregate of cells and the additional cell or the aggregate of additional cells are neurons. In some aspects, the method further comprises detecting junction formation or synapse formation between the cell or the aggregate of cells and the additional cell or the aggregate of additional cells.

[0015] In some aspects, the method further comprises contacting the cell or the aggregate of cells with a reagent, and determining whether the reagent alters the growth of the cell or the aggregate of cells. In some aspects, the method further comprises determining whether the reagent increases the growth, decreases the growth, directs a direction of the growth, or randomizes the direction of the growth of the cell or the aggregate of cells. In some aspects, the pathway comprises a higher concentration of the reagent than the chamber, or wherein the chamber comprises a higher concentration of the reagent than the pathway. In some aspects, the pathway comprises a chemical gradient of the reagent. In some aspects, the pathway comprises (i) a first end that comprises an opening to the chamber or a degradable wall of the chamber and (ii) a second end that comprises an opening to an additional chamber or to a space outside of the chamber; and wherein: (i) the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or (ii) the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway. In some aspects, the reagent is produced by the additional cell or the aggregate of additional cells.

[0016] In some aspects, the method further comprises degrading a portion of the chamber to form an opening that allows the growth of the cell or the aggregate of cells out of the chamber. In some aspects, the degradable wall of the first chamber is synthesized from a different polymer precursor than the chamber. In some aspects, the chamber comprises an opening through which the cell or the aggregate of cells can grow. In some aspects, the opening comprises a gap or a pore in a wall of the chamber. In some aspects, wherein the additional chamber comprises an opening through which the cell or the aggregate of cells canAttorney Docket No. 59528-732601 grow. In some aspects, the opening comprises a gap or a pore in a wall of the additional chamber.

[0017] In another aspect, provided herein is a method for measuring cellular movement, comprising: (a) inputting a cell or an aggregate of cells into a fluidic device; (b) inputting a polymer precursor into the fluidic device; (c) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells; (d) synthesizing a pathway adjacent to the chamber; (e) degrading a portion of the chamber to form an opening that allows movement of the cell or the aggregate of cells from the chamber to the pathway; and (f) measuring movement of the cell or the aggregate of cells through at least a portion of the pathway.

[0018] In some aspects, the synthesizing of the pathway in (d) uses the polymer precursor. In some aspects, the synthesizing of the pathway in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

[0019] In another aspect, provided herein is a method for measuring cellular movement, comprising: (a) inputting a cell or an aggregate of cells into a fluidic device; (b) inputting a polymer precursor into the fluidic device; (c) synthesizing a chamber with the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells; (d) synthesizing a pathway adjacent to the chamber; and (e) measuring movement of the cell or the aggregate of cells from the chamber through the pathway.

[0020] In some aspects, the chamber comprises an opening through which the cell or the aggregate of cells can move. In some aspects, the opening comprises a gap or a pore in a wall of the chamber. In some aspects, the method further comprises synthesizing an additional chamber, wherein the additional chamber is fluidically coupled to the pathway and comprises an opening from the pathway through which the cell or the aggregate of cells can move. In some aspects, the additional chamber is synthesized using the polymer precursor. In some aspects, the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor. In some aspects, the additional chamber comprises an opening through which the cell or the aggregate of the cells can move.

[0021] In some aspects, the measuring comprises determining a rate or a distance of the movement of the cell or the aggregate of cells through at least the portion of the pathway. In some aspects, the method further comprises determining whether the movement is stochastic or directed. In some aspects, the movement is towards: (i) an additional cell or an aggregateAttorney Docket No. 59528-732601 of additional cells located outside of the chamber; (ii) a bead located outside of the chamber; (iii) an additional chamber; or (iv) a combination thereof.

[0022] In some aspects, the additional cell, the aggregate of additional cells, a bead, or a combination thereof are disposed within the additional chamber. In some aspects, the method further comprises determining whether the additional cell, the aggregate of the additional cells, the bead, or the combination thereof increases the movement, decreases the movement, randomizes a direction of the movement, or directs the direction of the movement of the cell or the aggregate of cells. In some aspects, the cell is an effector cell and the additional cell or the aggregate of additional cells comprises a target cell, and the effector cell is configured to interact with the target cell. In some aspects, the method further comprises synthesizing a semipermeable matrix in at least a portion of the pathway, wherein the semipermeable matrix is configured to allow the cell or the aggregate of the cells to pass through at least a portion of the semipermeable matrix. In some aspects, the semipermeable matrix is synthesized using matrigel, gelatin, photocrosslinkable gelatin, collagen, a gel of an enzymatically degradable macro monomer, or a gel of an enzymatically cleavable PEG macromonomer. In some aspects, the semipermeable matrix is synthesized using the polymer precursor. In some aspects, the semipermeable matrix is synthesized using an additional polymer precursor, and wherein the additional polymer precursor is different from the polymer precursor. In some aspects, the semipermeable matrix or a portion of the semipermeable matrix is shorter than a height of the pathway, narrower than a width of the pathway, or a combination thereof. In some aspects, the semipermeable matrix is synthesized using a polymer precursor that is enzymatically cleavable, bioabsorbable, or enzymatically cleavable and bioabsorbable. In some aspects, the cell or the aggregate of cells passes through the semipermeable matrix from a first end to a second end of the pathway. In some aspects, the synthesizing the semipermeable matrix is subsequent to the synthesizing the pathway.

[0023] In some aspects, the method further comprises synthesizing an obstacle that partially blocks the pathway and is impermeable to the cell or the aggregate of cells. In some aspects, the obstacle is synthesized using the polymer precursor. In some aspects, the obstacle is synthesized using an additional polymer precursor, and wherein the additional polymer precursor is different from the polymer precursor. In some aspects, the measuring comprises determining whether the cell or the aggregate of cells moves past the obstacle.

[0024] In some aspects, the method further comprises contacting the cell or the aggregate of cells with a reagent, and determining whether the reagent alters the movement of the cell or the aggregate of cells. In some aspects, the method further comprises determining whetherAttorney Docket No. 59528-732601 the reagent increases the movement, decreases the movement, directs a direction of the movement, or randomizes the direction of the movement of the cell or the aggregate of cells. In some aspects, the pathway comprises a higher concentration of the reagent than the chamber, or wherein the chamber comprises a higher concentration of the reagent than the pathway. In some aspects, the pathway comprises a chemical gradient of the reagent. In some aspects, the pathway comprises (i) a first end that comprises an opening to the chamber or a degradable wall of the chamber and (ii) a second end that comprises an opening to an additional chamber or to a space outside of the chamber, and wherein: (i) the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or (ii) the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway. In some aspects, the reagent is produced by the second cell or the aggregate of the second cells.

[0025] In some aspects, the pathway comprises one or more polymer matrix walls that define an open space adjacent to the chamber through which the cell or the aggregate of cells can grow or move. In some aspects, the one or more polymer matrix walls comprise a polymer matrix wall of the chamber, a polymer matrix wall of an additional chamber, or a combination thereof. In some aspects, the pathway comprises (i) a first opening to the chamber or a degradable wall of the chamber and (ii) a second opening to a space outside of the pathway and the chamber. In some aspects, the pathway comprises a single route from the first opening to the second opening.

[0026] In some aspects, (i) the chamber and the pathway, (ii) the chamber and the additional chamber; (iii) the additional chamber and the pathway, or (iv) a combination thereof are synthesized simultaneously. In some aspects, the chamber, the pathway, or the additional chamber comprises an opening, and wherein the method further comprises synthesizing a polymer wall in the opening, thereby enclosing an analyte from the cell or the aggregate of cells within the chamber, the pathway, the additional chamber, or a combination thereof. In some aspects, the method further comprises lysing the cell or at least a subset of the aggregate of cells within the chamber, the pathway, or the additional chamber.

[0027] In some aspects, the measuring comprises imaging.

[0028] In some aspects, the bead comprises a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof. In some aspects, the bead comprises a nanoparticle, a microparticle, a lipid particle, a hydrogel particle, or a nanomaterial.Attorney Docket No. 59528-732601

[0029] In some aspects, the reagent comprises a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof. In some aspects, the synthesizing of the chamber comprises photopolymerization. In some aspects, the synthesizing of the pathway or the synthesizing of the additional chamber comprises photopolymerization. In some aspects, the degrading comprises photolysis, chemical degradation, or a combination thereof. In some aspects, the photolysis comprises inputting a photoinitiator into the fluidic device and directing a light to at least a portion of the chamber.

[0030] In some aspects, the pathway at least partially surrounds the chamber. In some aspects, the pathway is at least partially disposed within an additional chamber, and wherein the additional chamber at least partially encloses the chamber. In some aspects, the pathway forms at least a portion of an additional chamber, wherein the additional chamber at least partially encloses the chamber, and wherein the chamber comprises at least one opening to the additional chamber. In some aspects, the pathway comprises at least a portion of the wall of the additional chamber. In some aspects, the pathway comprises a maze. In some aspects, the pathway comprises: (i) a first opening adjacent to the chamber; (ii) a second opening to an additional chamber or a space outside of the chamber; and (iii) a plurality of interconnected paths of which only a subset lead from the first opening to the second opening. In some aspects, the pathway comprises a single route that connects the first opening to the second opening. In some aspects, the measuring comprises measuring a time between the cell moving or growing from the first opening to the second opening of the pathway. In some aspects, the pathway comprises a first end that is adjacent to the chamber and a second end that comprises an opening to a space outside of the chamber. In some aspects, the pathway comprises a first end that is adjacent to the chamber and a second end that comprises an opening to an additional chamber. In some aspects, a wall of the chamber comprises a greater thickness than a wall of the pathway, the wall of the pathway comprises a greater thickness than the wall of the chamber, the wall of the chamber comprises a greater thickness than a wall of the additional chamber, the wall of the additional chamber comprises a greater thickness than the wall of the chamber, the wall of the pathway comprises a greater thickness than the wall of the additional chamber, the wall of the additional chamber comprises a greater thickness than the wall of the pathway, the wall of the chamber comprises a greater porosity than the wall of the pathway, the wall of the pathway comprises a greater porosity than the wall of the chamber, the wall of the chamber comprises a greater porosity than the wall of the additional chamber, the wall of the additional chamber comprises a greaterAttorney Docket No. 59528-732601 porosity than the wall of the chamber, the wall of the pathway comprises a greater porosity the wall of the additional chamber, the wall of the additional chamber comprises a greater porosity than the wall of the pathway, or a combination thereof.

[0031] In some aspects, the method further comprises inputting a reagent into the fluidic device, wherein the reagent: diffuses across a wall of the chamber and does not diffuse across a wall of the pathway, diffuses across the wall of the pathway and does not diffuse across the wall of the chamber, diffuses across the wall of the chamber and does not diffuse across the wall of the additional chamber, diffuses across the wall of the additional chamber and does not diffuse across the wall of the chamber, diffuses across the wall of the pathway and does not diffuse across the wall of the additional chamber, diffuses across the wall of the additional chamber and does not diffuse across the wall of the pathway, or a combination thereof.

[0032] In some aspects, the pathway comprises a channel. In some aspects, the channel of the pathway comprises a cross-sectional area, wherein the cross-sectional area is less than a largest dimension of the cell. In some aspects, the cross-sectional area is less than the largest dimension of the cell by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. In some aspects, the channel of the pathway comprises a cross-sectional area, wherein the cross- sectional area is greater than a largest dimension of the cell. In some aspects, the cross- sectional area is greater than the largest dimension of the cell by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 500%, at least about 1000%, at least about 2000%, or at least about 5000%. In some aspects, the pathway comprises a length of about 10 to 25 pm, about 10 to 50 pm, about 10 to 100 pm, about 10 to 250 pm, about 10 to 500 pm, about 10 to 1000 pm, about 10 to 1500 pm, about 10 to 2000 pm, about 10 to 2500 pm, about 25 to 50 pm, about 25 to 100 pm, about 25 to 250 pm, about 25 to 500 pm, about 25 to 1000 pm, about 25 to 1500 pm, about 25 to 2000 pm, about 25 to 2500 pm, about 50 to 100 pm, about 50 to 250 pm, about 50 to 500 pm, about 50 to 1000 pm, about 50 to 1500 pm, about 50 to 2000 pm, about 50 to 2500 pm, about 100 to 250 pm, about 100 to 500 pm, about 100 to 1000 pm, about 100 to 1500 pm, about 100 to 2000 pm, about 100 to 2500 pm, about 250 to 500 pm, about 250 to 1000 pm, about 250 to 1500 pm, about 250 to 2000 pm, about 250 to 2500 pm, about 500 to 1000 pm, about 500 to 1500 pm, about 500 to 2000 pm, about 500 to 2500 pm, about 1000 to 1500 pm, about 1000 to 2000 pm, about 1000 to 2500 pm, or about 1500 to 2500 pm.Attorney Docket No. 59528-732601

[0033] In some aspects, the cell or the aggregate of cells is coupled to a surface of the fluidic device. In some aspects, an adherent substrate is coupled to a surface of the fluidic device. In some aspects, the adherent substrate comprises fibronectin, poly-1 -ornithine, an RGD peptide, actinin, collagen, fibrinogen, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, talin, VCAM- 1, vinculin, vitronectin, or a combination thereof. In some aspects, the adherent substrate promotes the movement or the growth of the cell or the aggregate of cells; the adherent substrate inhibits the movement or the growth of the cell or the aggregate of cells; the method further comprises determining whether the adherent substrate promotes or inhibits the movement or the growth of the cell or the aggregate first cells; or a combination thereof.

[0034] In some aspects, the cell or the aggregate of cells comprises an adipocyte, an antigen- presenting cell, a cancer cell, a cardiomyocyte, chondrocyte, a dendritic cell, an ectoderm, an effector cell, an embryonic stem cell, an endodermal cell, an endothelial cell, a fibroblast, a hematopoietic stem cell, a hepatocyte, an islet cell, a keratinocyte, a lymphocyte, a melanocyte, a mesenchymal cell, a mesenchymal stem cell, a mesenchymal cancer cell, a monocyte, a progenitor cell, a myoblast, a myocyte, a neural cell, an oligodendrocyte, an osteoblast, a pancreatic epithelial cell, a skeletal myocyte cell, a smooth muscle cell, or a white blood cell. In some aspects, the cell or the aggregate of cells comprises an adherent cell. In some aspects, the aggregate of the cells comprises a cell clump, an embryoid body, a spheroid, a neurosphere, a tumor, a tissue section, or an organoid. In some aspects, the cell comprises a neuron or the aggregate of cells comprises a neurosphere. In some aspects, the aggregate of cells comprises between about 5 and 25 cells, 5 and 50 cells, 5 and 100 cells, 5 and 250 cell, 5 and 500 cells, 5 and 1000 cells, 5 and 2000 cells, 25 and 50 cells, 25 and 100 cells, 25 and 250 cells, 25 and 500 cells, 25 and 1000 cells, 25 and 2000 cells, 50 and 100 cells, 50 and 250 cells, 50 and 500 cells, 50 and 1000 cells, 50 and 2000 cells, 100 and 250 cells, 100 and 500 cells, 100 and 1000 cells, 100 and 2000 cells, 250 and 500 cells, 250 and 1000 cells, 250 and 2000 cells, 500 and 1000 cells, 500 and 2000 cells, or 1000 and 2000 cells.

[0035] In some aspects, the method further comprises detecting a guide ribonucleic acid (RNA) associated with a genetic modification of the cell or the aggregate of cells. In some aspects, the guide RNA is coupled to an exogenous messenger ribonucleic acid (mRNA). In some aspects, the guide RNA is coupled to a barcode. In some aspects, the guide RNA is coupled to a polyAtail. In some aspects, the method further comprises generating a complementary DNA (cDNA) molecule comprising a complement of the guide RNA sequence, the exogenous mRNA, the barcode, or a combination thereof. In some aspects, theAttorney Docket No. 59528-732601 cDNA molecule is coupled to a spatial location tag corresponding to a unique location within the fluidic channel. In some aspects, the generating the cDNA molecule comprises capturing the guide RNA on the capture probe comprising the spatial location tag or the complement thereof, and reverse transcribing the guide RNA on the capture probe, thereby generating the cDNA molecule. In some aspects, the method further comprises sequencing the cDNA, thereby detecting the guide RNA associated with the genetic modification of the cell or the aggregate of cells.

[0036] In some aspects, the method further comprises determining a characteristic of the cell or the aggregate of cells. In some aspects, the characteristic is a messenger ribonucleic acid (mRNA) expressed by the cell or the aggregate of cells In some aspects, the determining comprises lysing the cell or at least a subset of the aggregate of cells, capturing the mRNA on a capture element coupled to a surface of the fluidic device, reverse transcribing the mRNA to generate a complementary DNA (cDNA) molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule. In some aspects, prior to the lysing, the method further comprises closing an opening in the chamber, closing an opening in the pathway, or a combination thereof.

[0037] In some aspects, the characteristic comprises an action potential. In some aspects, determining comprises calcium imaging, a microelectrode measurement, or a combination thereof.

[0038] In some aspects, the characteristic comprises a soluble factor secreted by the cell or the aggregate of cells. In some aspects, the detecting the soluble factor comprises disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell or the aggregate of cells, and detecting the soluble factor bound to the capture surface. In some aspects, the detecting the soluble factor bound to the capture surface comprises contacting the soluble factor bound to the capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. In some aspects, the capture surface is at least partially enclosed in the chamber, the pathway, the additional chamber, or the combination thereof. In some aspects, the soluble factor is a neurotransmitter.

[0039] In some aspects, the cell or the aggregate of cells comprises an effector cell, and the characteristic comprises cytotoxicity. In some aspects, the determining the cytotoxicity comprises measuring a rate or an occurrence of the effector cell killing the additional cell or at least a subset of the aggregate of additional cells.

[0040] In some aspects, the characteristic comprises activation. In some aspects, the cell or the aggregate of cells is activated by the additional cell or the aggregate of additional cells. InAttorney Docket No. 59528-732601 some aspects, the determining the activation comprises detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a soluble factor, a genomic sequence, an mRNA, or a combination thereof of the cell or the aggregate of cells.

[0041] In some aspects, the characteristic comprises proliferation.

[0042] In some aspects, the pathway is configured to permit the growth or the movement of the cell or the aggregate of cells through at least a portion of the pathway. In some aspects, the fluidic device comprises a fluidic channel.

[0043] In another aspect, provided herein is a system comprising: a fluidic device, and an instrument configured to: a) synthesize a chamber within the fluidic device, wherein the chamber at least partially encloses a cell or an aggregate of cells; b) synthesize a pathway adjacent to the chamber that is configured to permit movement or growth of the cell or the aggregate of the cells through the pathway, wherein: the pathway comprises (i) a first opening to the chamber or a degradable portion of the chamber and (ii) a second opening to an additional chamber or a space outside of the chamber, the chamber and the pathway comprise walls with different porosities, the chamber and the pathway comprise walls with different thicknesses, or a combination thereof; and c) measure movement or growth of the cell or the aggregate of cells through at least a portion of the pathway using a detector.

[0044] In some aspects, the pathway comprises a maze. In some aspects, walls of the first chamber and the second chamber comprise different thicknesses and / or porosities. In some aspects, the pathway comprises a plurality of chambers that partially enclose the first chamber, and wherein each chamber of the plurality of chambers comprises at least one opening to another chamber of the plurality of chambers or a space outside of the plurality of chambers.

[0045] In another aspect, provided herein is a method for measuring cellular growth or movement, comprising: inputting a cell or an aggregate of cells into a fluidic device; inputting a polymer precursor into the fluidic device; synthesizing a chamber with the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells; and measuring growth or movement of the cell or the aggregate of cells towards a target located within the fluidic device.

[0046] In some aspects, the target comprises an additional chamber, an additional cell or aggregate of additional cells, a bead, or a combination thereof. In some aspects, the target is a structure synthesized with the first polymer precursor or a second polymer precursor. In someAttorney Docket No. 59528-732601 aspects, the target is a location along a surface of the fluidic device. In some aspects, the target comprises a collection of points along a surface of the fluidic device.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0048] FIG. 1 is a schematic illustration of a portion of a channel disposed in a fluidic device, according to some embodiments.

[0049] FIG. 2A is an illustration of a portion of a system as provided herein including an energy source, according to some embodiments.

[0050] FIG. 2B is an illustration of a polymer matrix being formed around a biological component in a portion of a system as provided herein, according to some embodiments;

[0051] FIG. 2C is an illustration of a method of forming a polymer matrix around a biological component in a system as provided herein, according to some embodiments

[0052] FIG. 3A is an illustration of a top view of the bottom layer of a flow cell without any spatial barcoded oligonucleotides.

[0053] FIG. 3B is an illustration of a top view of the spacer layer with a cut-out region suitable for use as part of the flow cell.

[0054] FIG. 3C is an illustration of a top view of the top layer of the flow cell where the top layer has an inlet and outlet opening.

[0055] FIG. 3D is an illustration of a cross-sectional side view of a top layer and a bottom layer sandwiching a spacer layer to form multiple channels of a flow cell.

[0056] FIG. 4A is an illustration of a system with a flow cell and an imaging apparatus that are capable of implementing methods of the present disclosure.

[0057] FIG. 4B is an illustration of a flow cell, including a blown-up view of a portion of a channel containing cells disposed in hydrogel chambers.

[0058] FIG. 5 is a schematic of a computer system 1501 that may be programmed or otherwise configured to perform methods described herein.

[0059] FIG. 6 is a schematic of a method for monitoring the movement of a cell through a semipermeable matrix.Attorney Docket No. 59528-732601

[0060] FIG. 7 is a schematic of a method for simultaneously monitoring the movement of cells through pathways in obstacle courses synthesized around the cells.

[0061] FIG. 8 is a schematic of a method for measuring cell movement through a pathway that includes degrading a chamber enclosing the cell to create access to the pathway.

[0062] FIG. 9A is a schematic of a method for measuring cell growth through a pathway defined by two nested chambers.

[0063] FIG. 9B is a schematic of a method for measuring cell growth between two aggregates of cells in which one of the cells is enclosed in an obstacle course.

[0064] FIG. 9C is a schematic of a method for measuring cell growth between two aggregates of cells enclosed in separate chambers.

[0065] FIG. 10A is a schematic of a method for measuring cell movement through a pathway that contains a chemical gradient of a reagent.

[0066] FIG. 10B is a schematic of a method for measuring cell movement through a pathway that contains a chemical cleaved from a bead.

[0067] FIGS. 11A and 11B are brightfield images of neurospheres enclosed in hydrogel chambers taken 24 hours (FIG. 11A) and 48 hours (FIG. 11B) after chamber synthesis.

[0068] FIGS. 12A and 12B are brightfield images of neurospheres enclosed in hydrogel chambers taken 24 hours (FIG. 12A) and 48 hours (FIG. 11B) after chamber synthesis.

[0069] FIGS. 13A and 13B are fluorescence images of multiple neurospheres stained with DAPI, Tuj 1, HuC / D, and MAP2 separate by a hydrogel chamber. FIG. 13A is a fluorescence image of the neurospheres showing DAPI, Tuj 1, HuC / D, and MAP2 fluorescence. FIG. 13B is a fluorescence image of the neurospheres showing intensity from a MAP2 fluorescence channel.

[0070] FIGS. 14A-C are fluorescence images of multiple hydrogel chamber-enclosed neurospheres and one neurosphere not enclosed within a hydrogel chamber. FIG. 14A is a fluorescence image of two neurospheres that are each enclosed in a hydrogel chamber that are each in close proximity to a neurosphere not enclosed in a hydrogel chamber. FIG. 14B is a fluorescence image of one of the two hydrogel chamber-enclosed neurospheres from FIG. 14A. FIG. 14C is a fluorescence image of a hydrogel chamber-enclosed neurosphere that includes a neurite that extends beyond a wall of its hydrogel chamber.

[0071] FIGS. 15A-C are brightfield images of a neurosphere enclosed in a hydrogel chamber with multiple openings in its walls. FIG. 15A is an image collected immediately after hydrogel chamber synthesis. FIG. 15B is an image collected 24 hours after hydrogel chamber synthesis. FIG. 15C is an image collected 48 hours after hydrogel chamber synthesis.Attorney Docket No. 59528-732601

[0072] FIGS. 16A and 16B are brightfield images of a first hydrogel chamber enclosing three neurospheres and a second hydrogel chamber enclosing a single neurosphere. FIG. 16A was collected 24 hours after hydrogel chamber synthesis. FIG. 16B was collected 48 hours after hydrogel chamber synthesis.

[0073] FIGS. 17A-C are brightfield images of a neurosphere contained within an inner chamber enclosed by an outer chamber. FIG. 17A was collected immediately following hydrogel chamber synthesis. FIG. 17B was collected 24 hours after hydrogel chamber synthesis. FIG. 17C was collected 48 hours after hydrogel chamber synthesis.

[0074] FIGS. 18A and 18B are brightfield images of a neurosphere enclosed within three nested chambers. FIG. 18A was collected 24 hours after chamber synthesis. FIG. 18B was collected 48 hours after hydrogel chamber synthesis.

[0075] FIGS. 19A-C are brightfield images of neurospheres co-enclosed within hydrogel chambers with nucleic acid capture probes. FIG. 19A is a brightfield image of side-by-side hydrogel chambers that each contain a single neurosphere. FIG. 19B is a brightfield image of a single neurosphere enclosed within a hydrogel chamber. FIG. 19C is a brightfield image of a single neurosphere enclosed within and actively deforming a hydrogel chamber.

[0076] FIGS. 20 A and 20B are an image and time-dependent fluorescence of a hydrogel chamber-enclosed neurosphere generated using calcium imaging. FIG. 21A is an image of the neurosphere. FIG. 21B is time-dependent calcium imaging intensities at three positions indicated in FIG. 20A.

[0077] FIGS. 21A-D are illustrations of obstacle courses that include maze pathways. FIG. 21A is an illustration of a circular obstacle course with a central chamber surrounded by a maze. FIG. 21B is an illustration of the obstacle course shown in FIG. 21A, showing an aggregate of cells disposed in the central chamber and growing through the obstacle course. FIG. 21C is an illustration of an obstacle course with a rectangular chamber connected to an edge of a rectangular maze. FIG. 21D is an illustration that shows a cell moving through the maze of the obstacle course shown in FIG. 21C.

[0078] FIGS. 22A and 22B are images of a neurosphere enclosed in a hydrogel chamber. FIG. 22A is a fluorescence image of the neurosphere. FIG. 22B is a brightfield image of the neurosphere.

[0079] FIGS. 23A and 23B are fluorescence (FIG. 23A) and brightfield (FIG. 23B) images of a first neurosphere enclosed within a hydrogel chamber and a second neurosphere directly outside of the hydrogel chamber.Attorney Docket No. 59528-732601

[0080] FIGS. 24A and 24B are fluorescence (FIG. 24A) and brightfield (FIG. 24B) images of multiple neurospheres disposed within or nearby an obstacle course comprised of three nested chambers.

[0081] FIGS. 25A and 25B are fluorescence (FIG. 25A) and brightfield (FIG. 53B) images of a neurosphere disposed within an obstacle course comprised of four nested chambers and a neurosphere disposed directly outside of the obstacle course.

[0082] FIGS. 26A and 26B are fluorescence (FIG. 26A) and brightfield (FIG. 26B) images of a neurosphere disposed within an obstacle course comprised of four nested chambers.

[0083] FIGS. 27A and 27B are fluorescence (FIG. 27A) and brightfield (FIG. 27B) images of two neurospheres separately enclosed in adjacent hydrogel chambers that include openings permissive to neurite growth.

[0084] FIGS. 28A and 28B are fluorescence (FIG. 28A) and brightfield (FIG. 28B) images of a neurosphere enclosed in a hydrogel chamber that includes openings permissive to neurite growth.

[0085] FIGS. 29A and 29B are fluorescence (FIG. 29A) and brightfield (FIG. 29B) images of two neurospheres enclosed in adjacent hydrogel chambers that include openings permissive to neurite growth.

[0086] FIG. 30 is a schematic of a method for culturing and sequencing mRNA from a neurosphere enclosed within a hydrogel chamber.

[0087] FIG. 31 is an illustration of two neurons forming a synapse in an interstitial space between two obstacle courses.

[0088] FIGS. 32A-C are brightfield images of a hydrogel chamber with openings through its walls. FIG. 32A shows the hydrogel chamber shortly after its formation. FIG. 32B is an image of the hydrogel chamber collected at a later timepoint, after a cell has moved into an opening in the hydrogel chamber walls. FIG. 32C is an image of the hydrogel chamber collected after the cell has moved through the opening and into the interior of the hydrogel chamber.

[0089] FIGS. 33A and 33B are images of a hydrogel chamber that encloses a cell. FIG. 33A is an image of the hydrogel chamber shortly after its formation, at which point in time the hydrogel chamber includes multiple openings through its walls. FIG. 33B is an image of the hydrogel chamber collected at a later timepoint, after the openings through the hydrogel chamber walls were closed through a subsequent photopolymerization step.Attorney Docket No. 59528-732601DETAILED DESCRIPTION

[0090] Disclosed herein are systems and methods for studying the growth or motility of individual cells and cell aggregates. Contrasting conventional cell motility or growth assays (e.g., scratch-wound or fixation and staining assays) that measure aggregate movement or morphological changes within a population of cells, the presently disclosed methods allow cell movement and growth to be monitored at the single cell-level within custom-printed obstacle courses, mazes, and patterned pathways. For example, the present methods enable a unique obstacle course or maze to be rapidly and synthesized around tens, hundreds, or thousands of cells for massively parallelized growth or motility measurements. Further analyses can then be performed on individual cells to correlate their motility or growth with other phenotypic traits, such as surface marker expression, transcriptome, proliferative capacity, or cytotoxicity.Cell Growth Assays

[0091] An exemplary method for measuring cell growth can include inputting a first cell or an aggregate of first cells into a fluidic channel; inputting a first polymer precursor and optionally a second polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells; synthesizing a pathway adjacent to the first chamber with the first polymer precursor or the second polymer precursor, wherein the pathway is configured to permit growth of the cell or the aggregate of the first cells through the pathway; and measuring growth of the cell or the aggregate of the first cells through at least a portion of the pathway. The first chamber and pathway can collectively be referred to as an “obstacle course”, which hereinafter can denote structures that include at least one chamber and at least one pathway; at least two chambers; at least one pathway and at least one obstacle; or at least one pathway and at least one semipermeable matrix through which a cell can move or grow. As used herein, the term “obstacle” can denote a structure that partially blocks cell movement or growth through a chamber, pathway, opening, or other structure. As used herein, the term “semipermeable matrix” refers to a polymer matrix that is sufficiently porous and having an open space for a cell to move or grow through. The pores, for example, can be generated by the first cell or the aggregate of the first cells as they move through the semipermeable matrix. The first cell or the aggregate of the first cells may express an enzyme that cleaves a portion of the semipermeable matrix. For example, the semipermeable matrix may comprise a protease recognition sequence orAttorney Docket No. 59528-732601 oligosaccharide structure that is cleaved by the first cell or the aggregate of the first cells, thereby increasing the pore size of the semipermeable matrix. Alternatively or in addition thereto, the semipermeable matrix may be synthesized with pores that are sufficiently large for the first cell or the aggregate of the first cells to move through. For example, the semipermeable matrix may be synthesized from a polymer precursor that includes a micronsized porogen that is comparable in size to the first cell or the aggregate of the first cells.

[0092] In general, the term “obstacle” refers to a structure or a collection of structures that impede cell growth or movement. In some cases, an obstacle is impermeable to a cell, so that the cell can bypass but not grow or move through the obstacle. For example, an obstacle can be a pillar or wall that partially blocks a pathway or chamber opening. An obstacle can also comprise a semipermeable matrix, that is, a structure or collection of structures having an open space or pathway through which a cell can move or grow.

[0093] Two or more portions of an obstacle course can be synthesized simultaneously or at different times. In some cases, two or more of a first chamber, a pathway, a second chamber, an additional pathway, a third chamber, a semipermeable matrix, or a series of obstacles are synthesized at the same time, for example by projecting a pattern of light into a channel that simultaneously forms two or more structures (e.g., the first chamber and the pathway). In certain cases, every portion of an obstacle course is synthesized in a single, concerted photopolymerization step. In some cases, two or more structures are formed at different times and optionally from different polymer precursors. For example, following first chamber synthesis from a first polymer precursor, residual first polymer precursor can be removed from a fluidic device and replaced with a second polymer precursor that is used to generate a pathway and a semipermeable matrix in the pathway.

[0094] As used herein, a pathway can be a structure comprised of one or more polymer matrix walls that define an open space adjacent to the first chamber through which the first cell or the aggregate of the first cells can grow or move. A pathway can share a wall with one or more chambers or one or more additional pathways. In particular, one or more polymer matrix walls of a pathway can comprise a polymer matrix wall of the first chamber, a polymer matrix wall of the second chamber, or a combination thereof. A pathway can comprise a first opening to the first chamber or a degradable wall of the first chamber and a second opening to a space outside of the pathway and the first chamber (e.g., to a space outside of an obstacle course that contains the pathway). In some such cases, the pathway comprises a single route from the first opening to the second opening. While numerous pathway designs are applicable to the presently disclosed methods, exemplary pathwaysAttorney Docket No. 59528-732601 include channels, such as the narrow pathway (704) depicted in FIG. 7, and as detailed directly below, spaces formed between nested chambers, such as the pathway (903 A) depicted in FIG. 9A.

[0095] Turning to this latter figure, FIG. 9A depicts a method for measuring cell growth out of an obstacle course that includes a first chamber and a pathway. In this method, a first chamber (902) and a second chamber (903) surrounding the first chamber (902) are synthesized around a collection of cells (901).

[0096] As used herein, a collection of cells may refer to a plurality of cells of a single cell type or a plurality of cells with multiple cell types. The collection of cells may be connected in the form of one or more multicellular structures such as organoids, spheroids, neurospheres, and the like. As an example, a neurosphere may include a heterogeneous mixture of neural stem cells, neural progenitor cells, and neurons, as well as non-neuronal cells such as glial cells, astrocytes, oligodendrocytes, and Schwann cells. The ratio of cell types within the collection of cells (901) may be controlled, wherein collections of cells (901) with known compositions are input into a fluidic device or enclosed within an obstacle course. The cell types within a collection of cells (901) may be identified and quantified with fluorescently labeled antibodies targeted to surface markers for the cell types, and the collection of cells (901) can be enclosed within an obstacle course if the collection of cells (901) includes a desired number or ratio of cell types. Similarly, in some cases, the numbers of individual cell types may be determined based on cell morphologies determined through imaging. The types of cells present within a collection of cells may also be measured following analysis within an obstacle course. The numbers and types of cells present within a collection of cells can be determined using one or more methods disclosed herein, including brightfield imaging (e.g., to measure morphology), surface markers, transcriptome, or a combination thereof.

[0097] With continued reference to FIG. 9A, the first (902) and second (903) chambers each include multiple openings, such that the space between the walls of the first (902) and second (903) chambers defines a pathway (903 A) through which the collection of cells (901) can grow. The collection of cells may be trapped and / or adhered to a surface within the first chamber (902). The growth of structures from the collection of cells (901), such as neurites, may be measured (910). For example, a first set of structures (911 A, e.g., neurites) that do not grow out of the first chamber, a second set of structures (91 IB) that grow into but not through the pathway, and a third set of structures (911C) that grow out of the pathway may be counted using an imaging method.Attorney Docket No. 59528-732601

[0098] A pathway can be open to a space outside of an obstacle course or to another pathway or chamber. For example, the method can comprise synthesizing a second chamber with the first polymer precursor or the second polymer precursor, wherein the second chamber is fluidically coupled to the pathway. The method can similarly comprise inputting a third polymer precursor into the fluidic channel, and synthesizing a second chamber with the third polymer precursor, wherein the second chamber is fluidically coupled to the pathway. The first chamber, pathway, and optionally second chamber (or additional structures such as additional pathways) can be synthesized separately or in a single step, for example by projecting a single pattern of light that forms the first chamber, pathway, and optionally second chamber through photopolymerization. In photopolymerization-based syntheses, the relative thicknesses and porosities of the first chamber, pathway, and optionally second chamber, as well as portions of one or more of these structures, can be varied by modulating the intensity and duration of light applied during synthesis.

[0099] In certain implementations of the presently disclosed methods, a pathway can comprise a maze. For example, an obstacle course can include a first chamber that comprises an opening to an entrance of a maze and a second chamber that comprises an opening to an exit of the maze. Alternatively, an obstacle course can include a first chamber that comprises an opening to a maze, wherein the maze includes a second opening to a space outside of the obstacle course. In some cases, a maze comprises i) a first opening of a first chamber; ii) a second opening of a second chamber or a space outside of the first chamber (e.g., a space outside of the obstacle course of which a maze is part of), and iii) a plurality of interconnected paths of which only a subset lead from the first opening to the second opening. In other cases, a maze comprises a single opening to a chamber or a non-enclosed space. In such cases, the maze may be synthesized surrounding a cell or an aggregate of cells, and growth of the cell or the aggregate of the cells out of the cell can be measured. In general, the first and second openings are sufficiently large for the first cell or the aggregate of the first cells to grow through. In some cases, a maze includes a single route that connects its first opening to its second opening. In other cases, a maze includes a plurality of routes between the first and second openings. Furthermore, in certain designs, a maze can include a plurality of first openings and / or a plurality of second openings. A method comprising a maze can include measuring a time between a cell growing from the first opening to the second opening of the maze or a pathway comprising the maze. An illustration of a cell growth assay through a maze is provided in FIGS. 21C and 21D. In these figures, the maze pathway (2102) adjacent to a first chamber (2101), and includes a first opening (2104) to the firstAttorney Docket No. 59528-732601 chamber and a second opening (2103) to a space outside of the obstacle course. However, the second opening (2103) can alternatively lead to another pathway or another chamber of the obstacle course.

[0100] When a second chamber is present, the growth measured during the method can be growth towards or into the second chamber. The second chamber can include a second cell, an aggregate of second cells, a bead, or a combination of species that affect the growth of the first cell or the aggregate of the first cells. Similarly, the second chamber can include a reagent such as a soluble factor that alters cell growth. The growth can be towards the second cell, the aggregate of the second cells, the bead, the reagent, or another species in the second chamber. The method can also determine whether a species in the second chamber alters the growth of the first cell or the aggregate of the first cells. As examples, the bead can be a nanoparticle, a microparticle, a lipid particle, a hydrogel particle, or a nanomaterial, and can include a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof.

[0101] While embodiments of the present disclosure include measuring cellular growth through pathways, cell growth may also be measured outside of or between chambers. As an example, a method for measuring cellular growth can comprise inputting a first cell or an aggregate of first cells and a first polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells; and measuring growth of the first cell outside of the first chamber, for example by measuring the length or density of neurites that grow from the first cell or the aggregate of the first cells using imaging. For example, the growth of neurites can be imaged every 24 hours where the distance covered by the growth of the neurite can be measured per a unit of time or the time required for the neurite to reach a certain predetermined distance or location. In other examples, the time required for a first neurite from a first cell to grow and touch a second neurite from a second cell can be measured. The presently disclosed methods can also measure growth between cells. For example, a method for measuring cellular growth can comprise inputting a first cell or an aggregate of first cells, a second cell or an aggregate of second cells, and a first polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells, wherein the second cell or the aggregate of the second cells are outside of the first chamber; and measuring growth of the first cell or the aggregate of the first cells from the first chamberAttorney Docket No. 59528-732601 towards the second cell or the aggregate of the second cells. The method can include synthesizing a chamber around the second cell or the aggregate of the second cells, for example with the first polymer precursor used to synthesize the second chamber or with a second polymer precursor that is different from the first polymer precursor.

[0102] An example of a method for measuring growth between cell aggregates is provided in FIG. 9B. This figure depicts a cell growth assay that is similar to the cell growth assay depicted in FIG. 9A. In this assay, a first collection of cells (901) within a fluidic device is disposed within a first chamber (902) that is enclosed within a second chamber (903). The first (902) and second (903) chambers each comprise multiple openings, and thereby define a pathway (903 A) through which the first collection of cells (901) can grow. A second collection of cells (904) is disposed outside of the first (902) and second chambers (903). The second collection of cells (904) may have limited motility. For example, the second collection of cells (904) may be adhered to a surface of the fluidic device. The growth of structures from the first (901) and second (904) collections of cells (901) may be measured (910). In this example, the first collection of cells (901) grows a neurite (911C) that extends outside of the pathway (903 A) and the second collection of cells (904) grows a neurite (91 IB) that grows to and forms a synapse (911C) the neurite (911 A) from the first collection of cells (901).

[0103] As an additional example, a method for measuring cellular growth can comprise inputting a first cell or an aggregate of first cells into a fluidic channel; inputting a first polymer precursor and optionally a second polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells; synthesizing a second chamber with the first polymer precursor or the second polymer precursor; and measuring growth of the cell or the aggregate of the first cells from the first chamber towards the second chamber. A second cell, an aggregate of second cells, a bead, or a combination thereof can optionally be disposed within the second chamber. An example of such an assay is illustrated in FIG. 9C. This assay differs from the assays depicted in FIGS. 9A and 9B in that it does not include the synthesis of a pathway adjacent to a chamber or between cells. In this method, a first cell (901) and a second cell (904) are loaded into a fluidic device. A first chamber (902) is then synthesized around the first cell (901) and a second chamber (903) is synthesized around the second cell (904). The first cell (901), the second cell (904), or the combination thereof may adhere to a surface of the fluidic device, such as a bottom surface of the fluidic device that is coated with an adherent substrate. TheAttorney Docket No. 59528-732601 first cell (901) and / or the second cell (904) may adhere prior or subsequently to first chamber (902) and / or second chamber (903) formation. For example, the first and second cells (901, 904) can be in a suspended state at the time of first and second chamber (902, 903) formation, and may later adhere to a fluidic device surface, to a chamber surface, or a combination thereof. As some types of cell growths (e.g., neurites) primarily form following cell transitions to adherent states, such a method may ensure that the majority of cell growths (e.g., 911 A, 91 IB) form subsequently to chamber (902, 903) formation. However, in alternative embodiments, the first and / or second cells (901, 904) may adhere to fluidic device surfaces prior to first and second chamber (902, 903) formation. It is worth noting that chambers (902, 903) may be formed over portions of cells (901, 904) or cell growths (911 A, 91 IB), and that cell growths (911 A, 91 IB) may continue to develop and grow after a chamber (902, 903) forms over top of or around the cell growth (911 A, 91 IB).

[0104] With continued reference to FIG. 9C, the first (902) and second (903) chambers each include one or more openings that are sufficiently large for growths from the first (901) and second (904) cells to pass through, but prohibitively small for the first (901) and second (904) cells to pass through. Growths from the first cell (911 A) and second cell (91 IB) can then be measured (910), for example through brightfield or fluorescence imaging. The first (901) and second cells (904) can be subjected to further single cell analyses, such as surfaceome (surface marker) or transcriptomic analysis.

[0105] In general, cellular growth measured in a method of the present disclosure is growth of one or more structures or protrusions that are comparable or greater in length than a dimension of the cell from which they are derived. As non-limiting examples, a method of the present disclosure may measure axonal growth, dendritic growth, pseudopodium growth, filopodium growth, lamellipodium growth, a growth cone, acrosome growth, comet tail growth, or a combination of such structures. The measuring can comprise determining a rate or distance of the growth. The measuring can also comprise determining whether the direction of the growth is random or directed. For example, the measuring can comprise determining whether a second cell, an aggregate of second cells, a bead, or a combination of species present in a fluidic device increases growth, decreases growth, randomizes a direction of growth, or directs a direction of growth.

[0106] The measuring can also include determining whether a second cell or aggregate of second cells exhibits simultaneous growth towards the first cell or the aggregate of the first cells. For example, the first cell or the aggregate of the first cells and the second cell or the aggregate of the second cells can be neurons. In such cases, a method can includingAttorney Docket No. 59528-732601 measuring neurite growth between the first cell or the aggregate of the first cells and the second cell or the aggregate of the second cells, and can optionally include detecting synapse formation between the first cell or the aggregate of the first cells and the second cell or the aggregate of the second cells. Such methods can also include determining an amount of time required for synapse formation.

[0107] An example of such an assay is illustrated in FIG. 31. This figure depicts two neurons (3101 A and 3101B) disposed in separate obstacle courses comprised of chambers (3102A and 3102B) that are each connected to pathways (3103 A and 3103B), respectively. The pathways (3103 A and 3103B) are open to an interstitial space in between the obstacle courses. Neurite (3104A and 3104B) growth from each neuron (3101 A and 3103B) resulted in synapse formation.

[0108] Similarly, a method can include measuring a junction between the first cell or the aggregate of the first cells and the second cell or the aggregate of the second cells. The method may also include identifying a type of junction between the first cell or the aggregate of the first cells and the second cell or the aggregate of the second cells. Cell junctions are supramolecular structures that form between cells or between a cell and a support such as an extracellular matrix. Cell Junctions are typically classified into three functional categories: occluding junctions that seal gaps between adjacent cells, anchoring junctions that mechanically affix cells to structural supports and neighboring cells, and communicating junctions that facilitate intercellular signaling. Many cells are capable of forming more than one type of junction. Examples of cell junctions measurable with the disclosed methods include occluding junctions, anchoring junctions, communicating junctions, tight junctions, adherens junctions, desmosomes, gap junctions, channel forming junctions, plasmodesmata, and non-classical junctions. The junctions may be measured, for example, with brightfield imaging or fluorescence imaging (e.g., using antibody stains for junction structural proteins such as cadherin, catenin, claudin, occludin, desmoglein, desmocolin, desmoplakin, and / or keratin).

[0109] A method for measuring cellular growth can also include contacting a cell or an aggregate of cells with a reagent that affects the growth of the cell or the aggregate of cells. For example, a method may include contacting a neurosphere with an ephrin, a netrin, a neurotrophin, or a semaphorin with a known capacity to stimulate or inhibit neurite growth. Alternatively, a method can include contacting a cell or an aggregate of cells with a reagent, and determining whether the reagent alters the growth of the cell or the aggregate of cells. As examples, the method can include determining whether the reagent increases the growth,Attorney Docket No. 59528-732601 decreases the growth, directs a direction of the growth, or randomizes the direction of the growth of the cell or the aggregate of cells. As non-limiting examples, the reagent may be a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof.

[0110] A reagent may be added to portions of a chamber, pathway, or obstacle course. In methods that include a pathway and a first chamber, the pathway can have a higher concentration of the reagent than the first chamber, or the first chamber can have a higher concentration of the reagent than the pathway. Uneven reagent concentrations can be achieved by creating or providing an opening in a pathway or chamber, or by creating chamber and pathway walls with varying porosities. The pathway can also comprise a chemical gradient of the reagent. For example, the pathway can comprise a first end that with an opening to the first chamber or a degradable wall of the first chamber and a second end with an opening to a second chamber or to a space outside of the first chamber (e.g., open to the fluidic channel); wherein: the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway. A reagent can also be produced by a second cell or aggregate of second cells.[OHl] A method can also include degrading a portion of a chamber, pathway, or other polymeric structure to create an opening through which a cell or aggregate of cells can grow. In some cases, a method comprises degrading a portion of the first chamber comprising the first cell or the aggregate of the first cells to form an opening that allows the growth of the first cell or the aggregate of the first cells out of the first chamber. Alternatively, a chamber (e.g., the first chamber or the second chamber in the methods disclosed herein) can include an opening such as a gap or a pore that is sufficiently large to permit cell growth, such as neurite growth, to pass through.Cell Motility Assays

[0112] The present disclosure also provides methods for measuring the motility of individual cells and cell aggregates. Cellular motility has traditionally been challenging to measure at the single-cell level. Many motility measurements, such as scratch-wound assays, track average cell movement within a population of cells. Conversely, while live imaging methods (e.g., tracking a population of cells on a single slide or culture plate) can measure the movement of individual cells, these methods typically cannot control the local environment or pathways encountered by cells or facilitate downstream single-cell measurements that can be correlated to measured motilities. The presently disclosed methods overcome theseAttorney Docket No. 59528-732601 limitations by synthesizing custom obstacle courses around individual cells of interest, thereby trapping the cells for motility measurements and downstream analyses that connect measured motilities to other phenotypic traits.

[0113] In some embodiments, a method for measuring cellular movement comprises inputting a first cell or an aggregate of first cells into a fluidic channel; inputting a first polymer precursor and optionally a second polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells; synthesizing a pathway adjacent to the first chamber with the first polymer precursor or the second polymer precursor, wherein the pathway is configured to permit movement of the cell or the aggregate of the first cells through the pathway; degrading a portion of the first chamber to form an opening that allows movement of the first cell or the aggregate of the first cells from the first chamber to the pathway; and measuring movement of the first cell or the aggregate of the first cells through at least a portion of the pathway. In general, the obstacle course designs disclosed for cell growth measurements are also applicable for measuring cell movement, with chamber and pathway openings and sizes being expanded as needed to permit cell and cell aggregate movement. Accordingly, the method for measuring cellular movement can include synthesizing a second chamber, for example with the first polymer precursor, the second polymer precursor, or a third polymer precursor input with the first and / or second polymer precursor or separately from the first and / or second polymer precursor.

[0114] An example of such a method is provided in FIG. 8. In this assay, an “obstacle course” (801) is synthesized around a first cell (802A) and a second cell (803 A), such that the first (802A) and second (803 A) cells are disposed in different chambers (802 and 803, respectively) of the obstacle course. Specifically, the first cell (802A) is disposed in a first chamber (802) that is connected to a second chamber (803) containing the second cell (803 A) by a pathway (804). The first chamber (802) containing the first cell (802A) includes a degradable portion (802B) that prevents movement of the first cell into the pathway (804). At a selected time, the degradable portion (802B) of the first chamber (802) can be selectively degraded (810), for example by photocleaving the degradable portion (802B) or contacting the obstacle course (801) with a reagent that selectively degrades the first portion (802B) of the first chamber without degrading the remainder of the first chamber (802), pathway (804), or second chamber (803). Movement of the first cell (802A) towards the second cell (803 A), for example through the pathway (804), can then be monitored (820).Attorney Docket No. 59528-732601

[0115] As another example, a method for measuring cellular movement of the present disclosure can comprise inputting a first cell or an aggregate of first cells into a fluidic channel; inputting a first polymer precursor and optionally a second polymer precursor or the second polymer precursor and a third polymer precursor into the fluidic channel; synthesizing a first chamber with the first polymer precursor within the fluidic channel, the first chamber trapping and at least partially enclosing the first cell or the aggregate of the first cells; synthesizing a pathway adjacent to the first chamber with the first polymer precursor or the second polymer precursor, wherein the pathway is configured to permit movement of the cell or the aggregate of the first cells through the pathway to a second chamber; synthesizing the second chamber adjacent to the pathway with the first polymer precursor, the second polymer precursor, or the third polymer precursor, and measuring movement of the first cell or the aggregate of the first cells through the pathway towards the second chamber. The first chamber can comprise an opening through which the first cell or the aggregate of the first cells can move, such as a gap or a pore in a wall of the first chamber.

[0116] An example of such an assay is illustrated in FIG. 7. In this method, a plurality of cells (701) disposed in a fluidic channel (700) are each partially enclosed within first chambers (703) extending from the bottom to the top surface of the fluidic channel (700). Each first chamber is connected to a second chamber (705) by a pathway (704). The first chamber (703), pathway (704), and second chamber (705) can collectively comprise a dumbbell-like shape (702). However, alternate designs are contemplated herein, including nested chambers (e.g., as depicted in FIG. 9A), maze-like designs, and designs that include multiple and / or branching pathways. Furthermore, while the pathway in FIG. 7 is illustrated as a channel, other designs may be utilized in the obstacle courses disclosed herein. Movement of the cells (701) through the pathway (704) is then monitored (710). The cells (701) traverse the pathway (704) at varying rates, with a first subset of cells (711) remaining in the first chamber (703), a second subset of cells (712) having traversed a short distance within the first region (704), and a third subset of cells (713) having traversed a majority of the first region (704). The movement of the cells (701) can be correlated to a cellular characteristic (e.g., expression of a surface marker) or responsiveness to a stimulus (e.g., a chemoattractant or repellant). For example, the monitoring (710) may identify a third subset of cells (713) that strongly respond to and move towards a chemoattractant in the second chamber, a second subset of cells (712) that weakly respond to and move towards the chemoattractant, and a first subset of cells (711) that do not respond to the chemoattractant. Surfaceomes, secretomes, transcriptomes, genomes, or other phenotypic attributes of the cellsAttorney Docket No. 59528-732601 can also be identified as disclosed elsewhere herein. The phenotypes can be associated with the movement of the first (711), second (712), and third (713) subsets of the cells.

[0117] As depicted in FIG. 7, an obstacle course can include a dumbbell-like design that comprises a first chamber, a pathway that comprises a first end that is adjacent to the first chamber and a second end that comprises an opening to a second chamber. As with other designs, such an obstacle course can include a wall or opening that permits flow of a reagent into the obstacle course. In particular, a wall of the first chamber can comprise a greater thickness than a wall of the pathway, the wall of the pathway can comprise a greater thickness than the wall of the first chamber, the wall of the first chamber can comprise a greater thickness than a wall of the second chamber, the wall of the second chamber can comprise a greater thickness than the wall of the first chamber, the wall of the pathway can comprise a greater thickness than the wall of the second chamber, the wall of the second chamber c can comprise a greater thickness than the wall of the pathway, the wall of the first chamber can comprise a greater porosity than the wall of the pathway, the wall of the pathway can comprise a greater porosity than the wall of the first chamber, the wall of the first chamber can comprise a greater porosity than the wall of the second chamber, the wall of the second chamber can comprise a greater porosity than the wall of the first chamber, the wall of the pathway can comprise a greater porosity the wall of the second chamber, the wall of the second chamber can comprise a greater porosity than the wall of the pathway, or a combination thereof. A method can include inputting a reagent into the fluidic channel that contains the obstacle course that: diffuses across a wall of the first chamber and does not diffuse across a wall of the pathway, diffuses across the wall of the pathway and does not diffuse across the wall of the first chamber, diffuses across the wall of the first chamber and does not diffuse across the wall of the second chamber, diffuses across the wall of the second chamber and does not diffuse across the wall of the first chamber, diffuses across the wall of the pathway and does not diffuse across the wall of the second chamber, diffuses across the wall of the second chamber and does not diffuse across the wall of the pathway, or a combination thereof. An obstacle course can alternatively have a vial-like design, in which a pathway is connected to a chamber at its first end and open at its second end.

[0118] In many aspects, a pathway includes or is a channel. A pathway can include a cross- sectional area that is less than a largest dimension of a cell or an aggregate of cells, such as the pathway (704) in FIG. 7. For example, the cross-sectional area of a pathway can be less than the largest dimension of the first cell by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, atAttorney Docket No. 59528-732601 least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%. A pathway can also include a larger cross-sectional area than the cell or aggregate of cells. In some cases, the pathway has a cross-sectional area that is at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 500%, at least about 1000% larger than the larger dimension of the first cell. In certain cases, the pathway comprises a width that is similar to a largest dimension of the first cell, for example about 1 to 2 pm, about 1 to 5 pm, about 1 to 10 pm, about 1 to 15 pm, about 1 to 20 pm, about 1 to 25 pm, about 2 to 5 pm, about 2 to 10 pm, about 2 to 15 pm, about 2 to 20 pm, about 2 to 25 pm, about 5 to 10 pm, about 5 to 15 pm, about 5 to 20 pm, about 5 to 25 pm, about 10 to 15 pm, about 10 to 20 pm, about 10 to 25 pm, or about 15 to 25 pm. In some cases, the pathway is capable of accommodating multiple cells or aggregates of cells. For example, the pathway may have a width of about 25 to 50 pm, about 25 to 100 pm, about 25 to 250 pm, about 25 to 500 pm, about 25 to 1000 pm, about 25 to 1500 pm, about 25 to 2000 pm, about 25 to 2500 pm, about 50 to 100 pm, about 50 to 250 pm, about 50 to 500 pm, about 50 to 1000 pm, about 50 to 1500 pm, about 50 to 2000 pm, about 50 to 2500 pm, about 100 to 250 pm, about 100 to 500 pm, about 100 to 1000 pm, about 100 to 1500 pm, about 100 to 2000 pm, about 100 to 2500 pm, about 250 to 500 pm, about 250 to 1000 pm, about 250 to 1500 pm, about 250 to 2000 pm, about 250 to 2500 pm, about 500 to 1000 pm, about 500 to 1500 pm, about 500 to 2000 pm, about 500 to 2500 pm, about 1000 to 1500 pm, about 1000 to 2000 pm, about 1000 to 2500 pm, or about 1500 to 2500 pm. In some cases, the pathway comprises a width of about 500 to 5000 pm, about 500 to 10000 pm, about 1000 to 5000 pm, about 1000 to 10000 pm, about 2500 to 5000 pm, about 2500 to 10000 pm, or about 5000 to 10000 pm.

[0119] Furthermore, a pathway can be short or long relative to the size of a cell being measured. As non -limiting examples, a pathway can have a length of about 10 to 25 pm, about 10 to 50 pm, about 10 to 100 pm, about 10 to 250 pm, about 10 to 500 pm, about 10 to 1000 pm, about 10 to 1500 pm, about 10 to 2000 pm, about 10 to 2500 pm, about 25 to 50 pm, about 25 to 100 pm, about 25 to 250 pm, about 25 to 500 pm, about 25 to 1000 pm, about 25 to 1500 pm, about 25 to 2000 pm, about 25 to 2500 pm, about 50 to 100 pm, about 50 to 250 pm, about 50 to 500 pm, about 50 to 1000 pm, about 50 to 1500 pm, about 50 to 2000 pm, about 50 to 2500 pm, about 100 to 250 pm, about 100 to 500 pm, about 100 to 1000 pm, about 100 to 1500 pm, about 100 to 2000 pm, about 100 to 2500 pm, about 250 to 500 pm, about 250 to 1000 pm, about 250 to 1500 pm, about 250 to 2000 pm, about 250 to 2500 pm, about 500 to 1000 pm, about 500 to 1500 pm, about 500 to 2000 pm, about 500 toAttorney Docket No. 59528-7326012500 pm, about 1000 to 1500 pm, about 1000 to 2000 pm, about 1000 to 2500 pm, or about 1500 to 2500 pm. However, in obstacle courses with complex pathways, such as mazes, a pathway can be longer than 2500 pm. For example, in some cases, a pathway is about 500 to 5000 pm, about 500 to 10000 pm, about 1000 to 5000 pm, about 1000 to 10000 pm, about 2500 to 5000 pm, about 2500 to 10000 pm, or about 5000 to 10000 pm.

[0120] A cell motility measurement can determine a rate or a distance of the movement of a cell or an aggregate of cells through at least the portion of a pathway. A cell motility measurement can also determine whether movement is stochastic (i.e., random) or directed. For example, the method can include determining whether movement is towards a second cell or an aggregate of second cells located outside of the first chamber; a bead located outside of the first chamber; or a combination thereof.

[0121] Similarly, the method can include whether the movement is towards a reagent such as a bead or chemoattractant. For example, the method can include contacting a cell or an aggregate of cells with the reagent, and determining whether the reagent alters the movement of the cell or the aggregate of the cells. As examples, the method can include determining whether the reagent increases the movement, decreases the movement, directs a direction of the movement, or randomizes the direction of the movement of the cell or the aggregate of the cells. As in cell growth assays, a pathway can include a higher concentration of the reagent than a chamber (e.g., the first chamber), or a chamber can comprise a higher concentration of the reagent than the pathway. The pathway can also include a chemical gradient of the reagent. In particular, the pathway can comprise a first end that with an opening to the first chamber or a degradable wall of the first chamber and a second end with an opening to a second chamber or to a space outside of the first chamber (e.g., open to the fluidic channel); wherein: the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway. A reagent can also be produced by a second cell or aggregate of second cells. As non-limiting examples, the reagent may be a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof.

[0122] FIGS. 10A and 10B illustrate two chemoattractant-based motility assays consistent with the present disclosure. In FIG. 10A, an obstacle course (1001) is synthesized surrounding a cell (1005) within a fluidic device. The obstacle course 1001 includes a first chamber (1002) partially enclosing the cell (1005), a second chamber (1003), and a pathway (1004) connecting the first (1002) and second (1003) chambers. Walls of the second chamberAttorney Docket No. 59528-732601(1003) may include openings that are sufficiently large for reagents to flow through. The openings (1003 A) in the walls of the second chamber (1003) may be prohibitively small for the cell (1005) to move through. Any of the polymer matrix walls disclosed herein may include such openings. For example, openings in the nested chambers (902, 903) depicted in FIGS. 9A-B may be prohibitively small for a cell or a collection of cells (901) to move through, but may be large enough for one or more reagents such as nanoparticles, liposomes, viruses, or proteins to flow through. In some cases, an opening in a chamber wall is about 2 to about 5 pm, about 2 to about 10 pm, about 2 to about 25 pm, about 2 to about 50 pm, about 2 to about 100 pm, about 2 to about 250 pm, about 5 to about 10 pm, about 5 to about 25 pm, about 5 to about 50 pm, about 5 to about 100 pm, about 5 to about 250 pm, about 10 to about 25 pm, about 10 to about 50 pm, about 10 to about 100 pm, about 10 to about 250 pm, about 25 to about 50 pm, about 25 to about 100 pm, about 25 to about 250 pm, about 50 to about 100 pm, about 50 to about 250 pm, or about 100 to about 250 pm. It is also worth noting that polymer matrix walls can include pores which, separately from openings (1003 A), can allow reagents such as proteins and small molecules to diffuse through the polymer matrix walls.

[0123] In some embodiments, a method may involve measuring cell movement through pores or openings in a polymer matrix wall. In such a method, the size of the pores or openings can be varied across obstacle courses, and the ability of cells to move through the pores of openings can be associated with pore or opening size, as well as other cell characteristics (such as transcriptome, proteome, or morphology) that are measured during the method.

[0124] Returning to FIG. 10A, a chemoattractant 1011 is then input 1010 into the fluidic device. The chemoattractant (1011) flows through the openings (1003 A) in the second chamber (1003), but is blocked from flowing across the walls of the first chamber (1002) or pathway (1004). Accordingly, the pathway (1004) includes a gradient of the chemoattractant (1011) that is generated by the flow of the chemoattractant (1011) from the second chamber (1003) into the pathway (1004). Movement of the cell through the pathway (1004) is then measured (1020). Further analyses, such as surfaceomic or transcriptomic profiling, can optionally be performed on and correlated to the movement of the cell (1005).

[0125] In FIG. 10B, an obstacle course (1001) is synthesized surrounding a cell (1005) and a bead (1007A) within a fluidic device. The obstacle course (1001) includes a first chamber (1002) partially enclosing the cell (1005), a second chamber (1003) enclosing or partially enclosing the bead (1007A), and a pathway 1004 fluidically coupling the first (1002) and second (1003) chambers. The bead (1007A) is coupled to a chemoattractant (1007B) by aAttorney Docket No. 59528-732601 cleavable linker (1007C). The second chamber (1003) is separated from the pathway (1004) by a wall (1006) that includes openings or pores that are too small for the bead to flow through, but sufficiently large for cleaved chemoattractant (1007B) to cross. At a defined time, the chemoattractant (1007B) is cleaved from the bead (1007A), for example with light, heat, an enzyme, or a chemical reagent. Chemoattractant (1007B) liberated from the bead (1007 A) flows across the wall (1006), generating a gradient of the chemoattractant (1007B) in the pathway (1004). Movement of the cell (1005) through the pathway (1004) can then be measured (1020), for example with fluorescence or brightfield imaging. Further analyses can optionally be performed on and correlated with the movement of the cell (1005).

[0126] In some embodiments, the growth and / or motility of a first cell is assessed in the presence of a second cell. Such embodiments may, for example, be analogous to the method outlined in FIG. 10B, wherein the bead (1007A) is replaced with a second cell. The first and second cells can be separated by a polymer matrix wall with pores and / or openings that are prohibitively small for the second cell to transit but are large enough to permit secretions from the second cell to reach the first cell. A series of analyses may be performed (e.g., multiple instances of the obstacle course with separate instances of the first and second cells can be fabricated and then monitored) with varying pore and / or opening sizes in the wall or walls separating the first and second cell. The pore and / or opening size may then be related to the growth and / or movement of the first cell to determine the size of second cell secretions that affect first cell growth and / or movement Pore size may be modulated, for example, by varying porogen size in the polymer precursor used to generate the wall or walls separating the first and second cells.

[0127] As detailed above, the first chamber can be fluidically coupled to a second chamber. The second chamber can comprise an opening through which the first cell or the aggregate of the first cells can move, such as a gap or a pore. The second chamber can be synthesized with the opening or degraded to form the opening. The second chamber can include a second cell, an aggregate of second cells, a bead, or a combination of such species. The second cell, aggregate of second cells, or bead can function as a sort of reagent in the disclosed methods. For example, a method can include determining whether the second cell, the aggregate of the second cells, the bead, or the combination thereof increases the movement, decreases the movement, randomizes a direction of the movement, or directs the direction of the movement of the first cell or the aggregate of the first cells. Similarly, the disclosed methods may be used to study the movement of immune cells and their targets. As a specific example, the second cell or the aggregate of the second cells can comprise a target cell and the first cell orAttorney Docket No. 59528-732601 the aggregate of the first cells can comprise an effector cell configured to interact with the target cell.

[0128] A cell motility assay can include measuring movement through a semipermeable matrix. The semipermeable matrix may be sufficiently porous to allow passage of the first cell therethrough. Alternatively, the semipermeable matrix may be degradable by the first cell, thereby allowing the first cell to degrade a pathway through the semipermeable matrix. The semipermeable matrix can be disposed in a pathway, a chamber, or an opening therebetween. The semipermeable matrix or a portion of the semipermeable matrix can be shorter than a height of the pathway (e.g., shorter than a distance between top and bottom surfaces of a flow cell in which the semipermeable matrix is located), narrower than a width of the pathway, or a combination thereof. In some methods, the first cell or the aggregate of the first cells passes through the semipermeable matrix from a first end to a second end of the pathway. In other methods, the first cell or the aggregate of the first cells passes through the semipermeable matrix to enter or exit a pathway or chamber (e.g., to exit the first chamber) or to traverse a portion of the pathway.

[0129] The semipermeable matrix can be synthesized from a polymer precursor used in the synthesis of a pathway or chamber in the obstacle course (e.g., the first polymer precursor used to synthesize the first chamber), for example using lower power or shorter duration light during photopolymerization or lower heat during thermal polymerization. For example, when the first polymer precursor is structure 8 of Table 2B, a photopolymerization process may utilize about 3 seconds of illumination to generate a cell-impermeable polymer matrix wall of a pathway or chamber and less than 3 seconds of illumination to generate a semipermeable matrix through which a cell can grow and move. While polymerization rate typically varies depending on the polymer precursor, fluidic channel dimensions, and cell chemical tolerances, in many aspects disclosed herein, each component of an obstacle course (e.g., chambers, pathways, semipermeable matrices, etc.) are each independently synthesized in less than about 60 seconds, less than about 30 seconds, less than about 20 seconds, less than about 15 seconds, less than about 10 seconds, or less than about 5 seconds. Accordingly, complex obstacle courses can be rapidly synthesized around cells of interest. Using a spatial energy modulation element that is capable of generating spatially-controlled energy patterns, such as a digital micromirror device (DMD), hundreds, thousands, or tens of thousands of obstacle courses can be generated simultaneously or in rapid succession. While conventional cell growth and motility assays measure collections of cells in uniform environments, theAttorney Docket No. 59528-732601 presently disclosed methods enable tens of thousands of cells to be individually measured within unique environments within a single fluidic channel.

[0130] Semipermeable matrix, and more generally polymer matrix (e.g., semipermeable matrix as well as chamber and pathway wall) porosity can also be modulated by varying the polymer precursor used for polymer matrix synthesis. Accordingly, the semipermeable matrix may be synthesized concurrently or subsequently to the synthesis of a chamber (e.g., the first chamber) and / or a pathway. As used herein, a different polymer precursor can denote difference in an aspect of a polymer precursor formulation, for example identical but lower concentrations of monomers, different monomers, different photoinitiators, or different porogens. In some cases, the semipermeable matrix is formed from a polymer precursor that includes matrigel, gelatin, photocrosslinkable gelatin, collagen, a gel of an enzymatically degradable macro monomer, or a gel of an enzymatically cleavable PEG macromonomer. In certain cases, the polymer precursor used for semipermeable matrix synthesis is enzymatically cleavable or bioabsorbable.

[0131] As used herein, the term “porogen” can denote a species that modulates the porosity of a polymer matrix but does not incorporate into the polymer matrix during polymerization. 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.

[0132] As used herein, the term “photoinitiator” can denote a species that generates a radical upon photoexcitation. In many cases, a photoinitiator included in a polymer precursor formulation is a type I photoinitiator, that is a molecule that generates radicals through intramolecular cleavage (e.g., homolysis) upon photoexcitation, or a type II photoinitiator, that is a molecule that abstract an electron or hydrogen atom from a co-initiator following photoexcitation. Examples of photoinitiators utilizable in the present methods include acetophenone, anisoin, anthraquinone, anthraquinone-2-sulfonic acid, benzil, benzoin, benzophenone, 3,3’,4,4’-benzophenonetetracarboxylic dianydride, 4-benzoylbiphenyl, 2- benzyl-2-(dimethylamino)-4’-morpholinobutyrophenone, dibenzosuberenone, 2,2- diethoxyacetophenone, 2-ethylanthraquinone, ferrocene, 2-isopropylthioxanthone, lithium phenyl (2,4,6-trimethylbenzoyl) phosphinate, methyl-2-benzoylbenzoate, and thiooxanthen- 9-one.Attorney Docket No. 59528-732601

[0133] An example of a cell motility assay that measures cell movement through a semipermeable matrix is outlined in FIG. 6. The method can include synthesizing a first chamber (601 A) that encloses a cell (602) within a flow cell channel or other suitable fluidic space. A pathway (60 IB) can be synthesized adjacent to the chamber, either concurrently with the synthesis of the first chamber (601 A) or at an earlier or later time. The first chamber (601A) and pathway (601B) can be formed from the same polymer precursor or different polymer precursors. In FIG. 6, a first end of the pathway (60 IB) is enclosed by a portion of a wall (601C) of the first chamber (601 A). A second end of the pathway (601B) opposite the first end is open. However, alternate pathway designs are disclosed herein, including pathways with an opening disposed partway along an edge, pathways with multiple openings, multi-channel structures, nested channel structures (e.g., as depicted in FIGS. 9A-B), and maze-like structures.

[0134] A second chamber (611) is then synthesized (610) enclosing the open end of the pathway (601B). The second chamber (611) has greater porosity than the first chamber (601A) and pathway (601B), such that a polymer precursor (621) input (620) into the flow cell channel permeates the second chamber (611) but does not diffuse into the first chamber (601 A) which is fully closed and impermeable to the polymer precursor (621). As the pathway (601B) is open to the second chamber (611), the polymer precursor (621) fills the interiors of the second chamber and the pathway.

[0135] Next, the polymer precursor is polymerized (630) to form a semipermeable matrix (631) in a selected region of the flow cell, in this case within the pathway (601B) disposed between the first (601 A) and second (611) chambers. The semipermeable matrix (631) may extend from top to bottom surfaces of the flow cell channel, or may be formed at specified heights within the flow cell channel. Unreacted polymer precursors (621) can be removed from flow cell channel, for example in a wash step. At this stage, a portion (601C) of the first chamber wall separates the first chamber (601 A) from the pathway (601B) and the semipermeable matrix (631).

[0136] The portion of the first chamber wall (601C) can be degraded (640) to open the first chamber (601 A) to the pathway (601B) and second chamber (611). The cell (602) can then be induced to move through the semipermeable matrix (631) and into the second chamber (611). In this example, chemotaxis is initiated by loading (650) a chemoattractant (651) into the flow cell channel. A greater amount of the chemoattractant (651) diffuses into the second chamber (611) than the first chamber (601 A) or pathway (601B) due to the higher porosity of the second chamber (611), thereby creating a gradient of the chemoattractant in the chambersAttorney Docket No. 59528-732601(601 A, 611) and channel (611). The cell, which is drawn by the chemoattractant, moves (660, 670, 680) through the semipermeable matrix (631) and into the second chamber (611). The cell movement is recorded with an imaging apparatus (e.g., a brightfield or fluorescent microscope).

[0137] In some methods disclosed herein, a pathway includes one or more obstacles. For example, a method can include inputting a polymer precursor into a fluidic channel and synthesizing an obstacle that blocks or partially blocks the pathway. The obstacle can be a pillar, an array of pillars, wall, or another three-dimensional structure that partially blocks a pathway to impede cell movement or growth through the pathway. The obstacle can also be a semipermeable matrix that a cell can grow or move through. A method can include determining whether a cell or an aggregate of cells moves or grows past or through an obstacle.

[0138] A method for measuring cell motility can also include measuring cell movement through a maze. As detailed above, a maze can be a structure that includes i) a first opening adjacent to a first chamber; ii) a second opening to a second chamber or a space outside of the first chamber (e.g., a space outside of the obstacle course of which a maze is part of), and iii) a plurality of interconnected paths of which only a subset lead from the first opening to the second opening. Examples of a cell motility assay in a maze is provided in FIG. 21A and 21B. These figures depict a maze pathway (2102) surrounding a first chamber (2101). The maze pathway (2102) includes a first opening (2104) to the first chamber and a second opening (2103) to a space outside of the maze. As depicted in FIG. 21B, the obstacle course can be synthesized around a cell or a collection of cells (2105) such that the cell or the collection of cells (2105) are disposed within the first chamber (2101). Movement or growth of the cell (2106) through the maze pathway (2102) can then be monitored.

[0139] In addition to maze-designs, nested and concentric obstacle course designs are also contemplated herein. A simple example of a nested chamber design is provided in FIG. 9A, in which a pathway (903 A) is defined in a space between a first chamber (902) and a second chamber (903). In such designs, the pathway can at least partially surround the first chamber. The pathway can be at least partially disposed within a second chamber, wherein the second chamber at least partially encloses the first chamber. Similarly, the pathway can comprise a second chamber, the second chamber at least partially enclosing the first chamber, and the first chamber comprises at least one opening to the second chamber. The pathway can also comprise at least a portion of a wall of the second chamber.Attorney Docket No. 59528-732601

[0140] In some of the methods disclosed herein, a cell or aggregate of cells (e.g., the first cell or the aggregate of the first cells) is coupled to a surface of the fluidic device. The surface can include an adherent substrate, that is a species to which the cell or aggregate of cells binds. Examples of adherent substrates utilizable in the presently disclosed methods include fibronectin, poly-l-omithine, an RGD peptide, actinin, collagen, fibrinogen, ICAM-1, ICAM- 2, laminin, osteopontin, paxillin, talin, VCAM-1, vinculin, and vitronectin, as well as combinations and chemical derivatives of these species. As detailed further herein, an adherent substrate may improve cell survival within the fluidic device, and can also alter cell phenotype (e.g., promote cell spreading or growth). Accordingly, an adherent substrate may promote the movement or the growth of the first cell or the aggregate of the first cells or inhibit the movement or the growth of the first cell or the aggregate of the first cells. Similarly, a method can include determining whether the adherent substrate promotes or inhibits the movement or the growth of the first cell or the aggregate of the first cells.

[0141] A cell or cell aggregate can be subjected to further analysis prior to, during, or following its growth or movement to determine a characteristic of a cell or cell aggregate that may optionally be correlated to its growth or movement. As used herein, the phrase “determining a characteristic of a cell or cell aggregate” and the term “assay” refer to processes for detecting or measuring a cellular characteristic or property of a cell or cell aggregate. The assays disclosed herein can comprise a chemical, biochemical or molecular reaction (such as a cleavage of a bond, specific binding of complementary components, enzymatic reactions, dissolution of complementary components, or the like) or a change of physical state (such as an increase or decrease in temperature, change in energy level, or the like), along with a measurement of such reaction or change in physical state. The signals generated during an assay can include an electrical signal, an optical signal, a chemical signal, or a material output. A material signal comprises the production of a material that comprises information that can be extracted. For example, a material signal may be the amplification of a polynucleotide whose length, quantity, composition, or nucleotide sequence is indicative of a cellular characteristic. For example, a barcode oligonucleotide may be a material signal. Characteristics or properties of cells that are detected or measured may vary widely and include, but are not limited to, cytotoxicity, viability, proliferation capacity under selected conditions, size, shape, motility, types and profiles of cell surface, or cell membrane proteins, types and profiles of secreted proteins, production of metabolites, transcriptome, gene copy numbers, gene or allele identity, chromatin accessibility profiles, vector copy numbers for engineered or infected cells, and the like. In some cases,Attorney Docket No. 59528-732601 determining a characteristic of a cell or cell aggregate includes lysing the cell or cell aggregate to release analytes for capture and further analysis. In particular, the first cell or the aggregate of the first cells can be lysed in the first chamber, the pathway, or the second chamber. In particular embodiments, one or more cellular characteristics is selected from cytotoxicity, proliferative capacity or proliferation rate, activation status, guide RNA expression, cellular identity, purity, gene expression profile, transcriptome, epigenetic profile, sequence copy number (e.g., integrated viral copy number for transduced cells, plasmid copy number for transiently transfected cells, or gene copy number), or a combination thereof. Additional assays may include culture contamination assays including, but not limited to, viral, bacterial, yeast, mold, or mycoplasma assays, endotoxin assays, and cellular morphology assays.

[0142] A method disclosed herein can include detecting a guide ribonucleic acid (RNA) associated with a genetic modification of the first cell or the aggregate of the first cells. The first cell or the aggregate of the first cells can be transiently or stably transfected with a sequence encoding a guide RNA 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 first cell or the aggregate of the first cells can express a Cas protein that can utilize the guide RNA. Alternatively, a Cas protein can be delivered to the first cell or the aggregate of the first cells (e.g., in a liposome). Cell growth or movement can then be correlated with a genomic edit imparted by a particular guide RNA sequence. For example, the first cell or the aggregate of the first cells can be lysed to release guide RNA. The guide RNA can optionally be captured, 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 flow cell. For example, the spatial location tag can be present in a capture probe that hybridizes to the guide RNA and serves as a reverse transcription primer, and the method of generating the cDNA molecule can include capturing the guide RNA on the capture probe comprising the spatial location tag or the complement thereof, and reverse transcribing the guide RNA on the capture probe, thereby generating the cDNA molecule. The method can include sequencing the cDNA, thereby detecting the guide RNA associated with the genetic modification of the first cell or the aggregate of the first cells.Attorney Docket No. 59528-732601

[0143] A method can include determining an alternate or further characteristic of a cell or aggregate of cells (e.g., the first cell or the aggregate of the first cells). As an example, the characteristic can be an mRNA expressed by the first cell or the aggregate of the first cell, which may optionally be determined by lysing the first cell or at least a subset of the aggregate of the first cells, capturing the mRNA on a capture element coupled to a surface of the flow cell, reverse transcribing the mRNA to generate a cDNA molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule. In many cases, mRNA analysis comprises capturing mRNAs from cells or cell aggregates enclosed within a chamber, pathway, or obstacle course. The one or more capture elements can be coupled to a surface of the fluidic device. The fluidic device containing the cell or cell aggregate may be loaded with a lysing reagent so that messenger RNAs of the cells or cell aggregates are released and captured by the capture elements, and with reverse transcription reagents to copy the captured oligonucleotide labels to produce complementary DNAs thereof. The complementary DNAs may then be eluted from the fluidic device (e.g., cleaved from a surface of the top or bottom layer) and sequenced. It is understood that a sequencing step may comprise additional steps in particular embodiments including, but not limited to, tagmentation, adding adaptors, cleaving the cDNA to form appropriate lengths for sequencing, and the like. In some embodiments, an additional step may be implemented for depolymerizing or degrading the polymer matrix walls of the chambers after mRNA capture. Reverse transcription reagents comprise conventional reagents for reverse transcription; namely, a reverse transcriptase (such as, a Moloney murine leukemia virus (MMLV)), dNTPs, optional Rnase inhibitor, buffer. The sequencing step may be carried out at the sites of the captured mRNAs (in situ) or cDNAs may include a spatial barcode and be eluted and sequenced on a separate sequencing instrument (“external” sequencing). For in situ sequencing, further steps may include (i) amplifying the complementary DNAs, e.g. by bridge amplification, or like method, (ii) sequencing the amplified complementary DNAs, e.g. by a sequencing-by-synthesis technique, and (iii) determining relative expression of the mRNAs for the cells of each of the chambers. For external sequencing, further steps may include (i) providing capture elements comprising spatial barcodes, (ii) synthesizing cDNAs comprising spatial barcodes, and (iii) eluting and sequencing the cDNAs and correlating each cDNA with a chamber location by its spatial barcode.

[0144] The characteristic can also be an action potential, which, as used herein, may denote a change in voltage across a cell membrane. As examples, an action potential of a cell or anAttorney Docket No. 59528-732601 aggregate of cells may optionally be determined using calcium imaging, a microelectrode measurement, or a combination thereof.

[0145] The characteristic can also be a soluble factor secreted by a cell or cell aggregate. The soluble factor may be detected by disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell or cell aggregate, and detecting the soluble factor bound to the capture surface. Such a method can include disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell or cell aggregate and detecting the soluble factor bound to the capture surface. Disposing the capture surface adjacent to the cell or cell aggregate can denote enclosing or at least partially enclosing the capture surface with the cell or cell aggregate within a chamber, pathway, or obstacle course, and optionally removing non-enclosed capture surfaces from the fluidic system. The capture surface can be loaded into the fluidic device at a controlled density, for example 1 capture surface per about 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, or 0.001 mm2of the fluidic device.

[0146] In an exemplary embodiment, the capture surface comprises a bead. As used herein, the term “bead” can denote a microparticle or a nanoparticle, such as a ceramic, metal, metal oxide, polymer, or saccharide-based 30 to 10000 nm particle. However, further capture surfaces, including nanotubes, nucleic acid nanostructures, and antibody Fc domains. The capture surface affinity reagent can, as non-limiting examples, include antibodies, antibody fragments, aptamers, affimers, or a combination thereof.

[0147] In a further exemplary embodiment, the soluble factor comprises a cytokine such as interferon-y (IFN-y) and interferon-a (IFN-a), an interleukins such as interleukin-1 (IL-1), 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.

[0148] A soluble factor bound to a capture surface (e.g., an affinity reagent of a capture surface) can be detected by contacting the soluble factor bound to said capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody. Multiple soluble factors can be detected in a single assay by providing a capture surface or plurality of capture surfaces that comprise a plurality of affinity reagents configured to bind the plurality of soluble factors, contacting the plurality of soluble factors bound to the captureAttorney Docket No. 59528-732601 surface or plurality of capture surfaces with a plurality of labeled antibodies configured to bind to the plurality of soluble factors, and detecting a plurality of labels coupled to the plurality of antibodies. In this way, 2, 3, 4, 5, 6, or more soluble factors can be detected in a single assay. For example, an assay may utilize 2, 3, 4, 5, 6, or more beads that each include a different affinity reagent configured to bind to a different soluble factor and a commensurate number of antibodies configured to bind to the soluble factors and optionally containing distinguishable detectable labels (e.g., different fluorophores or oligonucleotide barcodes). In many cases, the soluble factor is a protein or a metabolite, such as a cytokine, immune active protein, hormone, or neurotransmitter.

[0149] In some cases, the first cell or the aggregate of the first cells comprises an effector cell, and the characteristic comprises cytotoxicity. In such cases, determining the cytotoxicity of the first cell or the aggregate of the first cells comprises measuring a rate or an occurrence of the effector cell killing a second cell or at least a subset of the aggregate of second cells. For example, an assay may measure the tumor-infiltration ability of a lymphocyte by measuring its ability to pass through a semipermeable polymer matrix, and then measure the lymphocyte’s ability to measure cancer cells trapped within or behind the semipermeable polymer matrix. As non-limiting examples, the second cell or aggregate of second cells may comprise a sample of tumor cells of a patient, or target cells may be from a cell line, e.g. tumor cell line, such as, hepatic tumor cell line, SK-HEP-1, Chava et al, J. Vis. Exp., 2020 Feb 22: (156): 10.3791 / 60714. Examples of effector cells include Tel cells, Tc2 cells, Tc9 cells, Tcl7 cells, Tc22 cells, and natural killer cells. In some embodiments, the effector cell is engineered for a therapeutic purpose, such as expression of a chimeric antigen receptor that confers cytotoxicity against a particular cancer cell.

[0150] The characteristic can also include activation. Cellular activation can be measured with numerous techniques disclosed herein, including surface marker expression, soluble factor secretion, transcriptomic analysis, proliferation, morphology, or a combination thereof. The disclosed methods are broadly amenable to detecting activation caused by contact between two or more cells or intercellular signaling mediated by secreted soluble factors. In a particular aspect of the present disclosure, determining activation comprises detecting a cell surface marker. For example, the method can comprise contacting the first cell or aggregate of the first cells with a binding agent configured to bind to the surface marker and detecting the binding agent. Activation can also be determined by detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a solubleAttorney Docket No. 59528-732601 factor, a genomic sequence, an mRNA, or a combination thereof of the first cell or the aggregate of the first cells.

[0151] In some aspects, determining a characteristic of a cell or cell aggregate includes determining a proliferation rate. As used herein, the term “proliferation” refers to cell division and development processes that increase cell number. It is understood that the term “proliferation rate” may include a measure of a lack of proliferation. For example, chambers enclosing one or more cells may be exposed to an agent (e.g., a drug candidate) to determine whether the agent promotes, kills, or retards the growth of the cells, for example in comparison to controls not exposed to the agent. Thus, in the case of the treated cells, a negative “proliferation rate” may be possible because the final numbers of cells counted in the chambers may be less than the original numbers; or a signal monotonically related to cell number may decline in value. In some embodiments, cells may be stained with a membrane or intracellular dye for determining proliferation by dye dilution so that an independent measure of cell proliferation may be obtained. Exemplary intracellular dyes for dye dilution include, but are not limited to, Hoechst 33342, carboxyfluorescein succinimidyl ester (CFSE), and the like. The desired number of single cells enclosed by chambers depends on statistical confidence desired in the measured values. If a subpopulation of interest is present as only a small fraction of a total population then a larger number chambers is required.

[0152] In some aspects, determining a characteristic of the one or more cells includes detecting a surface marker of the one or more cells. As used herein, the term “surface marker” denotes species that are expressed on the surface of a cell. Exemplary surface markers include surface proteins such as G protein-coupled receptors (GPCRs), ion channels, engineered receptors (e.g., chimeric antigen receptors), and cluster of differentiation (CD) molecules (e.g., CD3, CD4, CD5, CD6, CD7, CD8, etc.), as well as carbohydrates (e.g., sialic acids), glycolipids, and the like. However, in particular aspects of the present disclosure, the surface markers are proteins.

[0153] Surface marker detection can include contacting a cell or cell aggregate with a binding agent configured to bind to the surface marker and detecting the binding agent, thereby detecting the surface marker. A single surface marker or plurality of surface markers can be detected in a single assay. More precisely, the binding agent can include a single binding agent configured to bind to a single surface marker or a plurality of binding agents configured to bind to a plurality of surface markers. Multiple surface markers can be detected simultaneously, for example by contacting the cell or cell aggregate with multiple antibodies that bind different surface markers and are coupled to distinct detectable labels such asAttorney Docket No. 59528-732601 fluorophores that are simultaneously detectable on separate imaging channels. Alternatively or in addition thereto, two or more surface markers can be detected sequentially, for example by contacting a cell with a first antibody that binds to a first surface marker, detecting the first antibody, binding the cell with a second antibody that binds to a second surface marker, and detecting the second antibody. In particular cases, detecting a plurality of surface markers includes detecting relative expression levels of the plurality of surface markers. Exemplary binding agents include antibodies; antibody fragments such as single-chain antibody molecules, scFvs, Fab domains, diabodies, nanobodies, minibodies, linear antibodies, and cross-Fab fragments; aptamers; and affimers. A binding agent may be coupled to a detectable label such as a fluorescent label, an oligonucleotide label, a colorimetric label, an enzymatic label (e.g., pyrophosphatase), or a combination thereof. It is noted that a binding agent such as an antibody can be coupled to a cell prior to or following the cell’s introduction into a channel and / or encapsulation within a chamber.

[0154] The disclosed methods are applicable to a broad number of cells and cell aggregates. As non-limiting examples, a method for measuring cell movement or growth can utilize a cell or a cell aggregate that includes an adipocyte, an antigen-presenting cell, a cancer cell, a cardiomyocyte, chondrocyte, a dendritic cell, an ectoderm, an effector cell, an embryonic stem cell, an endodermal cell, an endothelial cell, a fibroblast, a hematopoietic stem cell, a hepatocyte, an islet cell, a keratinocyte, a lymphocyte, a melanocyte, a mesenchymal cell, a mesenchymal stem cell, a mesenchymal cancer cell, a monocyte, a progenitor cell, a myoblast, a myocyte, a neural cell, an oligodendrocyte, an osteoblast, a pancreatic epithelial cell, a skeletal myocyte cell, a smooth muscle cell, or a white blood cell. Exemplary cell aggregates include cell clumps, embryoid bodies, spheroids, neurospheres, tumors, tissue sections, and organoids. The cell aggregate can comprise between about 5 and 25 cells, 5 and 50 cells, 5 and 100 cells, 5 and 250 cell, 5 and 500 cells, 5 and 1000 cells, 5 and 2000 cells, 25 and 50 cells, 25 and 100 cells, 25 and 250 cells, 25 and 500 cells, 25 and 1000 cells, 25 and 2000 cells, 50 and 100 cells, 50 and 250 cells, 50 and 500 cells, 50 and 1000 cells, 50 and 2000 cells, 100 and 250 cells, 100 and 500 cells, 100 and 1000 cells, 100 and 2000 cells, 250 and 500 cells, 250 and 1000 cells, 250 and 2000 cells, 500 and 1000 cells, 500 and 2000 cells, or 1000 and 2000 cells. As detailed further herein, a cell or cell aggregate may comprise an adherent cell, such as a dendrite or neuron.

[0155] The methods of the present disclosure are particularly suitable for culturing and studying neurospheres. A method for analyzing a neurosphere can include inputting a neurosphere and a polymer precursor into a fluidic channel; synthesizing a chamber with theAttorney Docket No. 59528-732601 first polymer precursor within the fluidic channel, the chamber at least partially enclosing the neurosphere and trapping the neurosphere in the chamber; and incubating the neurosphere in the fluidic device under conditions that permit adherence of the neurosphere to a surface of the fluidic device or the chamber. One or more characteristics of the neurosphere or a cell of the neurosphere can then be measured. FIG. 30 illustrates a method for analyzing the transcriptome of a neurosphere in a fluidic device of the present disclosure. While this example is directed to a neurosphere, other collections of cells, such as tissue sections and tumor biopsies, are equally applicable to this method. The neurosphere 3001 may be introduced into a channel of the fluidic device 3000 and enclosed within a chamber 3002, such as an annular hydrogel matrix. Nucleic acid capture probes 3003 may be coupled to a surface of the fluidic device 3000 that is enclosed by the chamber 3002. As depicted in the top left portion of FIG. 30, the neurosphere 3001 may adhere to a surface of the fluidic device or to an adherent substrate coupled to the surface of the fluidic device, such as laminin. The neurosphere 3001 may adhere to the surface prior or subsequently to chamber 3002 formation. The neurosphere may grow neurites 3001 A within the chamber 3002. In a subsequent step, the neurosphere 3001 may be lysed 3010 to release nucleic acids 3021 (e.g., mRNA) that may be captured on the nucleic acid capture probes 3003. The chamber may then be degraded 3020 and the captured nucleic acids from the neurosphere can serve as templates for extension to produce, for example, cDNA coupled to the nucleic acid capture probes 3003. The nucleic acid capture probes 3003 (and thereby the cDNA coupled to the nucleic acid capture probes) can then be released 3030 from the surface of the fluidic device 3000, collected, and sequenced.

[0156] Further disclosed herein are systems for performing methods of the present disclosure. As examples, such a system can include a fluidic device that includes: a fluidic channel; a first chamber within the fluidic channel at least partially enclosing a first cell or an aggregate of first cells; a pathway adjacent to the first chamber that is configured to permit movement or growth of the cell or the aggregate of the first cells through the pathway; wherein: the pathway comprises a first opening to the first chamber or a degradable portion of the first chamber and a second opening to a second chamber or a space outside of the first chamber; the first chamber and the pathway comprise walls with different porosities; the first chamber and the pathway comprise walls with different thicknesses; or a combination thereof; and an instrument configured to synthesize a first chamber; and measure a movement or a growth of the first cell or the aggregate of the first cells through at least a portion of the pathway. In some cases, the pathway includes a maze. In some cases, walls of the first chamber and theAttorney Docket No. 59528-732601 second chamber comprise different thicknesses and / or porosities. In some cases, the pathway comprises a plurality of chambers that partially enclose the first chamber, and wherein each chamber of the plurality of chambers comprises at least one opening to another chamber of the plurality of chambers or a space outside of the plurality of chambers.Flow Cell Design

[0157] A channel or chamber of a fluidic device may receive or be configured to receive a biological sample. FIG. 1 shows a schematic illustration of a portion of a channel 100 that may be disposed in at least a portion of a fluidic device of a system as provided herein. The channel 100 may comprise a first surface 101, which may be a surface of the channel provided by the bottom layer. Further, the channel 100 may comprise a second surface 102, which may be a surface of the channel provided by the top layer. In some embodiments, the first surface 101 and the second surface 102 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 1). In some embodiments, the first surface 101 may be a lower surface. In certain embodiments, the second surface 102 may be an upper surface. The terms “lower” and “upper” are not intended to be limiting and are used herein for convenience when referring to the Figures. As a non-limiting example, a fluidic device, flow cell, or other apparatus disclosed herein may be operable under multiple orientations with respect to gravity. The channel 100 may receive a biological sample comprising one or more biological components 50, 51 (e.g., one or more cells or aggregates of cells). However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 101, the second surface 102, or both surfaces may couple to or receive, or be configured to couple to or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 101 may couple to or receive, or be configured to couple to or receive, a biological component (e.g., 50, 51). In certain cases, the second surface 102 may couple to or receive, or be configured to couple to or receive, a biological component (e.g., biological components 50, 51). The channel 100 may receive one or more polymer precursors.

[0158] A channel or chamber of a fluidic device (also sometimes referred to as a “flow chamber,” “flow channel,” or “reaction chamber”) may receive or be configured to receive a biological sample. FIG. 1 shows a simplified schematic cross-sectional side view illustration of a portion of the channel 100 of the fluidic device disclosed herein. The channel 100 may comprise a first surface 101 and a second surface 102. In some embodiments, the first surface 101 and the second surface 102 are disposed, placed, or positioned opposite of one another (e.g., as depicted in FIG. 1). For example, the first surface can be provided by the bottomAttorney Docket No. 59528-732601 layer and the second surface can be provided by the top layer that at least partially define the channel. In some embodiments, a middle spacer layer of double-sided adhesive with a cut-out portion can be used to position the first surface 101 and second surface 102 in a facing relationship to at least partly form the flow channel. 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 or obstacle courses are formed. In some embodiments, the perpendicular distance between a first surface and a second surface depends in part on the nature and size of the biological components to be analyzed. In some embodiments, such as, those adapted to analyzing mammalian cells, the perpendicular distance between a first surface and a second surface may be in the range of from 10 pm to 500 pm, or in the range of from 50 pm to 250 pm. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the biological component to be analyzed to five times the average size of the biological component to be analyzed. In some embodiments, the perpendicular distance between a first surface and a second surface may be in the range of from twice the average size of the largest biological component in the biological sample to five times the average size of the largest biological component in the biological sample. In some embodiments, the first surface 101 may be a lower surface. In certain embodiments, the second surface 102 may be an upper surface. The channel 100 may receive a biological sample comprising one or more biological components 50, 51. The channel 100 may receive one or more polymer precursors. As illustrated in FIG. 1, the biological components 50, 51 may include cells. However, as discussed herein, the biological components may include tissues, proteins, nucleic acids, etc. In some embodiments, the first surface 101, the second surface 102, or both surfaces may couple or receive, or be configured to couple or receive, at least one of the one or more biological components 50, 51. In some cases, the first surface 101 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In certain cases, the second surface, 102 may couple or receive, or be configured to couple or receive, a biological component (e.g., biological components 50, 51). In some embodiments, the first surface and / or second surface can be optically transmissive so that visible and UV light can transmit through one or both of the surface for the generation of polymeric hydrogels, imaging of the flow cell, and the measurement of the analyte and biological components.

[0159] 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 someAttorney Docket No. 59528-732601 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.

[0160] The first surface 101, the second surface 102, or both surfaces 101, 102 may be functionalized, for example with a coating. As a non-limiting example, a surface coating may be a surface polymer. Some non-limiting examples of surface coatings may include a capture reagent (e.g., pyridinecarboxaldehyde (PC A)), a functional group to capture one or more moi eties (e.g., a chemical moiety), an acrylamide, an agarose, a biotin, a streptavidin, a strep- tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an alkyne, an azide, an aldehyde dithiolane, or a combination thereof. In various embodiments, the surface coating may include a functional group to capture one or more moieties. For example, the acrylamide, the agarose, etc. may include such a functional group. In certain embodiments, the surface polymer may comprise polyethylene glycol (PEG), a thiol, an alkene, an alkyne, an azide, or combinations thereof. In various embodiments, the surface polymer may comprise a silane polymer. In some embodiments, the surface polymer may be functionalized with at least one of an oligonucleotide, an antibody, a cytokine, a chemokine, a protein, an antibody derivative, an antibody fragment, a carbohydrate, a toxin, or an aptamer. In particular embodiments, the surface coating comprises a material for which adherent cells have a binding affinity, such as fibronectin or laminin.

[0161] In some cases, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise one or more barcodes (e.g., nucleic acid capture probes). In some embodiments, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 1,000, 10,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,000,000, 5,000,000, 10,000,000, 15,000,000 barcodes, or any number of barcodes between any of the two numbers mentioned herein. The barcodes may cover an area of about 50 pm2to about 1000 pm2. In some embodiments, the first surface 101, the second surface 102, or both surfaces 101, 102 may comprise at most about 10,000,000 total number of barcodes. The barcodes may be different from one another (e.g., each barcode may be unique). In certain embodiments, a first portion or subset of the barcodes may be different from a second portion or subset of the barcodes. There may be 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, 100, 1,000, 10,000 portions or subsets of the barcodes, or any number of portions or subsets of the barcodesAttorney Docket No. 59528-732601 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.

[0162] 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 certain embodiments, the fluidic device may be a microfluidic device. In particular embodiments, the fluidic device may be a nanofluidic device.Hydrogel Chambers

[0163] FIGS. 2A-C illustrate an exemplary method for forming a chamber or other portion of an obstacle course enclosing one or more biological components in a fluidic device disclosed herein. FIG. 2A shows a portion of a system as provided herein (e.g., comprising a fluidic device as disclosed herein) including an energy source. FIG. 2B shows a polymer matrix being formed around a biological component in a portion of a system as provided herein. FIG. 2C shows a method of forming a polymer matrix around a biological component in a system as provided herein. FIG. 1 shows a schematic illustration of a portion of a channel disposed in a fluidic device. As illustrated in these figures, in some embodiments, the one or more chambers extend from the bottom layer to the top layer of a fluidic device.

[0164] FIG. 2A shows a portion of a system as provided herein including an energy source 203. The embodiment of FIG. 2A may include components that resemble components of FIG. 1 in some respects. For example, the embodiment of FIG. 2A includes a channel 200 that may resemble the channel 100 of FIG. 1. It will be appreciated that the illustrated embodiments may have analogous features. Accordingly, like features are designated with like reference numerals, with the leading digits incremented to “2.” RelevantAttorney Docket No. 59528-732601 disclosure set forth above regarding similarly identified features thus may not be repeated hereafter. Moreover, specific features of the system provided herein, and related components shown in FIG. 2A may not be shown or identified by a reference numeral in the drawings or specifically discussed in the written description that follows. However, such features may clearly be the same, or substantially the same, as features depicted in other embodiments and / or described with respect to such embodiments. Accordingly, the relevant descriptions of such features apply equally to the features of the system and related components of FIG. 2A. Any suitable combination of the features, and variations of the same, described with respect to the system and components illustrated in FIG. 1, can be employed with the system and components of FIG. 2A, and vice versa. This pattern of disclosure applies equally to further embodiments depicted in subsequent Figures and described hereafter.

[0165] With continued reference to FIG. 2A, the channel 200 of the system may include a first surface 201 provided by the bottom layer and a second surface 202 provided by the top layer of the fluidic device. The energy source 203 may comprise one or more energy emitting portions (e.g., an energy emitting portion 205). In some embodiments, the energy source 203 may comprise one or more non-emitting portions (e.g., a non-emitting portion 204). The nonemitting portion 204 may not emit, or be configured to emit, energy. In some embodiments, the emitting portion 205 can emit energy in the form of electromagnetic waves (e.g., microwaves, light, heat, etc.) to at least a portion of the fluidic device. For example, the energy source may comprise an LED array in which individual LEDs can be selectively activated (e.g., act as an energy emitting portion 205) to create light projections with specified patterns. In some embodiments, the fluidic channel may be coupled to or disposed on a movable stage. In other embodiments, light may be projected to or onto at least a portion of the first fluidic channel to generate one or more polymer matrices. The light may be directed to various parts of the first fluidic channel. The energy source (e.g., light source) may be coupled to the fluidic device via an objective (e.g., a microscope objective or lens). The energy source may be directed to a portion of the fluidic channel (e.g., via a movable objective). In some cases, the light source, the objective, and / or the fluidic channel are movable to allow emission of energy to the fluidic channel so as to generate a pattern on 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.

[0166] A channel of a fluidic device (e.g,. the channel) can comprise one or more polymer precursors for forming chambers. In some embodiments, the one or more polymer precursors are added the one or more cells. Such precursors may be selected from a wide variety ofAttorney Docket No. 59528-732601 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-glycolic acid) (PLGA), polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamic acid), polylysine, agar, agarose, alginate, heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan, cellulose, collagen, bisacrylamide, diacrylate, diallylamine, triallylamine, divinyl sulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate, polymethyleneglycol diacrylate, polyethyleneglycol diacrylate, trimethyl opropoane trimethacrylate, ethoxylated trimethylol triacrylate, or ethoxylated pentaerythritol tetraacrylate, or combinations or mixtures thereof. In some embodiments, the hydrogel comprises an enzymatically degradable hydrogel, PEGthiol / PEG-acrylate, acrylamide / N,N’-bis(acryloyl)cystamine (BACy), or PEG / PPO. In some embodiments, the following precursors and crosslinker may be used to form chambers with degradable polymer matrix (hydrogel) walls. Polymer precursors may be formed by using any hydrogel precursor and crosslinkers of Table 1 A (columns 1 and 3, respectively). The resulting polymer matrices may be degraded with the indicated degradation agents in Table 1 A (column 4). Representative crosslinkers useful for polymer synthesis are listed in Table IB.Table 1AAttorney Docket No. 59528-732601Table IBAttorney Docket No. 59528-732601Attorney Docket No. 59528-732601Attorney Docket No. 59528-732601Attorney Docket No. 59528-732601Attorney Docket No. 59528-732601

[0167] In some embodiments, the generation of a polymer matrix within said fluidic device comprises exposing the one or more polymer precursors to an energy source. In some embodiments, the energy source is a light generating device. In some embodiments, the light generating device generates light at 350 nm to 800 nm. In some embodiments, the light generating device generates light at 350 nm to 600 nm. In some embodiments, the light generating device generates light at 350 nm to 450 nm. In some embodiments, the light generating device generates UV light. In some embodiments, the generation of the polymer matrix comprises between about 1 and 3 seconds of illumination, between about 1 and 5 seconds of illumination, between about 1 and 10 seconds of illumination, between about 1 and 15 seconds of illumination, between about 1 and 20 seconds of illumination, between about 1 and 30 seconds of illumination, between about 1 and 50 seconds of illumination, between about 3 and 5 seconds of illumination, between about 3 and 10 seconds of illumination, between about 3 and 15 seconds of illumination, between about 3 and 20 seconds of illumination, between about 3 and 30 seconds of illumination, between about 3 and 50 seconds of illumination, between about 5 and 10 seconds of illumination, between about 5 and 15 seconds of illumination, between about 5 and 20 seconds of illumination, between about 5 and 30 seconds of illumination, between about 5 and 50 seconds of illumination, between about 10 and 20 seconds of illumination, between about 10 and 30 seconds of illumination, between about 10 and 50 seconds of illumination, between about 20 and 30 seconds of illumination, or between about 20 and 50 seconds of illumination. In some embodiments, the generation of a polymer matrix within said fluidic device is performed using a spatial light modulator (SLM) (i.e. a spatial energy modulation element that is capable of generating desired light intensity pattern spatially). In some embodiments, the SLM is a digital micromirror device (DMD). In some embodiments, the SLM is a laser beam steered using a galvanometer. In some embodiments, the SLM is liquid crystal based.

[0168] Optionally, a first chamber of the one or more chambers can be disposed inside of a second chamber of the one or more chambers. This design can be utilized to separately partition two species (e.g., a cell and a reagent or two cells) within close proximity. This design can also be used to control the timing with which two species are contacted. For example, a method can include forming a first chamber around a cell, flowing a bead (or other assay reagent incapable of diffusing into the first chamber) adjacent to the cell, forming a second chamber surrounding the first chamber and enclosing the bead, and selectively degrading the first chamber to allow the cell and bead to come into contact within the second chamber.Attorney Docket No. 59528-732601

[0169] In some embodiments, a functional group can be coupled to one or more chambers. Some non-limiting examples of functional group may include a capture reagent (e.g., pyridinecarboxaldehyde (PCA)), an acrylamide, an agarose, a biotin, a streptavidin, a strep- tag II, a linker, a functional group comprising an aldehyde, a phosphate, a silicate, an ester, an acid, an amide, an aldehyde dithiolane, PEG, a thiol, an alkene, an alkyne, an azide, or a combination thereof. In some cases, the functionalized chamber may be used to capture a biomolecule enclosed therein, thereby trapping the biomolecule in proximity to a biological component (e.g., a cell) enclosed within the chamber. The biomolecule may be produced by the biological component (e.g., secretome from a cell). The functionalized surface of the polymer matrix inside the compartment may be used to capture reagents or molecules from outside the compartment. The functionalized surface may increase surface area covered by a reagent, a molecular sensor, or any molecule of interest (e.g., an antibody).

[0170] With continued reference to FIG. 2A, the polymer matrix 208, 209, or at least a portion of the polymer matrix 208, 209, may be coupled to the first surface 201, the second surface 202, or both surfaces 201, 202. In certain embodiments, the polymer matrix, or at least a portion of the polymer matrix, may be coupled to a third surface, a fourth surface, a fifth surface, etc. as appropriate. In various embodiments, the polymer matrix 208, 209 may extend from the first surface 201 to the second surface 202 (e.g., through at least a portion of a lumen of the channel 200 or a cavity of a chamber) such that the polymer matrix surrounds, or substantially surrounds, the biological component 50. In some embodiments, two or more biological components (e.g., biological components 50, 51 of FIG. 2C) that are in close physical proximity may be separated (e.g., by agitating or shaking the fluidic device). The fluidic device may be agitated or shaken by physical movement, use of a sonic pulse, changing a flow in the channel, or any other suitable method of agitation. A polymer matrix may then be formed that surrounds (or partially surrounds) the biological components that are separated. FIG. 2B shows polymer matrices 208, 209 formed surrounding the biological component 50 after being separated from the biological component 51. FIG. 2C shows a process, according to various embodiments, of separating the two biological components 50, 51, which are in close proximity. That is, by agitating or shaking the fluidic device the biological components 50, 51 can be separated. In some embodiments, separation of the biological components is achieved through fluidic pressure, flow pulsation, dielectrophoresis, optothermal flow, or some combination thereof. In some cases, separation of the biological components is achieved through acoustic vibration. FIG. 2C also shows a polymer matrixAttorney Docket No. 59528-732601 being formed to generate a compartment 222 surrounding the biological component 50 after the separation of the biological components 50, 51.

[0171] A fluidic device disclosed herein can include a detector that is configured to detect one or more locations of one or more biological components contained within a channel. In certain embodiments, the energy source 203 can comprise, be coupled to, or be in communication with a detector that detects, or is configured to detect, a location of a biological component in the fluidic device. In various embodiments, a mask may 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 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).

[0172] In some embodiments, one or more chambers has sufficiently large pores to allow movement or transfer of a reagent (e.g., an enzyme, a chemical compound, a small molecule, an antibody, etc.) therethrough. Simultaneously, one or more chambers can have sufficiently small pores to allow movement or transfer of a reagent and / or biological component (e.g., DNA, RNA, a protein, a cell, etc.). In some embodiments, the pores have a diameter from 5 nm to 100 nm. In some embodiments, the pores have a diameter from 5 nm to 10 nm, 10 nm to 20 nm, 20 nm to 30 nm, 30 nm to 40 nm, 50 nm to 60 nm, 60 nm to 70 nm, 70 nm to 80 nm, 80 nm to 90 nm, 90 nm to 100 nm. In some embodiments, the pores may have a diameter larger than 100 nm. In some embodiments, the pores have a diameter smaller than 5 nm.

[0173] In particular embodiments, pores of the one or more chambers are formulated to encapsulate sufficiently large genetic material, nucleic acids with greater than 300 base pairs, but to allow smaller materials, such as reverse transcriptases and 50 base pair nucleic acidAttorney Docket No. 59528-732601 primers to pass through the pores, thereby passing in and out of the hydrogel structures. In some embodiments, the pore size of the hydrogel structures is tuned by varying the ratio of the concentrations of polymer precursors to the concentration of crosslinkers, varying pH, salt concentrations, temperature, light intensity, and the like. In some embodiments, the average diameter of pores of a chamber prevent passage of molecules having a molecular weight of 25 kiloDaltons (kDa) or greater; or having a molecular weight of 50 kDa or greater; or having a molecular weight of 75 kDa or greater; or having a molecular weight of 100 kDa or greater; or having a molecular weight of 150 kDa or greater. In some embodiments, DNA or RNA retained have lengths that are sequencable using conventional sequencing-by-synthesis techniques. For example, such DNA or RNA comprise at least 50 nucleotides, or in some embodiments, at least 100 nucleotides. In some embodiments, the pores may have an average diameter from 5 nm to 100 nm.

[0174] The pore sizes of the one or more chambers may be modulated using a chemical reagent, or by applying heat, electrical field, light, or another suitable stimulus. In other words, a chamber may comprise a tunable property (e.g., the pore size). In some cases, one or more chambers comprises a thermoresponsive or temperature-responsive polymer. A thermoresponsive polymer (e.g., poly(N-isopropylacrylamide) (NIPAAM)) may phase separate from a solution upon heating or upon cooling (e.g., polymer showing lower critical solution temperature (LCST) or upper critical solution temperature (UCST)). The polymer matrix may comprise polymer which may collapse at high temperature in order to, for example, control the pore size of the hydrogel or polymer matrix. Non-limiting examples of thermoresponsive polymers that may be used to form hydrogel / polymer matrix with tunable properties may include Poly(N-vinyl caprolactam), Poly(N-ethyl oxazoline), Poly(methyl vinyl ether), Poly(acrylic acid- coacrylamide), or a combination thereof. A change in temperature may enlarge or contract average pore size in the polymer matrix to allow selected molecules, such as a nucleic acid molecule, a protein, or any biomolecule or molecule smaller than the adjusted pore size to be released from a hydrogel chamber.Cell Culture and Incubation Protocols

[0175] The cells or cell aggregates and polymer precursors can optionally be mixed outside of the fluidic system before they are loaded into the channel. In some embodiments, cells are delivered to the channel, where they are dispersed over a surface of the channel. For example, the one or more cells may be randomly disposed on the surface. Alternatively, the one or more cells may be randomly disposed across or between structures or regions for which the one or more cells comprise a binding affinity. As an example, the one or more cellsAttorney Docket No. 59528-732601 may localize to regions that comprise fibronectin or laminin, and partition away from portions of the first surface composed of glass.

[0176] In various embodiments, a method of incubating one or more cells can include loading a channel (e.g., (100) in FIG. 1) with the one or more cells and one or more polymer precursors. The one or more polymer precursors can be polymerized to form one or more chambers within the channel that at least partially enclose the one or more cells. For example, after the location of the one or more cells are identified with a detector, a spatial energy modulated element may project light into the channel such that the projected light causes cross-linking of the one or more polymer precursors to form polymer matrix walls of the one or more chambers. Exemplary chamber designs include circular, elliptical, and polygonal shapes that fully enclose inner spaces (e.g., do not include a discontinuity or break). Furthermore, in many aspects, the one or more chambers extend from the bottom layer (400) to the top layer (404) of a flow cell, such that species enclosed within a chamber of the one or more chambers is isolated or partially isolated from species that are extrinsic to the one or more chambers or enclosed within other chambers. As used herein, “partially isolated” can denote that a chamber prevents a subset of species (e.g., cells and large nucleic acids) from entering or leaving a chamber enclosing a species but allows other species (e.g., small molecule analytes) to enter and leave the chamber.

[0177] An assay can include one or more assay components, which may be provided with or as part of a channel include, but are not limited to, capture elements such as capture oligonucleotides (e.g., nucleic acid capture probes), primers for captured nucleic acid amplification, antibodies, and detectable labels. In some embodiments, such assay components may be attached to any one of a first surface, a second surface or a polymer matrix wall exclusively, or on combinations of such surfaces, either exclusively, or in combination with other reagents. In some embodiments, assay components that may be provided after synthesizing gel chambers include, but are not limited to, lysing reagents, transcription reagents, reverse transcription reagents, antibodies, polymerases, primers, beads, and the like. In some embodiments, cellular or assay components may be attached or captured by capture elements on a polymer matrix wall.

[0178] In some embodiments, incubating under assay conditions may comprise only a single step of an assay comprising more than one step, such as, for example, a step of detecting a signal (or in the case of a material signal, generating a sequencing-ready nucleic acid), or such step of incubating under assay conditions may comprise a plurality of steps of a multi- step assay. In some embodiments, cells may be treated or subjected to assay steps prior toAttorney Docket No. 59528-732601 loading into a channel of a fluidic device, so that the step of incubating may comprise only a single step of a multi-step assay, such as signal generation and / or signal collection. In other embodiments, the step of incubating may comprise the implementation of an assay step or part of an assay, such as, cDNA synthesis, second strand synthesis, capture of an assay component or a cellular component, or the like. In some embodiments, assay conditions may comprise a series of steps each with different conditions (e.g. temperature, pH, presence or absence of particular reagents, such as, primers or an enzyme, e.g., a ligase, a polymerase, a transposase, or the like). For example, such steps may comprise loading primers so that they diffuse across chamber walls and anneal to target sites on a template strand in a sample in the chamber, extending the annealed primers, heating the channel so that the extended primers melt from its template strand, capturing the melted extended primers by capture agents attached to the first surface, and so on.Systems

[0179] FIG. 4A is an example system for carrying out the above method. Flow cell (500) is a component of a fluidic device that provides channels for carrying out a variety of assays and liquid handling components under programmable control for delivering samples and reagents to the channels. In this illustration, four channels (502, 504, 506, and 508) are shown. However, as detailed elsewhere herein, systems of the present disclosure can utilize flow cells with fewer or greater numbers of channels. Inlets, outlets and other features of the channels are not shown.

[0180] The system of FIG. 4A includes an optical system (521) for photosynthesizing chambers at locations of cells or other analytes in the channels (502, 504, 506, and 508) of the flow cell (500) and for collecting images and other optical signals. The optical system (521) includes a light source (522) that generates a light beam (523) of appropriate wavelength light (e.g. UV light) for synthesizing chambers (e.g., hydrogel chambers) in the flow cell (500). The light beam (523) that passes through an appropriate photo-mask or beam-shaping or beam steering (Galvo) system (524) for shaping a beam to synthesize a desired structure or structures in a channel. In some embodiments, this beam shaping system (524) includes a digital micromirror device (DMD). In other embodiments, a physical photomask may be employed. Reflected light from DMD (524) is shaped using conventional optics, e.g. collimating optics (528), and is directed through objective lens system (534) into channel 2 segment (510). In exemplary embodiments, the light is directed by one or more dichroic mirrors (530 and 531).Attorney Docket No. 59528-732601

[0181] Chamber position, shape and polymer matrix wall thickness is determined at least in part from cell position information determined from images collected by detector (532). Objective (534) and flow cell (500) move relative to one another in the xy-directions (536) to photosynthesize chambers at any position in any of the channels. In some embodiments, the flow cell (500) moves and optical system (521) is stationary. The system may utilize light from a light source (599), such as a homogenized light condenser, that is positioned on an opposite side of the flow cell as the optical system and directs light through the flow cell (500) to the objective (534). To achieve this functionality, the light source positioned on the opposite side of the flow cell (599) can be configured to move in tandem with the optical system (521), or the light source (599) and optical system can be stationary and the flow cell (500) can be moved to the region illuminated by the light source (599) and from which light is collected by the objective (534). In some embodiments, objective (534) may also direct light beam (527) from light source (529) to targets, such as cells, on first surface (514) and collect optical signals, such as fluorescent signals, from assays taking place on first surface (514). Optical signal collection can also be carried out with a separate objective.Information collected by detector (532), particularly cellular positions in their respective channels, is employed by computer (538) and / or subsidiary controllers to direct DMD (524) and translation devices controlling the relative positions of objective (534) and flow cell (500) to synthesize hydrogel chambers of the appropriate shape and size at the appropriate locations.

[0182] FIG. 4B provides a blown-up view the exemplary channel segment (510) of the flow cell of FIG. 4A. On first surface (514) of channel 2 (504) a plurality of cells, e.g. (518), are each enclosed by a hydrogel chamber, e.g. (516). In some embodiments, the porosity of polymer matrix walls of the hydrogel chambers is selected to be impermeable to the cells, but permeable to assay reagents. Thus, reagents may be introduced to, and removed from, the interiors of the hydrogel chambers by flowing (520) them through the channels, but cell are retained in.

[0183] One of ordinary skill in the art would recognize that optical systems with different configurations than those of FIG. 4A and 4B may be employed for carrying out these functions. In some embodiments, a plurality of DMD-objective subsystems for synthesizing hydrogel structures may be employed to increase the speed of synthesis by synthesizing multiple structures simultaneously.Computer SystemsAttorney Docket No. 59528-732601

[0184] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 5 shows a computer system 1501 that may be programmed or otherwise configured to perform methods described herein. The computer system 1501 can regulate various aspects of the present disclosure, such as, for example, identifying a biological component, detecting a barcode, generating a spatial modulating element (e.g., a mask), providing energy from an energy source, or detecting or measuring a local parameter using a sensor. The detector may be a camera (e.g., a fluorescent camera), such as a charged coupled device (CCD) camera capable of collecting optical signals and position information from a plurality of sources distributed over a planar region. The computer system 1501 can be an electronic device of a user or a computer system that may be remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0185] The computer system 1501 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 1505, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 1501 also includes memory or memory location 1510 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 1515 (e.g., hard disk), communication interface 1520 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1525, such as cache, other memory, data storage and / or electronic display adapters. The memory 1510, storage unit 1515, interface 1520 and peripheral devices 1525 are in communication with the CPU 1505 through a communication bus (solid lines), such as a motherboard. The storage unit 1515 can be a data storage unit (or data repository) for storing data. The computer system 1501 can be operatively coupled to a computer network (“network”) 1530 with the aid of the communication interface 1520. The network 1530 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that may be in communication with the Internet. The network 1530 in some cases may be a telecommunication and / or data network. The network 1530 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 1530, in some cases with the aid of the computer system 1501, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1501 to behave as a client or a server.

[0186] 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 theAttorney Docket No. 59528-732601 present disclosure. Examples of operations performed by the CPU 1505 can include fetch, decode, execute, and writeback.

[0187] 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).

[0188] 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.

[0189] 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.

[0190] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1501, such as, for example, on the memory 1510 or electronic storage unit 1515. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 1505. In some cases, the code can be retrieved from the storage unit 1515 and stored on the memory 1510 for ready access by the processor 1505. In some situations, the electronic storage unit 1515 can be precluded, and machine-executable instructions are stored on memory 1510.

[0191] 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.

[0192] 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 storageAttorney Docket No. 59528-732601 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.

[0193] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may beAttorney Docket No. 59528-732601 involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0194] 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.

[0195] 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.

[0196] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.EXAMPLES EXAMPLE 1 Neurite Growth Measurements

[0197] This example covers neurite growth measurements. Neurons were seeded into 200- cell neurospheres in aggrewells on day 19 of their differentiation. The resultant neurospheres were strained through a 70 pm filter and moved into media containing brain-derived neurotrophic factor (BDNF). The neurospheres were then transferred into a flow cell coated in poly-l-ornithine and laminin and incubated at 4°C. Hydrogel chambers of varying diameters were then synthesized around individual neurospheres. Neurite growth from individual neurospheres was then monitored with intermittent imaging.

[0198] Brightfield images of neurospheres enclosed in hydrogel chambers are shown in FIGS. HA (24 hours after enclosure in the hydrogel chambers) and 11B (the same neurospheres 48 hours after enclosure in the hydrogel chambers). In FIG. HA, a neurosphere, hydrogel chamber, and neurites are indicated with labels 1101, 1102, and 1103, respectively. The neurospheres increased in spread between the 24 and 48 hour timepoints, indicating adherence to the flow cell surface. Furthermore, more neurites are visible in FIG.Attorney Docket No. 59528-73260111B than in FIG. 11A, suggesting that the neurospheres actively grew neurites following hydrogel chamber enclosure. While many neurites visible in FIG. 11B follow the contours of the hydrogel chambers, no neurites project out of the chambers.

[0199] Additional brightfield images of hydrogel chamber-enclosed neurospheres are shown in FIGS. 12A (24 hours after enclosure in the hydrogel chambers) and 12B (the same neurospheres following an additional 24 hours of enclosure in the hydrogel chambers). In FIG. 12A, a neurosphere, hydrogel chamber, and neurites are indicated with labels 1201, 1202, and 1203, respectively. As with FIGS. 11A-B, increased neurosphere spread and neurite density are seen in FIGS. 12B relative to FIG. 12A. Hydrogel chamber deformation is exhibited in FIG. 12B, suggesting that the neurites adhered to and exerted force upon the hydrogel chambers. However, neurites were not observed outside of the chambers, indicating that neurites did not grow through the hydrogel walls of the chambers.

[0200] Neurospheres were then stained with DAPI and Tuj 1, HuC, HuD, and MAP2 antibodies to resolve the positions of nuclei, neurites, soma (HuC and HuD), and neurons (MAP2), respectively. An exemplary image of a DAPI, Tuj 1, HuC / D, and MAP2 stained neurosphere is provided in FIG. 13A. An image of the neurosphere in the MAP2 channel is shown in FIG. 13B. Images of hydrogel chamber-enclosed neurospheres stained with DAPI and Tuj l and HuC / D antibodies are shown in FIGS. 14A-C. In FIG. 14A, resolved axons 1401 A from a first neurosphere 1401 positioned directly outside of a hydrogel chamber 1402 enclosing a second neurosphere 1403. Similarly, an axon 1404 in the lower-left portion of FIG. 14B is positioned across a hydrogel chamber wall 1405 from axons 1406A of an enclosed neurosphere 1406. In FIG. 14C, a neurite 1407 from an enclosed neurosphere 1408 appears to cross through a hydrogel chamber 1409. Collectively, these results indicate the possibility that neurospheres formed synapses across hydrogel chamber walls, either by growing neurites through the hydrogel chamber walls or by having possessing neurites that extended beyond the boundaries of the hydrogel chamber walls prior to hydrogel chamber synthesis.EXAMPLE 2Neurite Growth Through Obstacle Courses

[0201] This example covers neurite growth measurements through hydrogel obstacle courses fabricated within flow cells. Neurospheres were generated as outlined in EXAMPLE 1. The neurospheres were then incubated at 4°C in flow cells coated with poly-l-ornithine and laminin. Hydrogel chambers and obstacle courses with varying diameters and manifolds ofAttorney Docket No. 59528-732601 openings were synthesized around individual neurospheres. Neurite growth from the neurospheres through obstacle courses and openings in the chambers were measured with brightfield, fluorescence, and calcium imaging.

[0202] FIGS. 15A-C are brightfield images a first neurosphere 1501 enclosed in a hydrogel chamber 1502 taken immediately after chamber synthesis (FIG. 15A), 24 hours after chamber synthesis (FIG. 15B), and 48 hours after chamber synthesis (FIG. 15C). FIG. 15A includes a second neurosphere 1503 positioned outside of but in close proximity to the hydrogel chamber that was subsequently removed from the fluidic device in a wash step. The hydrogel chamber included eight evenly-spaced openings 1502Athat were sufficietly small to prevent the neurospheres from traversing through but sufficiently large to permit neurite growth through one or more of the opentings. As shown in FIGS. 15B-C, the neurosphere exhibited progressively greater spread following 48 hours of incubation within the chamber, indicating increased adherence to the flow cell surface during incubation. The neurosphere also exhibited progressive neurite growth at 24 hours (FIG. 15B) and 48 hours (FIG. 15C). By 48 hours, multiple neurites 1501 A from the neurosphere extended through the chamber openings.

[0203] FIGS. 16A-B are brightfield images of a first hydrogel chamber 1601 enclosing three neurospheres 1602 and in close proximity to a second hydrogel chamber 1603 enclosing a single neurosphere 1604. The first and second hydrogel chambers 1601, 1603 each included regularly-spaced openings 1601 A, 1603 A that were sufficiently large to accommodate neurites, but prohibitively small for the neurospheres to pass through. FIGS. 16A and 16B were collected 24 and 48 hours after chamber synthesis, respectively. As shown in FIG. 16B, following 48 hours of incubation, neurites from neurospheres in separate chambers overlapped in multiple regions outside of the chambers 1605, indicating possible synapse formation between these neurospheres.

[0204] FIGS. 17A-C are brightfield images of a neurosphere 1701 contained within two chambers, namely an inner chamber 1702 enclosed by an outer chamber 1703. The inner chamber 1702 and outer chamber 1703 each included multiple openings (1702A and 1703 A, respectively) to a pathway in the region between the inner (1702) and outer (1703) chamber walls, and capable of accommodating neurite growth. FIGS. 17A-C were collected immediately after, 24 hours after, and 48 hours after chamber synthesis. Progressive neurite growth was observed during this timeframe. No neurites are visible in FIG. 17A, where the circular profile of the neurosphere 1701 suggests that it had not yet adhered to a surface of the fluidic device. In FIG. 17B, the neurosphere 1701 exhibits a spread-out profile indicativeAttorney Docket No. 59528-732601 of surface adherence, and included numerous neurites 1701 A that extend into the pathway disposed between the inner chamber 1702 and outer chamber 1703. By 48 hours (FIG. 17C), the neurosphere included multiple neurites 1701 A that extended out of the outer chamber 1703.

[0205] FIGS 18A and B are brightfield images of a neurosphere 1801 enclosed within three chambers: an inner chamber 1802, a middle chamber 1803, and an outer chamber 1804. Each chamber included multiple openings (1802A, 1803 A, 1804A) to the pathway between the inner chamber 1802 and the outer chamber 1804. FIGS. 18A and B were respectively collected 24 and 48 hours after chamber synthesis, and show different stages of neurite growth from the enclosed neurosphere 1801. Following 24 hours of incubation, the neurosphere 1801 included a small number of neurites 1801 A in the space between the inner chamber 1802 and middle chamber 1803. Following 48 hours of incubation, the neurosphere 1801 included multiple neurites 1801Athat extended outside of the outer chamber 1804.EXAMPLE 3Neurosphere Transcriptomic and Calcium Imaging Analysis

[0206] This example covers transcriptomic and calcium imaging analysis of single neurospheres enclosed in hydrogel chambers. Neurospheres were generated as outlined in EXAMPLE 1. The neurospheres were then incubated at 4°C in flow cells coated with poly-1- ornithine and laminin. Top surfaces of the flow cell included circular regions containing nucleic acid capture probes in the form of barcoded reverse transcription primers configured to bind mRNA. Hydrogel chambers were photosynthesized surrounding the neurospheres, and the neurospheres were then subjected to transcriptomic or calcium imaging analysis.

[0207] FIG. 19A-C are brightfield images of neurospheres 1901 enclosed within hydrogel chambers 1902. Circular regions containing the nucleic acid capture probes 1903 on the top surfaces of the flow cell are visible in these images. Neurosphere cells were lysed to release mRNA. Following mRNA capture on the capture probes, the hydrogel chambers were degraded, and the mRNA was reverse transcribed to generate cDNA coupled to the capture probes. Following cleavage from the flow cell surface, the cDNA was eluted from the flow cell and sequenced.

[0208] FIG. 20A is an image of a hydrogel chamber-enclosed neurosphere generated through calcium imaging. For these analyses, Neurospheres were incubated with Fluo-4AM dye for 1 hour at 37’C. The dye was then washed out and replaced with fresh media. Images wereAttorney Docket No. 59528-732601 acquired at 2 Hz. FIG. 20A provides calcium imaging traces in three distinct regions of interest (ROI) indicated in FIG. 20A.EXAMPLE 4Neurosphere Growth In Chambers and Obstacle Courses

[0209] This example covers neurosphere synapse formation through hydrogel chambers and obstacle courses. Neurospheres were generated according to the protocols outlined in EXAMPLE 1. The neurospheres were loaded into flow cells coated with poly-l-omithine and laminin and maintained at 4°C. Hydrogel chambers and obstacle courses were synthesized around select neurospheres located with a brightfield imaging detector. The neurospheres were then subjected to brightfield imaging and fluorescence imaging with DAPI (nuclei), Tuj 1 (neurite), HuC / D (soma) and MAP2 (neuron) stains.

[0210] FIGS. 22A and 22B are (respectively) fluorescence and brightfield images of a neurosphere 2201 enclosed in a hydrogel chamber 2202. As can be seen from the fluorescence image, in which neurites are resolved with Tuj 1 stains, neurites 2203 from the neurosphere are contained within the hydrogel chamber 2202, that is, they do not extend through the hydrogel chamber to a space outside of the chamber.

[0211] FIGS. 23A and 23B are fluorescence and brightfield images of two neurospheres, a first neurosphere 2301 enclosed within a hydrogel chamber 2302 and a second neurosphere 2303 disposed just outside of the chamber. While neurites 2301 A from the first neurosphere 2301 and neurites 2303 A from the second neurosphere 2303 largely appeared to be blocked by the hydrogel chamber, multiple neurites 2303 A from the second neurosphere 2303 projected around the hydrogel chamber wall.

[0212] FIGS. 24A and 24B are fluorescence and brightfield images that show multiple neurospheres 2401 enclosed within a hydrogel obstacle course 2402 and additional neurospheres 2403 disposed outside of the obstacle course 2402. These images highlight neurite growth through an obstacle course pathway defined by nested hydrogel chamber walls 2404 as well as neurites from separate neurospheres connecting with each other 2405.

[0213] FIGS. 25A and 25B are fluorescence and brightfield images of a first neurosphere 2501 enclosed within a hydrogel obstacle course comprised of a first chamber 2502A with a single opening to a pathway defined by three concentric chambers 2502B, 2502C, 2502D surrounding the first chamber 2502A, and a second neurosphere 2503 positioned immediately outside of the pathway. In the Tuj 1 fluorescence channel shown in the fluorescence imageAttorney Docket No. 59528-732601(FIG. 25A), a neurite 2504 from the second neurosphere 2503 grew through the pathway to the first neurosphere 2501 positioned inside of the first chamber 2502A.

[0214] FIGS. 26A and 26B are fluorescence and brightfield images of a neurosphere 2601 enclosed within a hydrogel obstacle similar to the one shown in FIGS. 25A and 25B. Contrasting FIGS. 25A and 25B, a second neurosphere is not positioned outside of the obstacle course. While multiple neurites are observable in the fluorescence image (FIG. 26A), none of these neurites extend through a pathway and outside of the obstacle course.

[0215] FIGS. 27A and 27B are fluorescence and brightfield images of two neurospheres 2701 enclosed in separate hydrogel chambers 2702 synthesized with four (left) and one (right) openings. Both neurospheres 2701 grew neurites 2703 outside of their respective chambers.

[0216] FIGS. 28A and 28B are fluorescence and brightfield images of a single neurosphere 2801 in a hydrogel chamber 2802 with multiple openings. Multiple neurites 2803 from the neurosphere 2801 grew out of the hydrogel chamber 2802 through its openings.

[0217] FIGS. 29A and 29B are fluorescence and brightfield images of two neurospheres 2901 enclosed in separate hydrogel chambers 2902 that each had seven openings. Both neurospheres 2901 projected neurites 2903 outside of their respective chambers. The neurites 2903 from the two neurospheres 2901 grew towards one another and connected in the space between the two hydrogel chambers 2902.EXAMPLE 5 Cell Movement Into a Chamber

[0218] This example covers the measurement of a cell moving through a chamber opening into a chamber. Human neurons derived from induced pluripotent stem cells were input into a fluidic device. Hydrogel chambers were then fabricated within the fluidic device by photopolymerizing a polymer precursor with light shaped by a digital micromirror device. The fluidic device was imaged periodically at 4x or lOx magnification.

[0219] Movement of a neuron into an annular hydrogel chamber was detected during this assay. FIG. 32A is an image of the annular hydrogel chamber 3200 collected shortly after its formation. The hydrogel chamber 3200 included annular walls surrounding an open interior. The walls included four openings 3201 spaced at even intervals around the annular walls. FIGS. 32B-C are images of the hydrogel chamber 3200 collected at later timepoints, with the image of FIG. 32C collected after the image of FIG. 32B. FIG. 32B includes a neuron 3202 that was not present in the image of FIG. 32A. At this timepoint, the neuron was present inAttorney Docket No. 59528-732601 one of the hydrogel chamber 3200 openings 3201. The neuron’s morphology suggested that the neuron was in an adherent state. In FIG. 32C (which was collected after FIG. 32B), the neuron 3202 had moved through the opening 3201 and into the interior of the hydrogel chamber 3200, demonstrating that the openings 3201 were sufficiently large for the neuron to move through.EXAMPLE 6 Chamber Closure Prior To Transcriptomic Analysis

[0220] This example is directed to repolymerization to close openings through hydrogel chamber walls. The bottom surface of a fluidic device was coated with a mixture of extracellular matrix proteins. A suspension of human cells was then input into the fluidic device to allow the cells to randomly disperse throughout the fluidic device. Cells were detected through brighffield imaging at lOx magnification. Hydrogel chambers were then formed around select cells by photopolymerizing a polymer precursor within the fluidic device with light shaped by a digital micromirror device. The hydrogel chambers had annular shapes with 45 pm radii, 10 pm wall thicknesses, and 3 windows at evenly spaced 120 degree intervals along the hydrogel chamber walls. An image of a hydrogel chamber 3300 with a cell 3302 is shown in FIG. 33A. An opening through the wall of the hydrogel chamber 3300 is indicated with label 3301.

[0221] A subset of the chambers were then closed through an additional photopolymerization step, thereby closing the openings from the chambers. FIG. 33B is an image of the hydrogel chamber from FIG. 33A following this closure process, with the closed chamber indicated by label 3303 and the cell indicated with label 3302.

[0222] The cells were then lysed. mRNA from the cells was captured on nucleic acid capture probes on the top surface of the fluidic device. The nucleic acid capture probes contained spatial barcode sequences that identified the locations of the nucleic acid capture probes within the fluidic device. cDNA was generated using the nucleic acid capture probes and captured mRNA. The cDNA was then eluted from the fluidic device and sequenced. cDNA sequences were associated with individual (cells detected with brighffield imaging) using the spatial barcode sequences from the nucleic acid capture probes. TABLE 2 provides a comparison of cDNA reads for chambers that were closed through the additional photopolymerization step (e.g., the chamber shown in FIG. 33B) and chambers whose openings were not closed prior to lysis. The chambers that were closed through a second photopolymerization step exhibited higher mRNA read counts than chambers that were notAttorney Docket No. 59528-732601 closed prior to cell lysis. However, approximately 1800 unique and 6000 total median mRNA reads were detected from the open chambers, indicating that mRNA measurements can be performed in chambers that have openings.TABLE 2

Claims

Attorney Docket No. 59528-732601CLAIMSWHAT IS CLAIMED IS:

1. A method for measuring cellular growth, comprising:(a) inputting a cell or an aggregate of cells into a fluidic device;(b) inputting a polymer precursor into the fluidic device;(c) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells;(d) synthesizing a pathway adjacent to the chamber; and(e) measuring growth of the cell or the aggregate of cells through at least a portion of the pathway.

2. The method of claim 1, wherein the synthesizing of the pathway in (d) uses the polymer precursor.

3. The method of claim 1, wherein the synthesizing of the pathway in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

4. The method of claim 1, further comprising synthesizing an additional chamber, wherein the additional chamber is fluidically coupled to the pathway.

5. The method of claim 4, wherein the additional chamber is synthesized using the polymer precursor.

6. The method of claim 4, wherein the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

7. The method of any one of the claims 4-6, wherein the growth of the cell or the aggregate of cells is towards the additional chamber.

8. The method of any one of claims 4-7, wherein an additional cell, an aggregate of additional cells, a bead, or a combination thereof are disposed within the additional chamber.

9. The method of claim 8, wherein the growth of the cell or the aggregate of cells is towards the additional cell, the aggregate of additional cells, the bead, or the combination thereof.Attorney Docket No. 59528-73260110. A method for measuring cellular growth, comprising:(a) inputting a cell or an aggregate of cells, an additional cell or an aggregate of additional cells, and a polymer precursor into a fluidic device;(b) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells, and wherein the additional cell or the aggregate of additional cells are outside of the chamber; and(c) measuring growth of the cell or the aggregate of cells from the chamber towards the additional cell or the aggregate of additional cells.

11. The method of claim 10, further comprising, prior to the measuring the growth of the cell or the aggregate of cells in (c), incubating the additional cell or the aggregate of additional cells at least until the additional cell or the aggregate of additional cells adhere to a position on a surface of the fluidic device.

12. The method of claim 10 or claim 11, further comprising synthesizing an additional chamber, wherein the additional chamber and at least partially encloses the additional cell or the aggregate of additional cells.

13. The method of claim 12, wherein the additional chamber is synthesized using the polymer precursor.

14. The method of claim 12, wherein the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

15. A method for measuring cellular growth, comprising:(a) inputting a cell or an aggregate of cells into a fluidic device;(b) inputting a polymer precursor into the fluidic device;(c) synthesizing a first chamber using the polymer precursor within the fluidic device, wherein the first chamber at least partially encloses the cell or the aggregate of cells;(d) synthesizing a second chamber; and(e) measuring growth of the cell or the aggregate of cells from the first chamber towards the second chamber.Attorney Docket No. 59528-73260116. The method of claim 15, wherein the synthesizing of the second chamber in (d) uses the polymer precursor.

17. The method of claim 15, wherein the synthesizing of the second chamber in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

18. The method of claim 15, wherein an additional cell, an aggregate of additional cells, a bead, or a combination thereof are disposed within the second chamber.

19. The method of any one of claims 1-18, wherein the growth comprises axonal growth, dendritic growth, neurite growth, pseudopodium growth, filopodium growth, lamellipodium growth, a growth cone, acrosome growth, comet tail growth, or a combination thereof.

20. The method of any one of claims 1-19, wherein the measuring comprises determining a rate or a distance of the growth of the first cell or the aggregate of the first cells.

21. The method of claim any one of claims 1-20, wherein the method further comprises determining whether a direction of the growth of the first cells or the aggregate of the first cells is random or directed.

22. The method of claim 18, further comprising determining whether the additional cell, the aggregate of additional cells, the bead, or the combination thereof increases the growth, decreases the growth, randomizes a direction of the growth, or directs the direction of the growth of the first cell or the aggregate of the first cells.

23. The method of any one of claims 8, 9, or 18-22, wherein the additional cell or the aggregate of additional cells grows towards the cell or the aggregate of cells.

24. The method of any one of claims 8, 9, or 18-23, wherein the cell or the aggregate of cells and the additional cell or the aggregate of additional cells are neurons.

25. The method of claim 23 or claim 24, wherein the method further comprises detecting junction formation or synapse formation between the cell or the aggregate of cells and the additional cell or the aggregate of additional cells.

26. The method of any one of claims 1-25, further comprising contacting the cell or the aggregate of cells with a reagent, and determining whether the reagent alters the growth of the cell or the aggregate of cells.Attorney Docket No. 59528-73260127. The method of claim 26, wherein the method further comprises determining whether the reagent increases the growth, decreases the growth, directs a direction of the growth, or randomizes the direction of the growth of the cell or the aggregate of cells.

28. The method of claim 26 or claim 27, wherein the pathway comprises a higher concentration of the reagent than the chamber, or wherein the chamber comprises a higher concentration of the reagent than the pathway.

29. The method of any one of claims 26-28, wherein the pathway comprises a chemical gradient of the reagent.

30. The method of claim 29, wherein the pathway comprises (i) a first end that comprises an opening to the chamber or a degradable wall of the chamber and (ii) a second end that comprises an opening to an additional chamber or to a space outside of the chamber; and wherein: i) the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or ii) the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway.

31. The method of any one of claims 26-30, wherein the reagent is produced by the additional cell or the aggregate of additional cells.

32. The method of any one of claims 1-31, further comprising degrading a portion of the chamber to form an opening that allows the growth of the cell or the aggregate of cells out of the chamber.

33. The method of claim 30, wherein the degradable wall of the first chamber is synthesized from a different polymer precursor than the chamber.

34. The method of any one of claims 1-33, wherein the chamber comprises an opening through which the cell or the aggregate of cells can grow.

35. The method of claim 34, wherein the opening comprises a gap or a pore in a wall of the chamber.

36. The method of any one of claims 1-35, wherein the additional chamber comprises an opening through which the cell or the aggregate of cells can grow.Attorney Docket No. 59528-73260137. The method of claim 36, wherein the opening comprises a gap or a pore in a wall of the additional chamber.

38. A method for measuring cellular movement, comprising:(a) inputting a cell or an aggregate of cells into a fluidic device;(b) inputting a polymer precursor into the fluidic device;(c) synthesizing a chamber using the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells;(d) synthesizing a pathway adjacent to the chamber;(e) degrading a portion of the chamber to form an opening that allows movement of the cell or the aggregate of cells from the chamber to the pathway; and(f) measuring movement of the cell or the aggregate of cells through at least a portion of the pathway.

39. The method of claim 38, wherein the synthesizing of the pathway in (d) uses the polymer precursor.

40. The method of claim 38, wherein the synthesizing of the pathway in (d) uses an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

41. A method for measuring cellular movement, comprising:(a) inputting a cell or an aggregate of cells into a fluidic device;(b) inputting a polymer precursor into the fluidic device;(c) synthesizing a chamber with the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells;(d) synthesizing a pathway adjacent to the chamber; and(e) measuring movement of the cell or the aggregate of cells from the chamber through the pathway.

42. The method of claim 41, wherein the chamber comprises an opening through which the cell or the aggregate of cells can move.

43. The method of claim 42, wherein the opening comprises a gap or a pore in a wall of the chamber.Attorney Docket No. 59528-73260144. The method of any one of claims 38-43, further comprising synthesizing an additional chamber, wherein the additional chamber is fluidically coupled to the pathway and comprises an opening from the pathway through which the cell or the aggregate of cells can move.

45. The method of claim 44, wherein the additional chamber is synthesized using the polymer precursor.

46. The method of claim 44, wherein the additional chamber is synthesized using an additional polymer precursor, wherein the additional polymer precursor is different from the polymer precursor.

47. The method of any one of claims 44-46, wherein the additional chamber comprises an opening through which the cell or the aggregate of the cells can move.

48. The method of any one of claims 38-47, wherein the measuring comprises determining a rate or a distance of the movement of the cell or the aggregate of cells through at least the portion of the pathway.

49. The method of any one of claims 38-48, wherein the method further comprises determining whether the movement is stochastic or directed.

50. The method of any one of claims 38-49, wherein the movement is towards: i) an additional cell or an aggregate of additional cells located outside of the chamber; ii) a bead located outside of the chamber; iii) an additional chamber; or iv) a combination thereof.

51. The method of claim 41, wherein the additional cell, the aggregate of additional cells, a bead, or a combination thereof are disposed within the additional chamber.

52. The method of claim 50 or claim 51, further comprising determining whether the additional cell, the aggregate of the additional cells, the bead, or the combination thereof increases the movement, decreases the movement, randomizes a direction of the movement, or directs the direction of the movement of the cell or the aggregate of cells.Attorney Docket No. 59528-73260153. The method of any one of claims 50-52, wherein the cell is an effector cell and the additional cell or the aggregate of additional cells comprises a target cell, wherein the effector cell is configured to interact with the target cell.

54. The method of any one of claims 38-53, further comprising synthesizing a semipermeable matrix in at least a portion of the pathway, wherein the semipermeable matrix is configured to allow the cell or the aggregate of the cells to pass through at least a portion of the semipermeable matrix.

55. The method of claim 54, wherein the semipermeable matrix is synthesized using matrigel, gelatin, photocrosslinkable gelatin, collagen, a gel of an enzymatically degradable macro monomer, or a gel of an enzymatically cleavable PEG macromonomer.

56. The method of claim 54, wherein the semipermeable matrix is synthesized using the polymer precursor.

57. The method of claim 54, wherein the semipermeable matrix is synthesized using an additional polymer precursor, and wherein the additional polymer precursor is different from the polymer precursor.

58. The method of any one of claims 54-57, wherein the semipermeable matrix or a portion of the semipermeable matrix is shorter than a height of the pathway, narrower than a width of the pathway, or a combination thereof.

59. The method of any one of claims 54-58, wherein the semipermeable matrix is synthesized using a polymer precursor that is enzymatically cleavable, bioabsorbable, or enzymatically cleavable and bioabsorbable.

60. The method of any one of claims 54-59, wherein the cell or the aggregate of cells passes through the semipermeable matrix from a first end to a second end of the pathway.

61. The method of any one of claims 54-60, wherein the synthesizing the semipermeable matrix is subsequent to the synthesizing the pathway.

62. The method of any one of claims 38-61, further comprising synthesizing an obstacle that partially blocks the pathway and is impermeable to the cell or the aggregate of cells.

63. The method of claim 62, wherein the obstacle is synthesized using the polymer precursor.Attorney Docket No. 59528-73260164. the method of claim 62, wherein the obstacle is synthesized using an additional polymer precursor, and wherein the additional polymer precursor is different from the polymer precursor.

65. The method of any one of claims 62-64, wherein the measuring comprises determining whether the cell or the aggregate of cells moves past the obstacle.

66. The method of any one of claims 38-65, further comprising contacting the cell or the aggregate of cells with a reagent, and determining whether the reagent alters the movement of the cell or the aggregate of cells.

67. The method of claim 66, wherein the method further comprises determining whether the reagent increases the movement, decreases the movement, directs a direction of the movement, or randomizes the direction of the movement of the cell or the aggregate of cells.

68. The method of claim 66 or claim 67, wherein the pathway comprises a higher concentration of the reagent than the chamber, or wherein the chamber comprises a higher concentration of the reagent than the pathway.

69. The method of any one of claims 66-68, wherein the pathway comprises a chemical gradient of the reagent.

70. The method of claim 69, wherein the pathway comprises (i) a first end that comprises an opening to the chamber or a degradable wall of the chamber and (ii) a second end that comprises an opening to an additional chamber or to a space outside of the chamber, and wherein: i) the first end of the pathway comprises a higher concentration of the reagent than the second end of the pathway, or ii) the second end of the pathway comprises a higher concentration of the reagent than the first end of the pathway.

71. The method of claim 66, wherein the reagent is produced by the second cell or the aggregate of the second cells.

72. The method of any one of claims 1-9 or 41-71, wherein the pathway comprises one or more polymer matrix walls that define an open space adjacent to the chamber through which the cell or the aggregate of cells can grow or move.Attorney Docket No. 59528-73260173. The method of claim 72, wherein the one or more polymer matrix walls comprise a polymer matrix wall of the chamber, a polymer matrix wall of an additional chamber, or a combination thereof.

74. The method of any one of claims 1-9 or 41-73, wherein the pathway comprises (i) a first opening to the chamber or a degradable wall of the chamber and (ii) a second opening to a space outside of the pathway and the chamber.

75. The method of claim 74, wherein the pathway comprises a single route from the first opening to the second opening.

76. The method of any one of claims 1-75, wherein (i) the chamber and the pathway, (ii) the chamber and the additional chamber; (iii) the additional chamber and the pathway, or (iv) a combination thereof are synthesized simultaneously.

77. The method of any one of claims 1-76, wherein the chamber, the pathway, or the additional chamber comprises an opening, and wherein the method further comprises synthesizing a polymer wall in the opening, thereby enclosing an analyte from the cell or the aggregate of cells within the chamber, the pathway, the additional chamber, or a combination thereof.

78. The method of claim 77, further comprising lysing the cell or at least a subset of the aggregate of cells within the chamber, the pathway, or the additional chamber.

79. The method of any one of claims 1-78, wherein the measuring comprises imaging.

80. The method of any one of claims 8, 9, 18-25, or 50-53, wherein the bead comprises a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof.

81. The method of any one of claims 8, 9, 18-25, or 50-53, wherein the bead comprises a nanoparticle, a microparticle, a lipid particle, a hydrogel particle, or a nanomaterial.

82. The method of any one of claims 26-31 or 66-71, wherein the reagent comprises a chemoattractant, a repellant, a signaling molecule, a protein, a nutrient, a cytokine, a hormone, a pheromone, a neurotransmitter, or a combination thereof.

83. The method of any one of claims 1-82, wherein the synthesizing of the chamber comprises photopolymerization.Attorney Docket No. 59528-73260184. The method of any one of claims 1-83, wherein the synthesizing of the pathway or the synthesizing of the additional chamber comprises photopolymerization.

85. The method of any one of claims 32 or 38-40, wherein the degrading comprises photolysis, chemical degradation, or a combination thereof.

86. The method of claim 85, wherein the photolysis comprises inputting a photoinitiator into the fluidic device and directing a light to at least a portion of the chamber.

87. The method of any one of claims 1-9 or 41-74, wherein the pathway at least partially surrounds the chamber.

88. The method of any one of claims 1-9 or 41-74, wherein the pathway is at least partially disposed within an additional chamber, and wherein the additional chamber at least partially encloses the chamber.

89. The method of any one of claims 1-9 or 41-74, wherein the pathway forms at least a portion of an additional chamber, wherein the additional chamber at least partially encloses the chamber, and wherein the chamber comprises at least one opening to the additional chamber.

90. The method of claim 89, wherein the pathway comprises at least a portion of the wall of the additional chamber.

91. The method of any one of claims 1-9, 41-74, or 88-90, wherein the pathway comprises a maze.

92. The method of any one of claims 1-9, 41-74, or 88-91, wherein the pathway comprises: i) a first opening adjacent to the chamber; ii) a second opening to an additional chamber or a space outside of the chamber; and iii) a plurality of interconnected paths of which only a subset lead from the first opening to the second opening.

93. The method of claim 92, wherein the pathway comprises a single route that connects the first opening to the second opening.

94. The method of claim 92 or claim 93, wherein the measuring comprises measuring a time between the cell moving or growing from the first opening to the second opening of the pathway.Attorney Docket No. 59528-73260195. The method of any one of claims 1-9, 41-74, or 88-94, wherein the pathway comprises a first end that is adjacent to the chamber and a second end that comprises an opening to a space outside of the chamber.

96. The method of any one of claims 1-9, 41-74, or 88-95, wherein the pathway comprises a first end that is adjacent to the chamber and a second end that comprises an opening to an additional chamber.

97. The method of claim 95 or claim 96, wherein: i) a wall of the chamber comprises a greater thickness than a wall of the pathway, ii) the wall of the pathway comprises a greater thickness than the wall of the chamber, iii) the wall of the chamber comprises a greater thickness than a wall of the additional chamber, iv) the wall of the additional chamber comprises a greater thickness than the wall of the chamber, v) the wall of the pathway comprises a greater thickness than the wall of the additional chamber, vi) the wall of the additional chamber comprises a greater thickness than the wall of the pathway, vii) the wall of the chamber comprises a greater porosity than the wall of the pathway, viii) the wall of the pathway comprises a greater porosity than the wall of the chamber, ix) the wall of the chamber comprises a greater porosity than the wall of the additional chamber, x) the wall of the additional chamber comprises a greater porosity than the wall of the chamber, xi) the wall of the pathway comprises a greater porosity the wall of the additional chamber, xii) the wall of the additional chamber comprises a greater porosity than the wall of the pathway, or xiii) a combination thereof.Attorney Docket No. 59528-73260198. The method of any one of claims 95-97, further comprising inputting a reagent into the fluidic device, wherein the reagent: i) diffuses across a wall of the chamber and does not diffuse across a wall of the pathway, ii) diffuses across the wall of the pathway and does not diffuse across the wall of the chamber, iii) diffuses across the wall of the chamber and does not diffuse across the wall of the additional chamber, iv) diffuses across the wall of the additional chamber and does not diffuse across the wall of the chamber, v) diffuses across the wall of the pathway and does not diffuse across the wall of the additional chamber, vi) diffuses across the wall of the additional chamber and does not diffuse across the wall of the pathway, or vii) a combination thereof.

99. The method of any one of claims 1-9, 41-74, or 88-98, wherein the pathway comprises a channel.

100. The method of claim 99, wherein the channel of the pathway comprises a cross- sectional area, wherein the cross-sectional area is less than a largest dimension of the cell.

101. The method of claim 100, wherein the cross-sectional area is less than the largest dimension of the cell by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

102. The method of any one of claims 99-101, wherein the channel of the pathway comprises a cross-sectional area, wherein the cross-sectional area is greater than a largest dimension of the cell.

103. The method of claim 102, wherein the cross-sectional area is greater than the largest dimension of the cell by at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 500%, at least about 1000%, at least about 2000%, or at least about 5000%.Attorney Docket No. 59528-732601104. The method of any one of claims 1-9, 41-74, or 88-103, wherein the pathway comprises a length of about 10 to 25 pm, about 10 to 50 pm, about 10 to 100 pm, about 10 to 250 pm, about 10 to 500 pm, about 10 to 1000 pm, about 10 to 1500 pm, about 10 to 2000 pm, about 10 to 2500 pm, about 25 to 50 pm, about 25 to 100 pm, about 25 to 250 pm, about 25 to 500 pm, about 25 to 1000 pm, about 25 to 1500 pm, about 25 to 2000 pm, about 25 to 2500 pm, about 50 to 100 pm, about 50 to 250 pm, about 50 to 500 pm, about 50 to 1000 pm, about 50 to 1500 pm, about 50 to 2000 pm, about 50 to 2500 pm, about 100 to 250 pm, about 100 to 500 pm, about 100 to 1000 pm, about 100 to 1500 pm, about 100 to 2000 pm, about 100 to 2500 pm, about 250 to 500 pm, about 250 to 1000 pm, about 250 to 1500 pm, about 250 to 2000 pm, about 250 to 2500 pm, about 500 to 1000 pm, about 500 to 1500 pm, about 500 to 2000 pm, about 500 to 2500 pm, about 1000 to 1500 pm, about 1000 to 2000 pm, about 1000 to 2500 pm, or about 1500 to 2500 pm.

105. The method of any one of claims 1-104, wherein the cell or the aggregate of cells is coupled to a surface of the fluidic device.

106. The method of any one of claims 1-105, wherein an adherent substrate is coupled to a surface of the fluidic device.

107. The method of claim 106, wherein the adherent substrate comprises fibronectin, poly- 1-ornithine, an RGD peptide, actinin, collagen, fibrinogen, ICAM-1, ICAM-2, laminin, osteopontin, paxillin, talin, VCAM-1, vinculin, vitronectin, or a combination thereof.

108. The method of claim 106 or claim 107, wherein: i) the adherent substrate promotes the movement or the growth of the cell or the aggregate of cells; ii) the adherent substrate inhibits the movement or the growth of the cell or the aggregate of cells; iii) the method further comprises determining whether the adherent substrate promotes or inhibits the movement or the growth of the cell or the aggregate first cells; or iv) a combination thereof.

109. The method of any one of claims 1-108, wherein the cell or the aggregate of cells comprises an adipocyte, an antigen-presenting cell, a cancer cell, a cardiomyocyte, chondrocyte, a dendritic cell, an ectoderm, an effector cell, an embryonic stem cell, anAttorney Docket No. 59528-732601 endodermal cell, an endothelial cell, a fibroblast, a hematopoietic stem cell, a hepatocyte, an islet cell, a keratinocyte, a lymphocyte, a melanocyte, a mesenchymal cell, a mesenchymal stem cell, a mesenchymal cancer cell, a monocyte, a progenitor cell, a myoblast, a myocyte, a neural cell, an oligodendrocyte, an osteoblast, a pancreatic epithelial cell, a skeletal myocyte cell, a smooth muscle cell, or a white blood cell.

110. The method of any one of claims 1-109, wherein the cell or the aggregate of cells comprises an adherent cell.

111. The method of any one of claims 1-110, wherein the aggregate of the cells comprises a cell clump, an embryoid body, a spheroid, a neurosphere, a tumor, a tissue section, or an organoid.

112. The method of any one of claims 1-111, wherein the cell comprises a neuron or the aggregate of cells comprises a neurosphere.

113. The method of any one of claims 1-112, wherein the aggregate of cells comprises between about 5 and 25 cells, 5 and 50 cells, 5 and 100 cells, 5 and 250 cell, 5 and 500 cells, 5 and 1000 cells, 5 and 2000 cells, 25 and 50 cells, 25 and 100 cells, 25 and 250 cells, 25 and 500 cells, 25 and 1000 cells, 25 and 2000 cells, 50 and 100 cells, 50 and 250 cells, 50 and 500 cells, 50 and 1000 cells, 50 and 2000 cells, 100 and 250 cells, 100 and 500 cells, 100 and 1000 cells, 100 and 2000 cells, 250 and 500 cells, 250 and 1000 cells, 250 and 2000 cells, 500 and 1000 cells, 500 and 2000 cells, or 1000 and 2000 cells.

114. The method of any one of claims 1-113, further comprising detecting a guide ribonucleic acid (RNA) associated with a genetic modification of the cell or the aggregate of cells.

115. The method of claim 114, wherein the guide RNA is coupled to an exogenous messenger ribonucleic acid (mRNA).

116. The method of claim 114 or claim 115, wherein the guide RNA is coupled to a barcode.

117. The method of any one of claims 114-116, wherein the guide RNA is coupled to a poly A tail.Attorney Docket No. 59528-732601118. The method of any one of claims 114-117, further comprising generating a complementary DNA (cDNA) molecule comprising a complement of the guide RNA sequence, the exogenous mRNA, the barcode, or a combination thereof.

119. The method of claim 118, wherein the cDNA molecule is coupled to a spatial location tag corresponding to a unique location within the fluidic channel.

120. The method of claim 118 or claim 119, wherein the generating the cDNA molecule comprises capturing the guide RNA on the capture probe comprising the spatial location tag or the complement thereof, and reverse transcribing the guide RNA on the capture probe, thereby generating the cDNA molecule.

121. The method of any one of claims 118-120, further comprising sequencing the cDNA, thereby detecting the guide RNA associated with the genetic modification of the cell or the aggregate of cells.

122. The method of any one of claims 1-121, further comprising determining a characteristic of the cell or the aggregate of cells.

123. The method of claim 122, wherein the characteristic is a messenger ribonucleic acid (mRNA) expressed by the cell or the aggregate of cells.

124. The method of claim 123, wherein the determining comprises lysing the cell or at least a subset of the aggregate of cells, capturing the mRNA on a capture element coupled to a surface of the fluidic device, reverse transcribing the mRNA to generate a complementary DNA (cDNA) molecule that optionally comprises a barcode sequence derived from the capture element, and sequencing the cDNA molecule.

125. The method of claim 124, wherein prior to the lysing, the method further comprises closing an opening in the chamber, closing an opening in the pathway, or a combination thereof.

126. The method of claim 122, wherein the characteristic comprises an action potential.

127. The method of claim 126, wherein the determining comprises calcium imaging, a microelectrode measurement, or a combination thereof.

128. The method of claim 122, wherein the characteristic comprises a soluble factor secreted by the cell or the aggregate of cells.Attorney Docket No. 59528-732601129. The method of claim 128, wherein the detecting the soluble factor comprises disposing a capture surface comprising an affinity reagent that binds the soluble factor adjacent to the cell or the aggregate of cells, and detecting the soluble factor bound to the capture surface.

130. The method of claim 129, wherein the detecting the soluble factor bound to the capture surface comprises contacting the soluble factor bound to the capture surface with a labeled antibody configured to bind to the soluble factor, and detecting the labeled antibody.

131. The method of claim 129 or claim 130, wherein the capture surface is at least partially enclosed in the chamber, the pathway, the additional chamber, or the combination thereof.

132. The method of any one of claims 129-131, wherein the soluble factor is a neurotransmitter.

133. The method of claim 122, wherein the cell or the aggregate of cells comprises an effector cell, and wherein the characteristic comprises cytotoxicity.

134. The method of claim 133, wherein the determining the cytotoxicity comprises measuring a rate or an occurrence of the effector cell killing the additional cell or at least a subset of the aggregate of additional cells.

135. The method of claim 122, wherein the characteristic comprises activation.

136. The method of claim 135, wherein the cell or the aggregate of cells is activated by the additional cell or the aggregate of additional cells.

137. The method of claim 135 or claim 136, wherein the determining the activation comprises detecting expression of a surface marker, a morphology change, a cytotoxicity increase, a proliferation rate change, a soluble factor, a genomic sequence, an mRNA, or a combination thereof of the cell or the aggregate of cells.

138. The method of claim 122, wherein the characteristic comprises proliferation.

139. The method of any one of claims 1-9, 41-74, or 88-104, wherein the pathway is configured to permit the growth or the movement of the cell or the aggregate of cells through at least a portion of the pathway.Attorney Docket No. 59528-732601140. The method of any one of claims 1-139, wherein the fluidic device comprises a fluidic channel.

141. A system for performing the method of any one of claims 1-140.

142. A system comprising: a fluidic device, and an instrument configured to: a) synthesize a chamber within the fluidic device, wherein the chamber at least partially encloses a cell or an aggregate of cells; b) synthesize a pathway adj acent to the chamber that is configured to permit movement or growth of the cell or the aggregate of the cells through the pathway, wherein: i) the pathway comprises (i) a first opening to the chamber or a degradable portion of the chamber and (ii) a second opening to an additional chamber or a space outside of the chamber, ii) the chamber and the pathway comprise walls with different porosities, iii) the chamber and the pathway comprise walls with different thicknesses, or iv) a combination thereof; and c) measure movement or growth of the cell or the aggregate of cells through at least a portion of the pathway using a detector.

143. The system of claim 142, wherein the pathway comprises a maze.

144. The system of claim 142 or claim 143, wherein walls of the first chamber and the second chamber comprise different thicknesses and / or porosities.

145. The system of any one of claims 142-144, wherein the pathway comprises a plurality of chambers that partially enclose the first chamber, and wherein each chamber of the plurality of chambers comprises at least one opening to another chamber of the plurality of chambers or a space outside of the plurality of chambers.

146. A method for measuring cellular growth or movement, comprising:Attorney Docket No. 59528-732601 inputting a cell or an aggregate of cells into a fluidic device; inputting a polymer precursor into the fluidic device; synthesizing a chamber with the polymer precursor within the fluidic device, wherein the chamber at least partially encloses the cell or the aggregate of cells; and measuring growth or movement of the cell or the aggregate of cells towards a target located within the fluidic device.

147. The method of claim 146, wherein the target comprises an additional chamber, an additional cell or aggregate of additional cells, a bead, or a combination thereof.

148. The method of any one of claims 146-147, wherein the target is a structure synthesized with the first polymer precursor or a second polymer precursor.

149. The method of any one of claims 146-148, wherein the target is a location along a surface of the fluidic device.

150. The method of any one of claims 146-149, wherein the target comprises a collection of points along a surface of the fluidic device.

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