Methods of producing tissue-derived epithelial organoids and uses thereof

Abstract

The present disclosure provides methods of preparing tissue-derived epithelial organoids. In particular, the present disclosure provides tissue-derived epithelial organoids embedded in hydrogel suspended in a medium. The present disclosure further provides methods of using such organoids.

Description

[0001] METHODS OF PRODUCING TISSUE-DERIVED EPITHELIAL ORGANOIDS AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] The application claims priority to U.S. Provisional Application No. 63 / 508,132, filed June 14, 2023, the contents of which is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of producing and using such organoids.

[0006] BACKGROUND

[0007] Tissue-derived epithelial organoids are three-dimensional (3D) multicellular spheroids that recapitulate the complexity and functions of the tissue in vivo, and have emerged as a physiologically relevant in vitro model of the tissue. For example, intestinal organoids, also termed “enteroids” or “colonoids,” are derived from adult stem cells isolated from primary intestinal tissue, and can be easily propagated and cryopreserved for long term storage. Intestinal organoids can differentiate into various intestinal cell types, perform epithelial functions such as barrier maintenance, absorption, secretion and digestion, and recapitulate biological characteristics and clinical responses of the patients from which they are derived (Clevers (2016) Cell 165, 1586-1597; Zachos et al. (2016) J Biol Chem 291, 3759-3766). Thus, many intestinal organoids have been widely adopted in place of traditional transformed and immortalized intestinal cell lines, leading to basic science discoveries and facilitating translational applications in numerous fields including cancer biology, infectious diseases and cystic fibrosis (Clevers (2016); Schutgens and Clevers (2019) Annu Rev Pathology Meeh Dis 15, 1-24).

[0008] A challenge that has limited implementation of tissue-derived epithelial organoids in areas such as drug development is that the existing organoid culture technique is difficult to scale, requiring tedious manual approaches or development of advanced automation infrastructure (Louey et al. (2021) Sias Discov 26, 1138-1147). In the existing technique, tissue-derived epithelial stem cells (either isolated from primary tissue or passaged from established organoid cultures) are resuspended in a cold extracellular matrix (ECM) solution, most often CULTREX® Basement Membrane Extract (BME) or MATRIGEL® hydrogels. The ECM is deposited onto the surface of a plate, then warmed to solidify the ECM-cell solution into a surface-attached hydrogel dome, and overlaid with media. Media in each well is changed every few days and organoids form over the course of 1-2 weeks (Mahe et al. Curr Protoc Mouse Biology 3, 217-240; Pleguezuelos-Manzano et al. (2020) Curr Protoc Immunol 130, el06; Sato et al. (2009) Nature 459, 262-265; Sato et al. (2011) Gastroenterology 141, 1762-1772). This technique is difficult to scale up because it is limited by available surface area for hydrogel dome formation, it is timeconsuming and labor-intensive and can be prone to user error, and diffusion limitations of the hydrogel cause organoid growth and morphological heterogeneity (Park et al. (2022) Nat Methods 19, 1449-1460; Ringel et al. (2020) Cell Stem Cell 26, 43 l-440.e8; Shin et al. (2020) iScience 23, 101372). Accordingly, there is a need in the art for more efficient and high-throughput methods of generating tissue-derived epithelial organoids.

[0009] SUMMARY

[0010] The subject matter disclosed herein relates to tissue-derived epithelial organoids and methods of producing such organoids. The present disclosure further provides methods of using the tissue-derived epithelial organoids and systems for performing the methods disclosed herein.

[0011] In certain embodiments, a method of generating tissue-derived epithelial organoids includes (a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture, (b) suspending the hydrogel- tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel- tissue-derived epithelial stem cell mixture and (c) culturing the suspended hydrogel-tissue- derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids. In certain embodiments, a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method further includes fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0012] In certain embodiments, the hydrogel is solidified upon contact with the medium. In certain embodiments, suspending the hydrogel-tissue-derived epithelial stem cell mixture in the medium includes submerging a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the medium. In certain embodiments, the temperature of the medium is from about 25°C to about 50°C. In certain embodiments, the temperature of the medium is from about 30°C to about 50°C. In certain embodiments, the temperature of the medium is from about 30°C to about 40°C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is less than about 20°C. In certain embodiments, the temperature of the hydrogel -tissue- derived epithelial stem cell mixture is from about 2°C to about 25°C. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 20°C. In certain embodiments, the temperature of the hydrogel- tissue-derived epithelial stem cell mixture is from about 2°C to about 10°C.

[0013] The present disclosure further provides a method of generating a suspension culture of tissue-derived epithelial organoids. In certain embodiments, the method includes (a) introducing a mixture comprising a hydrogel and a tissue-derived epithelial stem cell into a medium to generate a suspended mixture and (b) culturing the suspended mixture in the medium to generate the tissue-derived epithelial organoids in suspension. In certain embodiments, the mixture introduced into the medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells. In certain embodiments, the method includes (a) introducing a mixture comprising a hydrogel and a plurality of tissue-derived epithelial stem cells into a medium to generate a suspended mixture and (b) culturing the mixture in the medium to generate the tissue-derived epithelial organoids in suspension. In certain embodiments, the hydrogel is solidified upon contact with the medium. In certain embodiments, introducing a mixture in the medium comprises submerging a dispensing device containing the mixture in the medium and dispensing the mixture into the medium. In certain embodiments, the temperature of the medium is from about 25°C to about 50°C. In certain embodiments, the temperature of the medium is from about 30°C to about 50°C. In certain embodiments, the temperature of the medium is from about 25°C to about 40°C. In certain embodiments, the temperature of the medium is from about 30°C to about 40°C. In certain embodiments, the temperature of the mixture is less than about 20°C. In certain embodiments, the temperature of the mixture is from about 2°C to about 25°C. In certain embodiments, the temperature of the mixture is from about 2°C to about 20°C. In certain embodiments, the temperature of the mixture is from about 2°C to about 10°C. In certain embodiments, the method further includes fragmenting the suspended mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0014] In certain embodiments, the method for generating a suspension culture of tissue-derived epithelial organoids includes (a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture; (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture; (d) suspending the solidified hydrogel- tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel- tissue-derived epithelial stem cell mixture; and (e) culturing the suspended hydrogel-tissue- derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids. In certain embodiments, a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture. For example, but not by way of limitation, the method includes (a) contacting a plurality of tissue-derived epithelial stem cells with a hydrogel to generate a hydrogel-tissue- derived epithelial stem cell mixture, (b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate, (c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture, (d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture and (e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids. In certain embodiments, the method further includes dislodging the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the medium.

[0015] In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet. In certain embodiments, the hydrogel- tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filamentlike structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the method further includes fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0016] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture or the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter greater than about 0.1 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture or the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter of about 0.1 mm to about 1,000 mm, e.g., about 0.1 mm to about 20 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture or the solidified hydrogel-tissue-derived epithelial stem cell mixture is in droplets. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture, the suspended mixture or the solidified hydrogel-tissue-derived epithelial stem cell mixture has a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape.

[0017] In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is contained within a tissue fragment, an organoid fragment or a combination thereof. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue. In certain embodiments, the tissue-derived epithelial stem cell is obtained from a fragment of a tissue selected from the group consisting of the lacrimal gland, the tonsils, the salivary gland, gastrointestinal tissue, the thyroid, the lung, the mammary gland, the liver, the bile duct, the stomach, the kidney, the pancreas, the endometrium, the fallopian tube, the cervix, the prostate, the bladder, the ovary, the taste bud, the placenta and a combination thereof. Alternatively or additionally, the tissue-derived epithelial stem cell is obtained from a fragment of an organoid selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

[0018] In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

[0019] In certain embodiments, the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel and a combination thereof. In certain embodiments, the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component. In certain embodiments, the hydrogel has a protein concentration greater than about 1 mg / ml. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at a w / v % greater than about 1 w / v %. In certain embodiments, the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0020] In certain embodiments, the medium is present in a container. In certain embodiments, the container is a petri dish, a multi-well plate, a conical tube, a reservoir, a culture bag, a bioreactor or a flask.

[0021] The present disclosure further provides a tissue-derived epithelial organoid generated by a method disclosed herein.

[0022] In certain embodiments, the tissue-derived epithelial organoids produced by methods of the present disclosure have uniform morphology compared to reference tissue- derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. In certain embodiments, the tissue-derived epithelial organoids have uniform size. In certain embodiments, the average diameter of the tissue-derived epithelial organoids is more uniform than the reference tissue-derived epithelial organoids.

[0023] In certain embodiments, a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids produced by the methods of the present disclosure at a higher level compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, a differentiation marker is expressed in a population of the tissue-derived epithelial organoids produced by the methods of the present disclosure at a lower level compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUR0G3, NKX6.1, SM0C2, PDX1, CD44 and a combination thereof. In certain embodiments, the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

[0024] The present disclosure further provides a composition comprising a tissue- derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium. In certain embodiments, the hydrogel has a geometric shape comprising a length, width and / or diameter greater than about 0.1 mm. In certain embodiments, the hydrogel has a geometric shape comprising a length, width and / or diameter of about 0.1 mm to about 1,000 mm, e.g., about 0.1 mm to about 20 mm. In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44 and a combination thereof. In certain embodiments, a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALP I, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof. In certain embodiments, the reference tissue-derived epithelial organoids are tissue- derived epithelial organoids embedded within hydrogels attached to a substrate.

[0025] The present disclosure further provides a method for screening an agent, e.g., a therapeutic agent. In certain embodiments, the method includes (a) contacting a tissue- derived epithelial organoid or a population of the tissue-derived epithelial organoid or a composition of a tissue-derived epithelial organoid with an agent, e.g., a therapeutic agent, and (b) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids that is indicative of the effectiveness, disposition, and / or toxicity of the agent, e.g., therapeutic agent. In certain embodiments, the agent, e.g., the therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 1 minute to about 3 years, e.g., about 15 minutes to about 3 years. In certain embodiments, the agent is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell-based therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redux potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

[0026] The present disclosure further provides a method for performing a genomic screen. In certain embodiments, the method includes (a) providing a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid or a composition of a tissue-derived epithelial organoid, (b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid and (c) analyzing a change in the tissue- derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation. In certain embodiments, the mutation is generated using a gene-regulating system. In certain embodiments, the gene-regulating system is a geneediting system. In certain embodiments, the gene-editing system is a CRISPR system. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redux potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

[0027] The present disclosure further provides a method for generating an epithelial cell model. In certain embodiments, the method includes (a) providing a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid or a composition of a tissue-derived epithelial organoid, (b) digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells and (c) culturing the single cells in a medium to generate a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the medium is a differentiation medium. In certain embodiments, the medium is a cell growth medium. In certain embodiments, the medium is a stem cell promoting medium.

[0028] The present disclosure further provides a screening method using a cell monolayer generated by a method described herein. In certain embodiments, the screening method is a method for screening an agent, e.g., a therapeutic agent. For example, but not by way of limitation, the method can include (a) contacting a cell monolayer generated a method described herein with an agent, e.g., a therapeutic agent; and (b) analyzing a change in the cell monolayer that is indicative of the effectiveness, disposition, and / or toxicity of the agent, e.g., therapeutic agent. In certain embodiments, the agent, e.g., the therapeutic agent, is contacted with the cell monolayer for about 1 minute to about 3 years, e.g., about 15 minutes to about 3 years. In certain embodiments, the agent is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell-based therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof. In certain embodiments, the screening method is a genomic screen. In certain embodiments, the method for performing a genomic screen can include (a) providing a cell monolayer generated by a method described herein; (b) generating a mutation in the genome of one or more cells of the cell monolayer; and (c) analyzing a change in the cell monolayer associated with the mutation. In certain embodiments, the mutation is generated using a gene-regulating system. In certain embodiments, the gene-regulating system is a gene-editing system. In certain embodiments, the gene-editing system is a CRISPR system. In certain embodiments, the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redux potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post- translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

[0029] In certain embodiments, one or more steps of a method of the present disclosure can be performed using one or more robotic and / or automated components. For example, but not by way of limitation, one or more steps of a method of generating tissue- derived epithelial organoids, a method of generating a suspension culture of tissue-derived epithelial organoids, a method for screening an agent, a method for performing a genomic screen, a method for generating an epithelial cell model and / or a screening method using a cell monolayer as described herein can be performed using one or more robotic and / or automated components. In certain embodiments, the one or more robotic and / or automated components are selected from the group consisting of liquid handling robots, 3D printers, syringe pumps and combinations thereof. In certain embodiments, the one or more robotic and / or automated components comprise a liquid handling robot.

[0030] The present disclosure further provides systems for culturing a tissue-derived epithelial organoid. In certain embodiments, the system includes a tissue-derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium. In certain embodiments, the hydrogel has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm, e.g., about 0.1 mm to about 20 mm. In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake, or spiral shape. In certain embodiments, a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. In certain embodiments, the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44 and a combination thereof. In certain embodiments, a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. In certain embodiments, the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (K14), Smooth muscle Actin (SMA) and a combination thereof. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof. In certain embodiments, the system includes one or more robotic and / or automated components for generating and / or culturing the tissue-derived epithelial organoid. In certain embodiments, the one or more robotic and / or automated components are selected from the group consisting of liquid handling robots, 3D printers, syringe pumps and combinations thereof. In certain embodiments, the one or more robotic and / or automated components comprise a liquid handling robot.

[0031] The present disclosure further provides systems for performing the methods disclosed herein. For example, but not by way of limitation, the present disclosure provides systems for performing a method of generating tissue-derived epithelial organoids, a method of generating a suspension culture of tissue-derived epithelial organoids, a method for screening an agent, a method for performing a genomic screen, a method for generating an epithelial cell model and / or a screening method using a cell monolayer. In certain embodiments, the system includes one or more robotic and / or automated components for generating and / or culturing the tissue-derived epithelial organoid. In certain embodiments, the one or more robotic and / or automated components are selected from the group consisting of liquid handling robots, 3D printers, syringe pumps and combinations thereof. In certain embodiments, the one or more robotic and / or automated components comprise a liquid handling robot.

[0032] BRIEF DESCRIPTION OF THE FIGURES

[0033] FIGs. 1 A-1H show a scale up of intestinal organoids in suspended BME hydrogels. (A) A schematic, (B) photographs and (C) brightfield microscopy of human colon organoids in conventional Dome culture (top) and suspended BOBA (BME- embedded Organoid Bead Assembly) culture (bottom). Scale bars for micrographs are 500 pm. (D) Organoid diameter for dome and BOBA cultures in brightfield images. (E) Percent Ki67-positive cells in confocal images. Data represented are mean ± SD, Student’s t-test, n = 3 with 10 fields of view each. (F) 3D-reconstructed confocal images of colon organoids in Dome or BOBA cultures. Ki67 in green, nuclei in blue, actin in white and scale bars are 20 pm. (G, H) Total viable cells, cells per cm2surface area or cells per pL BME for (G) colon and (H) ileum organoids. All data represented are mean ± SD, Student’s t-test *p < 0 05,****p < 0.0001, n=3 experiments.

[0034] FIGs. 2A-2C show organoid differentiation in BOBA culture. (A) Brightfield images of colon organoids in Dome (top) and BOBA (bottom) culture show comparable morphology of proliferative and differentiated organoids. Scale bars are 100 pm. (B) Bulk RNA-seq shows that Dome- and BOBA-cultured colon organoids similarly downregulate stem and progenitor markers and upregulate differentiation markers after transition from Growth Media to Differentiation media. (C) Markers for differentiated epithelial cell types (MUC2 for goblet cells, FABP1 for enterocytes and CHGA for enteroendocrine cells) are expressed in organoids cultured in both Dome (top) and BOBA (bottom) formats. Nuclei in blue, Actin in white and scale bars are 10 pm.

[0035] FIGs. 3A-3C show that BME volume and culture vessel can impact organoid growth in suspended BME hydrogel culture. (A) Brightfield images of colon organoids in surface-attached Dome in a 24-well plate (50 pL BME in 0.5 mL media), or in BOBA cultures in a 6-well plate well (0.5, 1 or 2 mL BME in 5 mL media). Scale bars are 200 pm. (B, C) Quantification of organoid diameters, total viable cells, viable cells per cm2surface area and viable cells per pL of BME in (B) 6-well plates or (C) 25 cm2flasks. Data represented are mean ± SD, One-way ANOVA multiple comparison test, n = 3 experiments; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0036] FIGs. 4A-4E show organoid size uniformity in BOBA cultures. (A) Schematic describing imaging strategy for uniformity analysis. (B, C) Brightfield images of colon organoids at the deepest plane in a 50 pl BME dome or a 10 pl suspended BOBA hydrogel. Scale bars are (B) 200 pm and (C) 1 mm. (D) Quantification of organoid diameter in a horizontal ROI of a Dome or BOBA hydrogel by position across the X-axis. Data represented are mean ± SD and n=3 replicates in a representative experiment of 3 experiments. (E) Quantification of average organoid diameter at the edge or core of Dome and BOBA cultures. All data represented are mean ± SD, Two-way ANOVA and n=3 replicates in a representative experiment of 3 experiments.

[0037] FIGs. 5A-5B show gene expression of organoids in Dome and BOBA culture. (A, B) Bulk RNA-seq analysis shows differences in gene expression of (A) stem cell and proliferation markers and (B) enterocyte markers between colon organoids in dome culture or suspended BOBA culture after 7d culture in Growth Media. Data represented are mean ± SD for normalized counts determined by DESeq2, n = 3 replicates and statistical analysis by the negative binomial distribution model with BH adjusted p-values, *padj < 0.05, ** padj < 0.01.

[0038] FIGs. 6A-6D show that alternate suspended BME hydrogel culture formats have comparable organoid growth. (A) Photographs and (B) brightfield images of colon organoids in 6-well plate cultures in BOBA, SOBA (Syringe-extruded Organoid BME Assembly) or SOBA fragment formats. Alternate formats enable expedited culture preparation for scale up. Scale bars are 1 mm. (C) Quantification of organoid diameter and (D) viable cells per well. Data represented are mean ± SD, One-way ANOVA multiple comparison test, n = 3 experiments.

[0039] FIGs. 7A-7E show the application of suspended BME hydrogel organoid cultures in medium-throughput screen. (A) Schematic of experiment. SOBA fragment organoid culture in a 225 cm2flask was triturated to achieve a uniform organoid suspension, then seeded in 96-well plates. (B) Brightfield images of a SOBA fragment culture cultured in a 225 cm2flask. Scale bars are 1 mm. (C) Brightfield images of wells chosen at random across a 96-well plate show comparable well-to-well organoid densities. Scale bars are 1 mm. (D) Cell Titer Gio 3D (CTG) viability readout shows similar well-to-well variability between Dome and suspended BME cultures in a 96-well plate. Data represented are mean ± SD. Student’s t-test. (E) Representative dose response viability curves (CTG assay) for suspended BME organoids treated with diacerin, sorafenib, SN-38 or docetaxel for 3 d. Data represented are mean ± SD, n = 4.

[0040] FIGs. 8A-8C show the use of suspended BME hydrogel organoids to generate Transwell monolayers. (A) Schematic of experiment. SOBA fragment organoid culture in a 225 cm2flask was digested to a single cell suspension, then seeded in 96-well BME-coated Transwell inserts at confluence. (B) Brightfield images of SOBA fragment organoid-derived Transwell monolayers 3d post-seeding. Transwells were established with Monolayer Growth Media or Monolayer Differentiation Media. Scale bars are 100 pm. (C) Transepithelial Electrical Resistance (TEER) of Transwell monolayers cultured in Monolayer Growth Media (filled circles) or Monolayer Differentiation Media (empty circles). Data represented are mean ± SD and n = 6 wells.

[0041] FIG. 9 provides an image of lung AT2 organoids embedded in hydrogel suspended in medium in a BOB A culture.

[0042] FIG. 10 provides a schematic depicting exemplary conditions for generating hydrogel droplets, referred to herein as Basement membrane Organoid Bead Assemblies (BOBA). Organoids or dissociated organoid cells suspended in cold liquid hydrogel are dispensed as droplets into media that is room temperature or warmer, which enables the hydrogel to immediately cure when dispensed into the media.

[0043] FIG. 11 provides a schematic depicting exemplary conditions for generating hydrogel filaments, referred to herein as Syringe-extruded Organoid Basement membrane Assemblies (SOBA). Organoids or dissociated organoid cells suspended in cold liquid hydrogel are extruded (by syringe, pipette or any other dispensing apparatus) as filaments into media that is room temperature or warmer, which enables the hydrogel to immediately cure when dispensed into the media.

[0044] DETAILED DESCRIPTION

[0045] The present disclosure provides tissue-derived epithelial organoids in suspension culture and methods of generating such tissue-derived epithelial organoids. The presently disclosed methods enable the production of large-scale organoid cultures without the need for tedious manual handling, specialized equipment or automation. By growing organoid cells in suspended hydrogels instead of the conventional surface-attached hydrogel domes, the hydrogel volume and, therefore, the number of organoid cells that can grow in a culture vessel can be significantly increased. In addition, because the presently disclosed methods do not require hydrogels to be deposited on a two-dimension (2D) surface, this method is compatible with various culture vessels, e.g., culture flasks and culture bags, which allows further culture scale-up, resulting in high throughput.

[0046] The present disclosed method allows the use of varying geometries of suspended hydrogels, which can expedite the hands-on culture preparation time. Moreover, because the organoids are in suspension, they can be sampled, divided or collected at various times during culture, which is difficult for conventional organoid cultures immobilized in a plate. As shown in Example 1, organoids in suspended BME hydrogels grow more uniformly than in conventional surface-attached hydrogel domes, where limited molecular diffusion results in nutrient gradients (Park et al. (2022); Shin et al. (2020)). The suspended hydrogel culture methods disclosed herein result in tissue-derived epithelial organoid models that are more amenable to high-throughput studies that will benefit both basic science and translational fields.

[0047] For clarity, but not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0048] I. Definitions;

[0049] II. Organoids and Compositions Thereof;

[0050] III. Methods of Producing Organoids;

[0051] IV. Methods of Use;

[0052] V. Systems; and

[0053] VI. Exemplary Embodiments

[0054] I. DEFINITIONS

[0055] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the subject matter of the present disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in the present disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0056] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0057] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold or within 2-fold, of a value.

[0058] The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0059] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv) and multispecific antibodies formed from antibody fragments.

[0060] As used herein, the term “culture medium” or “medium” refers to a liquid that covers cells in a culture vessel (such as a culture flask and a multi-well plate) and contains nutrients to nourish the cells. In certain embodiments, the culture medium can also include growth factors or differentiation factors to produce desired changes in the cells.

[0061] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0062] As used herein, the term “contacting” cells with a compound, e.g., a therapeutic agent, refers to exposing cells to a compound, for example, placing the compound in a location that will allow it to touch the cell. The contacting can be accomplished using any suitable methods. For example, but not by way of limitation, “contacting” can be accomplished by adding the compound to a container containing cells. Contacting can also be accomplished by adding the compound to a culture medium comprising the cells. In certain embodiments, “contacting” refers to exposing a cell, e.g., a cell within a tissue-derived epithelial organoid or in a cell monolayer, to an agent or compound. In certain embodiments, “contacting” refers to the exposure of a tissue-derived epithelial organoid or a cell monolayer to a potential therapeutic agent or therapeutic agent of interest.

[0063] As used herein, the term “derived from” or “established from” or “differentiated from” when made in reference to any cell disclosed herein refers to a cell that was obtained from (e.g., isolated or purified) a parent cell in a cell line, tissue (such as a dissociated tissue) or fluids using any manipulation. Non-limiting examples of such manipulation include single cell isolation, cultured in vitro, treatment and / or mutagenesis using, e.g., proteins, chemicals, radiation, viral infection and transfection with nucleic acids. In certain embodiments, a derived cell can be selected from a mixed population by response to a growth factor, cytokine, selected progression of cytokine treatments, adhesiveness, lack of adhesiveness, a sorting procedure or a combination thereof.

[0064] The term “detection” or “detecting” include any means of detecting, including direct and indirect detection.

[0065] As used herein, the term “droplet” when used in reference to a geometric shape refers to a spherical or a spherical-like shape.

[0066] The terms “embed” or “embedded,” as used herein, refers to at least partially covering or surrounding a cell. In certain embodiments, the terms “embed” or “embedded,” as used herein, refer to the complete covering or surrounding of a tissue-derived epithelial organoid, e.g, with a hydrogel.

[0067] The term “expression” or “express,” as used herein, refers to the transcription and / or translation of a nucleotide sequence.

[0068] The term “expression vector” is used to denote a nucleic acid molecule that is either linear or circular, into which another nucleic acid sequence fragment of appropriate size can be integrated. Such nucleic acid fragment(s) can include additional segments that provide for transcription of a gene encoded by the nucleic acid sequence fragment. The additional segments can include and are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication and such, as known in the art. Expression vectors are often derived from plasmids, cosmids, and viral vectors; vectors are often recombinant molecules containing nucleic acid sequences from several sources.

[0069] As used herein, the term “gastrointestinal” refers to the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine and rectum.

[0070] As used herein, the term “gastrointestinal stem cell” refers to a stem cell of the gastrointestinal system.

[0071] As used herein, the term “individual” or “subject” refers to a vertebrate or an invertebrate, such as a human or non-human animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, apes and monkeys. In certain embodiments, the individual or subject is a human.

[0072] As used herein, the term “intestinal” or “intestines” refers to the rectum, small intestine and large intestine.

[0073] As used herein, the term “intestinal stem cell” refers to a stem cell of the intestines.

[0074] As used herein, the term “z z vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments exemplified, but are not limited to, cell cultures.

[0075] As used herein, the term “z z vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[0076] As used herein, the term “linear” when used in reference to a geometric shape refers to a shape that resembles a line. In certain embodiments, the line can be a straight line, a curved line or a line of any shape.

[0077] As used herein, the term “isolated” in reference to a cell, e.g, a gastrointestinal stem cell, refers to a call that has been separated from a component of its natural environment.

[0078] As used herein, a “marker” refers to an agent that allows for direct or indirect detection. Markers include, but are not limited to, fluorescent labels, chromogenic labels, electron dense labels, chemiluminescent labels and radioactive labels. Non-limiting examples of markers include green fluorescent protein (“GFP”), mCherry, dtTomato, or other fluorescent proteins known in the art e.g., Shaner et al., A Guide to Choosing Fluorescent Proteins, Nature Methods 2(12):905-909 (2005) incorporated by reference herein),32P,14C,125I,3H and13 JI, fluorogens (such as Rare Earth Chelate or lucifer yellow and its derivatives), Rhodamine (rhodamine) and its derivatives, dansyl, umbelliferone, luciferase (such as firefly luciferase and bacterial fluorescence plain enzyme) (U.S. Patent number 4,737,456), fluorescein, 2,3-dihydros phthalazine diketone, as well as enzymes producing detectable signals, e.g., horseradish peroxidase (HRP), alkaline phosphatase, beta galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (such as glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0079] The term “nucleic acid” or “polynucleotide” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (z.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (z.e., deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. The term nucleic acid encompasses deoxyribonucleic acid (DNA) including, e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), e.g. messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular. In addition, the term nucleic acid includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues.

[0080] The term “operably linked,” when applied to nucleic acid sequences, for example in an expression vector, indicates that the sequences are arranged so that they function cooperatively in order to achieve their intended purposes, i.e., a promoter sequence allows for initiation of transcription that proceeds through a linked coding sequence as far as the termination signal.

[0081] The term “organoid,” as used herein, refers to a three-dimensional cellular structure obtained by expansion of stem cells, e.g, adult stem cells, that self-organize and can differentiate into functional cell types. See Corro et al. (2020) Am. J. Physiol. Cell Physiol. 319:C151-C165. As used herein, the term “plurality” refers to a number larger than one. In certain embodiments, the term “plurality of tissue-derived epithelial stem cells” refers to a number of tissue-derived epithelial stem cells larger than one. For example, but not by way of limitation, a plurality of tissue-derived epithelial stem cells includes at least two tissue- derived epithelial stem cells. In certain non-limiting embodiments, a plurality of tissue- derived epithelial stem cells can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000 or at least about 1,000,000,000 tissue-derived epithelial stem cells.

[0082] As used herein, the term “population of tissue-derived epithelial organoids” refers to a group of at least two tissue-derived epithelial organoids. In certain embodiments, a “population of tissue-derived epithelial organoids” refers to a group of tissue-derived epithelial organoids produced by the same method. In certain non-limiting embodiments, a population of tissue-derived epithelial organoids can include at least about 10, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 5,000, at least about 10,000, at least about 100,000, at least about 1,000,000, at least about 10,000,000, at least about 100,000,000 or at least about 1,000,000,000 tissue- derived epithelial organoids.

[0083] As used herein, the term “proliferation” refers to an increase in cell number. The term “promoter” as used herein denotes a region within a gene to which transcription factors and / or RNA polymerase can bind so as to control expression of an associated coding sequence. Promoters are commonly, but not always, located in the 5' noncoding regions of genes, upstream of the translation initiation codon. The promoter region of a gene can include one or more consensus sequences that act as recognizable binding sites for sequence specific nucleic acid binding domains of nucleic acid binding proteins. Nevertheless, such binding sites can also be located in regions outside of the promoter, for example in enhancer regions located in introns or downstream of the coding sequence.

[0084] As used herein, the term “solidify” or “solidified” refers to the hardening, thickening, polymerization and / or increased stiffness of a substance.

[0085] As used herein, the term “snake” when used in reference to a geometric shape refers to a serpentine shape. As used herein, the term “spiral” when used in reference to a geometric shape refers to a continuous curving line that spirals around a center point or around an axis.

[0086] As used herein, the term “subset” refers to a small portion of a larger quantity of material.

[0087] As used herein, “tissue-derived epithelial stem cell” refers to an epithelial stem cell obtained from a tissue. In certain embodiments, tissue-derived epithelial stem cells do not include pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)).

[0088] As used herein, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more signs or symptoms, diminishment of extent of disease, stabilized (ie., not worsening) state of disease, prevention of disease, delay or slowing of disease progression, remission of the disease (e.g., cancer) and / or amelioration or palliation of the disease state. The decrease can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% decrease in severity of complications, signs or symptoms or in likelihood of progression to another grade. In certain embodiments, “treatment” can also refer to inhibiting proliferation of a cancer or progression to a higher grade by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%.

[0089] II. ORGANOIDS AND COMPOSITIONS THEREOF

[0090] The present disclosure provides tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids are embedded in hydrogel not attached to a substrate, e.g., a substrate of a culture vessel. The present disclosure further provides compositions including such organoids. In certain embodiments, the tissue- derived epithelial organoids are produced by the methods disclosed herein, e.g., the methods disclosed in Section III.

[0091] In certain embodiments, the present disclosure provides compositions that include a tissue-derived epithelial organoid and a medium, where the tissue-derived epithelial organoid is embedded within a hydrogel. In certain embodiments, the hydrogel is not attached to the surface of a substrate, e.g., surface of a cell culture dish and / or a multiwell plate. In certain embodiments, the tissue-derived epithelial organoid embedded within the hydrogel is suspended in the medium. In certain embodiments, the tissue-derived epithelial organoid is a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid or a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is a lacrimal gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a tonsil organoid. In certain embodiments, the tissue-derived epithelial organoid is a salivary gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid. In certain embodiments, the tissue-derived epithelial organoid is a thyroid organoid. In certain embodiments, the tissue-derived epithelial organoid is a lung organoid. In certain embodiments, the tissue-derived epithelial organoid is a mammary gland organoid. In certain embodiments, the tissue-derived epithelial organoid is a liver organoid. In certain embodiments, the tissue-derived epithelial organoid is a bile duct organoid. In certain embodiments, the tissue-derived epithelial organoid is a gastric organoid. In certain embodiments, the tissue-derived epithelial organoid is a kidney organoid. In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In certain embodiments, the tissue-derived epithelial organoid is an endometrial organoid. In certain embodiments, the tissue-derived epithelial organoid is a fallopian tube organoid. In certain embodiments, the tissue-derived epithelial organoid is a cervix organoid. In certain embodiments, the tissue-derived epithelial organoid is a prostate organoid. In certain embodiments, the tissue-derived epithelial organoid is a bladder organoid. In certain embodiments, the tissue-derived epithelial organoid is an ovarian organoid. In certain embodiments, the tissue-derived epithelial organoid is a taste bud organoid. In certain embodiments, the tissue-derived epithelial organoid is a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreatic organoid, a mammary organoid and a combination thereof.

[0092] In certain embodiments, the tissue-derived epithelial organoid is a lung organoid. In certain embodiments, the tissue-derived epithelial organoid is an alveolar type II (ATII) organoid.

[0093] In certain embodiments, the tissue-derived epithelial organoid is a gastrointestinal organoid. In certain embodiments, the gastrointestinal organoid is an intestinal organoid. For example, but not by way of limitation, the tissue-derived epithelial organoid is a colon or an ileum organoid. In certain embodiments, the tissue-derived epithelial organoid is a colon organoid. In certain embodiments, the tissue-derived epithelial organoid is an ileum organoid. In certain embodiments, the tissue-derived epithelial organoid is a rectal organoid. In certain embodiments, the tissue-derived epithelial organoid is an esophageal organoid. In certain embodiments, the tissue-derived epithelial organoid is an oral and maxillofacial organoid. In certain embodiments, the gastrointestinal organoid comprises goblet cells and enterocytes.

[0094] In certain embodiments, the tissue-derived epithelial organoid is a mammary gland organoid.

[0095] In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In certain embodiments, the pancreatic organoid comprises ductal cells, e.g., as disclosed in Example 6.

[0096] In certain embodiments, the tissue-derived epithelial organoid is a liver organoid. In certain embodiments, the liver organoid comprises hepatocytes, e.g., as disclosed in Example 7.

[0097] In certain embodiments, a composition of the present disclosure can include one or more different types of tissue-derived epithelial organoids. For example, but not by way of limitation, a composition of the present disclosure can include colon and ileum organoids. In certain embodiments, a composition of the present disclosure can include liver and bile duct organoids.

[0098] In certain embodiments, a composition of the present disclosure includes about 1 or more tissue-derived epithelial organoids, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 50,000 or more, about 100,000 or more, about 500,000 or more, about 1,000,000 or more, about 10,000,000 or more, about 100,000,000 or more or about 1,000,000,000 or more tissue-derived epithelial organoids, embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 50 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 100 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 1,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 5,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 10,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 100,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 500,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 1,000,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 10,000,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium. In certain embodiments, a composition of the present disclosure includes about 100,000,000 or more tissue-derived epithelial organoids embedded within hydrogel suspended in the medium.

[0099] In certain embodiments, the hydrogel is a three-dimension (3D) scaffold. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 10°C. For example, but not by way of limitation, the hydrogel is composed of a material that solidifies at temperatures greater than about 15°C, greater than about 20°C, greater than about 25°C, greater than about 30°C, greater than about 35°C, greater than about 40°C, greater than about 45°C or greater than about 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 25°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 30°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 35°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 40°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 45°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures greater than about 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 25°C to about 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 25°C to about 40°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 30°C to about 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature from about 30°C to about 40°C, e.g., about 37°C.

[0100] In certain embodiments, the hydrogel is a synthetic hydrogel, a native hydrogel or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a native hydrogel. In certain embodiments, the hydrogel can be a mixture of synthetic and native hydrogels. In certain embodiments, the hydrogel does not include chemically crosslinked proteins and / or polymers.

[0101] In certain embodiments, the hydrogel is a native hydrogel. In certain embodiments, a native hydrogel includes one or more components that are naturally occurring. For example, but not by way of limitation, a native hydrogel can include one or more proteins, e.g., glycoproteins, and / or polysaccharides. Non-limiting examples of glycoproteins include collagen (e.g., Type I collagen, Type II collagen, Type III collagen, Type IV collagen, Type V collagen, Type VI collagen, Type VII collagen, Type VIII collagen, Type IX collagen, Type X collagen, Type XI collagen and / or Type XII collagen), fibronectin, entactin, tenascin, vitronectin, fibrillin, hyaluronic acid and laminin. In certain embodiments, the native hydrogel can further include one or more components such as, but not limited to, polysaccharides, water and / or elastin. In certain embodiments, a native hydrogel of the present disclosure includes laminin, entactin and Type IV collagen. In certain embodiments, a native hydrogel of the present disclosure includes laminin, entactin, Type IV collagen and heparin sulfate proteoglycan. In certain embodiments, the native hydrogel includes extracellular matrix (ECM) secreted by and / or derived from epithelial cells, endothelial cells, parietal endoderm like cells and / or connective tissue cells.

[0102] In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers such as ProNectin (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethyleneimine) and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31 (10):2253 -2271 ; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, the contents of each are incorporated by reference herein in their entireties.

[0103] In certain embodiments, the hydrogel of the present disclosure does not include alginate. In certain embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECMs include ECM proteins and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In certain embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin and collagen IV. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV and heparin sulfate proteoglycan.

[0104] In certain embodiments, the hydrogel has a protein concentration, e.g., a glycoprotein concentration, greater than about 0.7 mg / ml. In certain embodiments, the hydrogel has a protein concentration, e.g., a glycoprotein concentration, greater than about 1 mg / ml. In certain embodiments, the hydrogel has a protein concentration, e.g., a glycoprotein concentration, greater than about 2 mg / ml. In certain embodiments, the hydrogel has a protein concentration greater than about 3 mg / ml. In certain embodiments, the hydrogel has a protein concentration greater than about 4 mg / ml. In certain embodiments, the hydrogel has a protein concentration greater than about 5 mg / ml. In certain embodiments, the hydrogel has a protein concentration greater than about 6 mg / ml, greater than about 7 mg / ml, greater than about 8 mg / ml, greater than about 9 mg / ml or greater than about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration from about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration from about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration from about 5 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration from about 6 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration that is not less than 0.7 mg / ml. In certain embodiments, the hydrogel has a protein concentration that is not less than 1 mg / ml. In certain embodiments, the hydrogel has a protein concentration that is not less than 5 mg / ml. In certain embodiments, the hydrogel has a protein concentration that is not less than 6 mg / ml.

[0105] In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % greater than about 1 w / v %. For example, but not by way of limitation, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % greater than about 1 w / v %, greater than about 1.5 w / v %, greater than about 2 w / v %, greater than about 2.5 w / v %, greater than about 3 w / v %, greater than about 3.5 w / v %, greater than about 4 w / v %, greater than about 4.5 w / v %, greater than about 5 w / v %, greater than about 5.5 w / v %, greater than about 6 w / v %, greater than about 6.5 w / v %, greater than about 7 w / v %, greater than about 7.5 w / v %, greater than about 8 w / v %, greater than about 8.5 w / v %, greater than about 9 w / v %, greater than about 9.5 w / v %, greater than about 10 w / v %, greater than about 10.5 w / v %, greater than about 11 w / v %, greater than about 11.5 w / v %, greater than about 12 w / v %, greater than about 12.5 w / v %, greater than about 13 w / v %, greater than about 13.5 w / v %, greater than about 14 w / v %, greater than about 14.5 w / v %, greater than about 15 w / v %, greater than about 15.5 w / v %, greater than about 16 w / v %, greater than about 16.5 w / v %, greater than about 17 w / v %, greater than about 17.5 w / v %, greater than about 18 w / v %, greater than about 18.5 w / v %, greater than about 19 w / v %, greater than about 19.5 w / v % or greater than about 20 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 1 w / v % to about 10 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 2 w / v % to about 10 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 5 w / v % to about 10 w / v %.

[0106] In certain embodiments, the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0107] In certain embodiments, the hydrogel comprising the tissue-derived epithelial organoids and suspended in the medium has a geometric shape. In certain embodiments, the geometric shape of the hydrogel has a length, width and / or diameter greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a length greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a width greater than about 0.1 mm. In certain embodiments, the geometric shape of the hydrogel has a diameter greater than about 0.1 mm. For example, but not by way of limitation, the hydrogel has a geometric shape that has a length, width and / or diameter greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm, greater than about 750 mm, greater than about 800 mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm or greater than about 1,000 mm.

[0108] In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm or about 1 mm to about 50 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometric shape having a length of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometric shape having a width of about 0.1 mm to about 20.0 mm. In certain embodiments, the hydrogel has a geometric shape having a diameter of about 0.1 mm to about 20.0 mm. For example, but not by way of limitation, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 5 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 1 mm to about 5 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 1 mm to about 4 mm.

[0109] In certain embodiments, the geometric shape of the hydrogel suspended in the media is a droplet, e.g., as shown in FIG. 6. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 4 mm. In certain embodiments, each hydrogel droplet includes about 1 or more tissue-derived epithelial organoids, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more or about 10,000 or more tissue-derived epithelial organoids.

[0110] In certain embodiments, the hydrogel has a filament-like structure, e.g., as shown in FIG. 6. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the filament-like structure has a linear shape. In certain embodiments, the filament-like structure has a snake shape. In certain embodiments, the filament-like structure has a spiral shape. In certain embodiments, the filament-like structure has a length and / or width of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm or about 1 mm to about 50 mm. In certain embodiments, the filament-like structure has a length and / or width of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a length of about 0.1 mm to about 1 mm. In certain embodiments, the filament-like structure has a length of about 0.1 mm to about 4 mm. In certain embodiments, the filament-like structure has a length of about 1 mm to about 20 mm. In certain embodiments, the filamentlike structure has a length of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a length of about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a width of about 0.1 mm to about 1 mm. In certain embodiments, the filament-like structure has a width of about 0.1 mm to about 4 mm. In certain embodiments, the filament-like structure has a width of about 1 mm to about 20 mm. In certain embodiments, the filam ent-like structure has a width of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a width of about 1 mm to about 4 mm.

[0111] In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 20 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 10 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 5 mm. In certain embodiments, the filament-like structure has a diameter of about 1 mm to about 20 mm. In certain embodiments, the filam ent-like structure has a diameter of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a diameter of about 1 mm to about 5 mm.

[0112] In certain embodiments, each filament-like structure includes about 1 or more tissue-derived epithelial organoids, e.g., about 2 or more, about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more or about 10,000 or more tissue-derived epithelial organoids.

[0113] In certain embodiments, the composition includes any suitable cell culture medium. In certain embodiments, the cell culture medium contains components that are important to support maintenance of the cultured cells. In certain embodiments, a cell culture medium for use in the present disclosure can be a nutrient solution that includes standard cell culture ingredients such as, but not limited to, amino acids, vitamins, inorganic salts, a carbon energy source (e.g., glucose) and a buffer. In certain embodiments, the medium is a differentiation medium. In certain embodiments, the medium is a growth medium. In certain embodiments, the medium is a stem cell promoting medium. Nonlimiting examples of cell culture media, e.g., organoid growth media, are provided in the examples.

[0114] In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 : 1 or greater. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 : 1 to about 1 : 100. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 :5 to about 1 : 100, about 1 : 10 to about 1 : 100, about 1 : 15 to about 1 : 100, about 1 :20 to about 1 : 100, about 1 :25 to about 1 : 100, about 1 :30 to about 1 : 100, about 1 :35 to about 1 : 100, about 1 :40 to about 1 : 100, about 1 :45 to about 1 : 100, about 1 :45 to about 1 : 100, about 1 :45 to about 1 : 100, about 1 :45 to about 1 : 100, about 1 :50 to about 1 : 100, about 1 :55 to about 1 : 100, about 1 :60 to about 1 : 100, about 1 :65 to about 1 : 100, about 1 :70 to about 1:100, about 1:75 to about 1:100, about 1:80 to about 1:100, about 1:85 to about 1:100, about 1 :90 to about 1 : 100, about 1 :95 to about 1 : 100, 1 :5 to about 1:50, about 1 : 10 to about 1:50, about 1:15 to about 1:50, about 1:20 to about 1:50, about 1:25 to about 1:50, about 1:30 to about 1:50, about 1:35 to about 1:50, about 1:40 to about 1:50, about 1:45 to about 1:50, about 1:1 to about 1:95, about 1:1 to about 1:90, about 1:1 to about 1:85, about 1:1 to about 1:80, about 1:1 to about 1:75, about 1:1 to about 1:70, about 1:1 to about 1:65, about 1:1 to about 1:60, about 1:1 to about 1:55, about 1:1 to about 1:50, about 1:1 to about 1:45, about 1 : 1 to about 1 :40, about 1 : 1 to about 1 :35, about 1 : 1 to about 1 :30, about 1 : 1 to about 1:35, about 1:1 to about 1:30, about 1:1 to about 1:25, about 1:1 to about 1:20, about 1:1 to about 1:15, about 1:1 to about 1:10, about 1:1 to about 1:5, about 1:5 to about 1:75, about 1:5 to about 1:60, about 1:5 to about 1:50, about 1:1 to about 1:40, about 1:5 to about 1:30 or about 1 :5 to about 1 :20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is from about 1 : 1 to about 1:50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is from about 1 :2 to about 1:50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is from about 1 : 1 to about 1 :20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is from about 1:1 to about 1:15. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:1. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:2. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:5. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:10, e.g., as shown in Example 8. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 :30. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:40. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1:60. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 :70. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 :80. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 :90. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) in the composition is about 1 : 100.

[0115] In certain embodiments, the tissue-derived epithelial organoids are more uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue- derived epithelial organoids embedded within hydrogels attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by a method of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids because of the differences in nutrient availability as described in Example 1. For example, but not by way of limitation, tissue-derived epithelial organoids produced by a method of the present disclosure are more uniform in size over the width of the suspension culture droplet (e.g., the suspended hydrogel droplet) compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within hydrogels attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoids are produced in hydrogel domes as disclosed in Example 1.

[0116] In certain embodiments, the tissue-derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). For example, but not by way of limitation, the tissue-derived epithelial organoids (e.g., a population of tissue- derived epithelial organoids) of the present disclosure express markers at a higher level than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue- derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express markers at a lower level than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell and / or proliferation marker, e.g., a gene associated with stem cell and / or proliferation, as shown in FIG. 5A. For example, but not by way of limitation, the stem cell and / or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1 and / or CD44. In certain embodiments, the marker is a differentiation marker, e.g., a gene associated with differentiation, as shown in FIG. 5B. For example, but not by way of limitation, the differentiation marker is Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (K18), Cytokeratin 5 (K5), Cytokeratin 14 (K14) and / or Smooth muscle Actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. For example, but not by way of limitation, reference tissue-derived epithelial organoids are tissue-derived epithelial organoids that are produced in hydrogel domes as disclosed in Example 1.

[0117] In certain embodiments, the tissue-derived epithelial organoids are gastrointestinal organoids, and the differentially expressed markers in gastrointestinal organoids of the present disclosure are MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0118] In certain embodiments, the tissue-derived epithelial organoids are mammary organoids, and the differentially expressed markers in mammary organoids of the present disclosure are EpCAM, CD49f, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (K14), and / or Smooth muscle Actin (SMA).

[0119] In certain embodiments, the tissue-derived epithelial organoids are pancreatic organoids, and the differentially expressed markers in pancreatic organoids of the present disclosure are CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, Keratin 19 (KRT19), MUC1, INS, GCG and / or AMY.

[0120] In certain embodiments, the tissue-derived epithelial organoids are liver organoids, and the differentially expressed markers in liver organoids of the present disclosure are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0121] In certain embodiments, a stem cell and / or proliferation marker is expressed by a population of tissue-derived epithelial organoids of the present disclosure (e.g., a population of tissue-derived epithelial organoids in a composition of the present disclosure) at a higher level compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44 and a combination thereof. In certain embodiments, the stem cell and / or proliferation markers are selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44 and a combination thereof. In certain embodiments, the stem cell and / or proliferation marker is MKI67. In certain embodiments, the stem cell and / or proliferation marker is ASCL2. In certain embodiments, the stem cell and / or proliferation marker is LGR5. In certain embodiments, the stem cell and / or proliferation marker is SOX9. In certain embodiments, the stem cell and / or proliferation marker is SM0C2. In certain embodiments, the stem cell and / or proliferation marker is CD44. In certain embodiments, the stem cell and / or proliferation marker is EpCAM. In certain embodiments, the stem cell and / or proliferation marker is CD49f. In certain embodiments, the stem cell and / or proliferation marker is CD133. In certain embodiments, the stem cell and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell and / or proliferation marker is NEUR0G3. In certain embodiments, the stem cell and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell and / or proliferation marker is PDX1. In certain embodiments, the stem cell and / or proliferation marker is BMI1. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least

[0122] 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least

[0123] 180% greater, at least 190% greater, at least 200% greater, at least 210% greater, at least

[0124] 220% greater, at least 230% greater, at least 240% greater, at least 250% greater, at least

[0125] 260% greater, at least 270% greater, at least 280% greater, at least 290% greater or at least 300% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 50% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 100% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 200% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 300% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids.

[0126] In certain embodiments, a differentiation marker is expressed by a population of tissue-derived epithelial organoids of the present disclosure (e.g., a population of tissue- derived epithelial organoids in a composition of the present disclosure) at a lower level compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of differentiation markers include Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (K14), Smooth muscle Actin (SMA) and a combination thereof. In certain embodiments, the differentiation markers are selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7 and a combination thereof. In certain embodiments, the differentiation marker is Keratin 20 (KRT20). In certain embodiments, the differentiation marker is FABP1. In certain embodiments, the differentiation marker is MUC2. In certain embodiments, the differentiation marker is MUC5B. In certain embodiments, the differentiation marker is TFF3. In certain embodiments, the differentiation marker is ALPI. In certain embodiments, the differentiation marker is SI. In certain embodiments, the differentiation marker is CEACAM7. In certain embodiments, the differentiation marker is Keratin 19 (KRT19). In certain embodiments, the differentiation marker is Keratin 7 (KRT7). In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is MUC 1. In certain embodiments, the differentiation marker is INS. In certain embodiments, the differentiation marker is GCG. In certain embodiments, the differentiation marker is AMY. In certain embodiments, the differentiation marker is ALB. In certain embodiments, the differentiation marker is CYP3A4. In certain embodiments, the differentiation marker is HNF4A. In certain embodiments, the differentiation marker is Cytokeratin 8 (K8). In certain embodiments, the differentiation marker is Cytokeratin 18 (KI 8). In certain embodiments, the differentiation marker is Cytokeratin 5 (K5). In certain embodiments, the differentiation marker is Cytokeratin 14 (K14). In certain embodiments, the differentiation marker is Smooth muscle Actin (SMA). In certain embodiments, the differentiation marker is MUC5AC. In certain embodiments, the differentiation marker is MUC6. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 100% less, at least 110% less, at least 120% less, at least 130% less, at least 140% less, at least 150% less, at least 160% less, at least 170% less, at least 180% less, at least 190% less, at least 200% less, at least 210% less, at least 220% less, at least 230% less, at least 240% less, at least 200% less, at least 250% less, at least 260% less, at least 270% less, at least 280% less, at least 290% less or at least 300% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 50% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 100% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 200% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids of the present disclosure is at least 300% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids.

[0127] III. METHODS OF PRODUCING ORGANOIDS

[0128] The present disclosure provides methods of producing tissue-derived epithelial organoids. In certain embodiments, the present disclosure provides methods for producing tissue-derived epithelial organoids that are embedded in a hydrogel suspended in a medium. The present disclosure further provides methods for producing suspension culture of tissue-derived epithelial organoids. In certain embodiments, the organoids can be generated using the methods described in Examples 1 and 4-8 and FIGs. 10 and 11.

[0129] In certain embodiments, the present disclosure provides methods for producing lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, gastric organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervix organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids or cytotrophoblast organoids. In certain embodiments, the present disclosure provides methods for producing lacrimal gland organoids. In certain embodiments, the present disclosure provides methods for producing tonsil organoids. In certain embodiments, the present disclosure provides methods for producing salivary gland organoids. In certain embodiments, the present disclosure provides methods for producing gastrointestinal organoids. In certain embodiments, the present disclosure provides methods for producing thyroid organoids. In certain embodiments, the present disclosure provides methods for producing lung organoids. In certain embodiments, the present disclosure provides methods for producing mammary gland organoids. In certain embodiments, the present disclosure provides methods for producing liver organoids. In certain embodiments, the present disclosure provides methods for producing bile duct organoids. In certain embodiments, the present disclosure provides methods for producing gastric organoids. In certain embodiments, the present disclosure provides methods for producing kidney organoids. In certain embodiments, the present disclosure provides methods for producing pancreatic organoids. In certain embodiments, the present disclosure provides methods for producing endometrial organoids. In certain embodiments, the present disclosure provides methods for producing fallopian tube organoids. In certain embodiments, the present disclosure provides methods for producing cervix organoids. In certain embodiments, the present disclosure provides methods for producing prostate organoids. In certain embodiments, the present disclosure provides methods for producing bladder organoids. In certain embodiments, the present disclosure provides methods for producing ovarian organoids. In certain embodiments, the present disclosure provides methods for producing cytotrophoblast organoids. In certain embodiments, the present disclosure provides methods for producing cytotrophoblast organoids.

[0130] In certain embodiments, the present disclosure provides methods for producing a tissue-derived epithelial organoid is selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreatic organoid, a mammary organoid and a combination thereof.

[0131] In certain embodiments, the present disclosure provides methods for producing gastrointestinal organoids. In certain embodiments, the present disclosure provides methods for producing intestinal organoids. For example, but not by way of limitation, the intestinal organoids are colon or ileum organoids. In certain embodiments, the intestinal organoids are colon organoids. In certain embodiments, the intestinal organoids are ileum organoids. In certain embodiments, the present disclosure provides methods for producing rectal organoids. In certain embodiments, the present disclosure provides methods for producing esophageal organoids. In certain embodiments, the present disclosure provides methods for producing oral and maxillofacial organoids. In certain embodiments, the methods of the present disclosure can produce two or more different types of tissue-derived epithelial organoids, e.g., colon and ileum organoids.

[0132] In certain embodiments, the present disclosure provides methods for producing lung organoids.

[0133] In certain embodiments, the present disclosure provides methods for producing liver organoids.

[0134] In certain embodiments, the present disclosure provides methods for producing pancreatic organoids.

[0135] In certain embodiments, the present disclosure provides methods for producing mammary organoids.

[0136] In certain embodiments, a method for producing tissue-derived epithelial organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture.

[0137] In certain embodiments, the plurality of tissue-derived epithelial stem cells includes at least two or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes at least about 10 or more tissue- derived epithelial stem cells, at least about 100 or more tissue-derived epithelial stem cells, at least about 1,000 or more tissue-derived epithelial stem cells, at least about 10,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 100,000 or more tissue-derived epithelial stem cells, at least about 1,000,000 or more tissue-derived epithelial stem cells, at least about 10,000,000 or more tissue-derived epithelial stem cells, or at least about 100,000,000 or more tissue- derived epithelial stem cells or at least about 1,000,000,000 or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes at least about 10 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 100 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 1,000 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 10,000 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 100,000 or more tissue- derived epithelial stem cells / ml of hydrogel, at least about 100,000 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 1,000,000 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 10,000,000 or more tissue-derived epithelial stem cells / ml of hydrogel, at least about 100,000,000 or more tissue-derived epithelial stem cells / ml of hydrogel or at least about 1,000,000,000 or more tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes at least about 10,000 or more tissue-derived epithelial stem cells / ml of hydrogel.

[0138] In certain embodiments, the plurality of tissue-derived epithelial stem cells includes from about 1 x io4to about 1 x io10tissue-derived epithelial stem cells / ml of hydrogel. For example, but not by way of limitation, the plurality of tissue-derived epithelial stem cells includes from about 1 x 104to about 1 x 109tissue-derived epithelial stem cells / ml of hydrogel, about 1 x 104 to about 1 x 108tissue-derived epithelial stem cells / ml of hydrogel, about 1 x io4to about 1 x io7tissue-derived epithelial stem cells / ml of hydrogel, about 1 x 104to about 1 x io6tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 104to about 1 x 105tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 105to about 1 x 1010tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 106to about 1 x 1010tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 107to about 1 x 1010tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 108to about 1 x 1010tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 109to about 1 x io10tissue-derived epithelial stem cells / ml of hydrogel, from about 1 x 105to about 1 x io7tissue-derived epithelial stem cells / ml of hydrogel or from about 1 x 105to about 1 x io8tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes from about 1 x io4tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes from about 1 x io4tissue-derived epithelial stem cells / ml of hydrogel to about 1 x io6tissue-derived epithelial stem cells / ml of hydrogel.

[0139] In certain embodiments, the method can further include suspending the hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, suspending the hydrogel -tissue-derived epithelial stem cell mixture in the medium includes submerging a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the medium. In certain embodiments, dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the medium can be repeated to generate multiple and separated hydrogel- tissue-derived epithelial stem cell mixtures suspended in the medium. In certain embodiments, the dispensing device can be any device that allows the delivery of a mixture. In certain embodiments, the delivery device dispenses mixtures in an accurate and controlled manner. Non-limiting examples of dispensing devices include pipettes, droppers and syringes. In certain embodiments, the dispensing device can be manually operated or automatically operated. For example, but not by way of limitation, the dispensing device can be automatically operated. In certain embodiments, the dispensing device can be an automated liquid handler. In certain embodiments, the dispensing device can be a liquid handling robot.

[0140] In certain embodiments, the volume of hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 1 pl. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 5 pl, at least about 10 pl, at least about 50 pl, at least about 100 pl, at least about 500 pl, at least about 1 ml, at least about 10 ml, at least about 50 ml, at least about 100 ml, at least about 500 ml, at least about 1 L, at least about 1.5 L, at least about 2 L, at least about 5 L or at least about 10L. In certain embodiments, the volume of hydrogel -tissue-derived epithelial stem cell mixture dispensed into the medium can be from about 1 pl to about 1 L. In certain embodiments, the volume of hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be from about 1 pl to about 1 ml. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium is from about 1 pl to about 900 pl, about 1 pl to about 800 pl, about 1 pl to about 700 pl, about 1 pl to about 600 pl, about 1 pl to about 500 pl, about 1 pl to about 400 pl, about 1 pl to about 300 pl, about 1 pl to about 200 pl, about 1 pl to about 100 pl, about 1 pl to about 10 pl, about 1 pl to about 900 pl, from about 10 pl to about 1 ml, from about 100 pl to about 1 ml, from about 200 pl to about 1 ml, from about 300 pl to about 1 ml, from about 400 pl to about 1 ml, from about 500 pl to about 1 ml, from about 600 pl to about 1 ml, from about 700 pl to about 1 ml, from about 800 pl to about 1 ml, from about 900 pl to about 1 ml, from about 1 pl to about 50 pl, from about 5 pl to about 20 pl, from about 10 pl to about 100 pl, from about 10 pl to about 500 pl, from about 100 pl to about 200 pl or from about 100 pl to about 500 pl. In certain embodiments, the volume of hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be from about 1 pl to about 100 pl.

[0141] In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue- derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 10°C. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 15°C, greater than about 20°C, greater than about 25°C, greater than about 30°C, greater than about 35°C, greater than about 40°C, greater than about 45°C or greater than about 50°C. In certain embodiments, the hydrogel of the hydrogel- tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 25°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 30°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 35°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 40°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 45°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature from about 25°C to about 50°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature from about 30°C to about 50°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature from about 25°C to about 40°C, e.g., about 37°C. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at a temperature from about 30°C to about 40°C, e.g., about 37°C.

[0142] In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C or lower before contacting the medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 20°C or lower before contacting the medium. In certain embodiments, the hydrogel- tissue-derived epithelial stem cell mixture is at a temperature of about 15°C or lower before contacting the medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 10°C or lower before contacting the medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 5°C or lower before contacting the medium. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is at a temperature of about 25°C, about 24°C, about 23°C, about 22°C, about 21°C, about 20°C, about 19°C, about 18°C, about 17°C, about 16°C, about 15°C, about 14°C, about 13°C, about 12°C, about 11°C, about 10°C, about 9°C, about 8°C, about 7°C, about 6°C, about 5°C, about 4°C, about 3°C or about 2°C before contacting the medium. In certain embodiments, the temperature of the hydrogel- tissue-derived epithelial stem cell mixture is from about 2°C to about 25°C before contacting the medium. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 20°C before contacting the medium. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 15°C before contacting the medium. In certain embodiments, the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 10°C before contacting the medium.

[0143] In certain embodiments, the hydrogel can include any material that solidifies. In certain embodiments, the hydrogel is a synthetic hydrogel, a native hydrogel or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a native hydrogel. In certain embodiments, the hydrogel can be a mixture of synthetic and native hydrogels. In certain embodiments, the hydrogel does not include chemically crosslinked proteins and / or polymers.

[0144] In certain embodiments, the hydrogel is a native hydrogel. In certain embodiments, a native hydrogel includes one or more components that are naturally occurring. For example, but not by way of limitation, a native hydrogel can include one or more proteins, e.g., glycoproteins, and / or polysaccharides. Non-limiting examples of glycoproteins include collagen (e.g., Type I collagen, Type II collagen, Type III collagen, Type IV collagen, Type V collagen, Type VI collagen, Type VII collagen, Type VIII collagen, Type IX collagen, Type X collagen, Type XI collagen and / or Type XII collagen), fibronectin, entactin, tenascin, vitronectin, fibrillin, hyaluronic acid and laminin. In certain embodiments, the native hydrogel can further include one or more components such as, but not limited to, polysaccharides, water and / or elastin. In certain embodiments, a native hydrogel of the present disclosure includes laminin, entactin and Type IV collagen. In certain embodiments, a native hydrogel of the present disclosure includes laminin, entactin, Type IV collagen and heparin sulfate proteoglycan. In certain embodiments, the native hydrogel includes extracellular matrix (ECM) secreted by and / or derived from epithelial cells, endothelial cells, parietal endoderm like cells and / or connective tissue cells.

[0145] In certain embodiments, the hydrogel is a synthetic hydrogel. Non-limiting examples of synthetic hydrogels include synthetic polymers such as ProNectin (Sigma Z378666), polyethylene glycol (PEG), poly(hydroxyethyl methacrylate), poly(ethyleneimine) and polyvinyl alcohol (PVA). Additional non-limiting examples of synthetic hydrogels and polymers of such synthetic hydrogels are disclosed in Unal and West (2020) Bioconjugate Chem. 31 (10):2253 -2271 ; and Madduma-Bandarage and Madihally (2020) J. of Applied Polymer Science 138(19):e50376, the contents of each are incorporated by reference herein in their entireties.

[0146] In certain embodiments, the hydrogel of the present disclosure does not include alginate.

[0147] In certain embodiments, the hydrogel can be a commercially available ECM. Non-limiting examples of commercially available ECMs include ECM proteins and basement membrane preparations from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In certain embodiments, the ECM is MATRIGEL™ (BD Biosciences), which includes laminin, entactin and collagen IV. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV and heparin sulfate proteoglycan.

[0148] In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration, e.g., a glycoprotein concentration, greater than about 0.7 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue- derived epithelial stem cell mixture has a protein concentration, e.g., a glycoprotein concentration, greater than about 1 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration, e.g., a glycoprotein concentration, greater than about 2 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 3 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 4 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 5 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration greater than about 6 mg / ml, greater than about 7 mg / ml, greater than about 8 mg / ml, greater than about 9 mg / ml or greater than about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration from about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration from about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration from about 5 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration from about 6 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of the hydrogel- tissue-derived epithelial stem cell mixture has a protein concentration that is not less than 0.7 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration that is not less than 1 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration that is not less than 5 mg / ml. In certain embodiments, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture has a protein concentration that is not less than 6 mg / ml.

[0149] In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % greater than about 1 w / v %. For example, but not by way of limitation, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % greater than about 1 w / v %, greater than about 1.5 w / v %, greater than about 2 w / v %, greater than about 2.5 w / v %, greater than about 3 w / v %, greater than about 3.5 w / v %, greater than about 4 w / v %, greater than about 4.5 w / v %, greater than about 5 w / v %, greater than about 5.5 w / v %, greater than about 6 w / v %, greater than about 6.5 w / v %, greater than about 7 w / v %, greater than about 7.5 w / v %, greater than about 8 w / v %, greater than about 8.5 w / v %, greater than about 9 w / v %, greater than about 9.5 w / v %, or greater than about 10 w / v %, greater than about 10.5 w / v %, greater than about 11 w / v %, greater than about 11.5 w / v %, greater than about 12 w / v %, greater than about 12.5 w / v %, greater than about 13 w / v %, greater than about 13.5 w / v %, greater than about 14 w / v %, greater than about 14.5 w / v %, greater than about 15 w / v %, greater than about 15.5 w / v %, greater than about 16 w / v %, greater than about 16.5 w / v %, greater than about 17 w / v %, greater than about 17.5 w / v %, greater than about 18 w / v %, greater than about 18.5 w / v %, greater than about 19 w / v %, greater than about 19.5 w / v % or greater than about 20 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 1 w / v % to about 10 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 2 w / v % to about 10 w / v %. In certain embodiments, the hydrogel comprises a BME component, an ECM component or a polymer at w / v % from about 5 w / v % to about 10 w / v %.

[0150] In certain embodiments, the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0151] In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) is about 1 : 1 to about 1 :50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium (volume ratio) is about 1 :2 to about 1 :50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :5 to about 1 :50, about 1 : 10 to about 1 :50, about 1 : 15 to about 1 :50, about 1 :20 to about 1 :50, about 1 :25 to about 1 :50, about 1 :30 to about 1 :50, about 1 :35 to about 1 :50, about 1 :40 to about 1 :50, about 1 :45 to about 1 :50, about 1 :2 to about 1 :45, about 1 :2 to about 1 :40, about 1 :2 to about 1 :35, about 1 :2 to about 1 :30, about 1 :2 to about 1 :35, about 1 :2 to about 1 :30, about 1 :2 to about 1 :25, about 1 :2 to about 1 :20, about 1 :2 to about 1 : 15, about 1 :2 to about 1 : 10 or about 1 :2 to about 1 :5. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the medium is from about 1 :2 to about 1 :20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is from about 1 :2 to about 1 : 15. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :2. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :5. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :10, e.g., as shown in Example 8. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :30. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :40. In certain embodiments, the ratio of the volume of the hydrogel to the volume of medium is about 1 :50.

[0152] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. For example, but not by way of limitation, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter greater than about 0.1 mm. In certain embodiments, the geometric shape of the suspended hydrogel- tissue-derived epithelial stem cell mixture has a length greater than about 0.1 mm. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture has a width greater than about 0.1 mm. In certain embodiments, the geometric shape of the suspended hydrogel-tissue-derived epithelial stem cell mixture has a diameter greater than about 0.1 mm. For example, but not by way of limitation, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape that has a length, width and / or diameter greater than about 0.5 mm, greater than about 1 mm, greater than about 1.5 mm, greater than about 2 mm, greater than about 2.5 mm, greater than about 3 mm, greater than about 3.5 mm, greater than about 4 mm, greater than about 4.5 mm, greater than about 5 mm, greater than about 5.5 mm, greater than about 6 mm, greater than about 6.5 mm, greater than about 7.5 mm, greater than about 8 mm, greater than about 8.5 mm, greater than about 9 mm, greater than about 9.5 mm, greater than about 10 mm, greater than about 10.5 mm, greater than about 11 mm, greater than about 11.5 mm, greater than about 12 mm, greater than about 12.5 mm, greater than about 13 mm, greater than about 13.5 mm, greater than about 14 mm, greater than about 14.5 mm, greater than about 15 mm, greater than about 15.5 mm, greater than about 16 mm, greater than about 16.5 mm, greater than about 17.5 mm, greater than about 18 mm, greater than about 18.5 mm, greater than about 19 mm, greater than about 19.5 mm, greater than about 20 mm, greater than about 50 mm, greater than about 100 mm, greater than about 150 mm, greater than about 200 mm, greater than about 250 mm, greater than about 300 mm, greater than about 350 mm, greater than about 400 mm, greater than about 450 mm, greater than about 500 mm, greater than about 550 mm, greater than about 600 mm, greater than about 650 mm, greater than about 700 mm, greater than about 750 mm, greater than about 800 mm, greater than about 850 mm, greater than about 900 mm, greater than about 950 mm or greater than about 1,000 mm.

[0153] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm or about 1 mm to about 50 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 1 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape having a length, width and / or diameter of about 1 mm to about 20 mm. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape having a length, width and / or diameter of about 1 mm to about 10 mm. In certain embodiments, the suspended hydrogel -tissue- derived epithelial stem cell mixture has a geometric shape having a length, width and / or diameter of about 1 mm to about 4 mm.

[0154] In certain embodiments, the geometric shape of a suspended hydrogel-tissue- derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the filament-like structure has a linear shape. In certain embodiments, the filament-like structure has a snake shape. In certain embodiments, the filament-like structure has a spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets (see, e.g., FIG. 10). In certain embodiments, the hydrogel droplet has a diameter greater than about 0.1 mm. In certain embodiments, the hydrogel droplet has a diameter from about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter from about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel droplet has a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel droplet has a diameter of about 1 mm to about 4 mm. In certain embodiments, each hydrogel droplet includes about 1 or more tissue-derived epithelial stem cells, e.g., about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000 or more, about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more or about 1,000,000,000 or more tissue-derived epithelial stem cells.

[0155] In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture has a filament-like structure (see, e.g., FIG. 11). In certain embodiments, the filament-like structure has a length and / or width greater than about 0.1 mm. In certain embodiments, the filament-like structure has a length greater than about 0.1 mm. In certain embodiments, the filament-like structure has a width greater than about 0.1 mm. In certain embodiments, the filament-like structure has a length and / or width of about 0.1 mm to about 1000 mm, e.g., about 0.1 mm to about 500 mm, about 0.1 mm to about 100 mm, about 0.1 mm to about 50 mm, about 0.1 mm to about 20 mm, about 0.1 mm to about 10 mm, about 1 mm to about 1000 mm, about 20 mm to about 1000 mm, about 50 mm to about 1000 mm, about 100 mm to about 1000 mm, about 500 mm to about 1000 mm, about 1 mm to about 100 mm or about 1 mm to about 50 mm. In certain embodiments, the filament-like structure has a length and / or width of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about

[0156] 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about

[0157] 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a length of about 0.1 mm to about 1 mm. In certain embodiments, the filament-like structure has a length of about 0.1 mm to about 4 mm. In certain embodiments, the filament-like structure has a length of about 1 mm to about 20 mm. In certain embodiments, the filament-like structure has a length of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a length of about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a width of about 0.1 mm to about 1 mm. In certain embodiments, the filament-like structure has a width of about 0.1 mm to about 4 mm. In certain embodiments, the filament-like structure has a width of about 1 mm to about 20 mm. In certain embodiments, the filament-like structure has a width of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a width of about 1 mm to about 4 mm.

[0158] In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 20 mm, e.g., from about 0.1 mm to about 19 mm, from about 0.1 mm to about 18 mm, from about 0.1 mm to about 17 mm, from about 0.1 mm to about 16 mm, from about 0.1 mm to about 15 mm, from about 0.1 mm to about 14 mm, from about 0.1 mm to about 13 mm, from about 0.1 mm to about 12 mm, from about 0.1 mm to about 11 mm, from about 0.1 mm to about 10 mm, from about 0.1 mm to about 9 mm, from about 0.1 mm to about 8 mm, from about 0.1 mm to about 7 mm, from about 0.1 mm to about 6 mm, from about 0.1 mm to about 5 mm, from about 0.1 mm to about 4 mm, from about 0.1 mm to about 3 mm, from about 0.1 mm to about 2 mm, from about 0.1 mm to about 1 mm, from about 0.5 mm to about 20 mm, from about 1 mm to about 20 mm, from about 2 mm to about 20 mm, from about 3 mm to about 20 mm, from about 4 mm to about 20 mm, from about 5 mm to about 20 mm, from about 6 mm to about 20 mm, from about 7 mm to about 20 mm, from about 8 mm to about 20 mm, from about 9 mm to about 20 mm, from about 10 mm to about 20 mm, from about 11 mm to about 20 mm, from about 12 mm to about 20 mm, from about 13 mm to about 20 mm, from about 14 mm to about 20 mm, from about 15 mm to about 20 mm, from about 16 mm to about 20 mm, from about 17 mm to about 20 mm, from about 18 mm to about 20 mm, from about 19 mm to about 20 mm, from about 1 mm to about 15 mm, from about 1 mm to about 10 mm, from about 1 mm to about 5 mm or from about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 20 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 10 mm. In certain embodiments, the filament-like structure has a diameter of about 0.1 mm to about 5 mm. In certain embodiments, the filament-like structure has a diameter of about 1 mm to about 20 mm. In certain embodiments, the filam ent-like structure has a diameter of about 1 mm to about 10 mm. In certain embodiments, the filament-like structure has a diameter of about 1 mm to about 5 mm, e.g., as disclosed in Example 8.

[0159] In certain embodiments, each filament-like structure includes about 1 or more tissue-derived epithelial stem cells, e.g., about 5 or more, about 10 or more, about 50 or more, about 100 or more, about 500 or more, about 1,000 or more, about 5,000 or more, about 10,000, more or about 100,000 or more, about 200,000 or more, about 300,000 or more, about 400,000 or more, about 500,000 or more, about 600,000 or more, about 700,000 or more, about 800,000 or more, about 900,000 or more about 1,000,000 or more, about 2,000,000 or more, about 3,000,000 or more, about 4,000,000 or more, about 5,000,000 or more, about 6,000,000 or more, about 7,000,000 or more, about 8,000,000 or more, about 9,000,000 or more, about 10,000,000 or more, about 100,000,000 or more, about 1,000,000,000 or more or about 10,000,000,000 or more tissue-derived epithelial stem cells.

[0160] In certain embodiments, the method can further include culturing the tissue- derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids. In certain embodiments, the cell culture medium contains components that are important to support maintenance of the tissue-derived epithelial stem cells and / or organoids. In certain embodiments, a cell culture medium for use in the present disclosure can be a nutrient solution that includes standard cell culture ingredients such as, but not limited to, amino acids, vitamins, inorganic salts, a carbon energy source (e.g., glucose) and a buffer. In certain embodiments, the medium is a stem cell promoting medium. In certain embodiments, the medium is a cell growth medium. In certain embodiments, the medium is a differentiation medium. Media known to support growth of specific tissue and cell types are known in the art and can be used in connection with the subject matter disclosed herein. For example, but not by way of limitation, examples of media that can be used in the present disclosure are disclosed in Cala et al., Front. Bioeng. Biotechnol. 11 : 1058970 (2023) (e.g., Table 1 of Cala et al.), the entire contents of which is hereby incorporated by reference. Additional non-limiting examples of media for use in the present disclosure are provided in the examples, e.g., in Example 8.

[0161] In certain embodiments, the medium is present in a container. Non-limiting examples of containers include a petri dish, a multi-well plate, a conical tube, a reservoir, a culture bag, a bioreactor or a flask. In certain embodiments, the conical tube is a 50 ml conical tube. In certain embodiments, the multi-well plate is a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate or a 384-well plate. In certain embodiments, the reservoir is a custom reservoir. In certain embodiments, the flask is a 25 ml flask, a 50 ml flask, a 250 ml flask or a 600 ml flask. In certain embodiments, the flask is a single flask or a hotel flask. In certain embodiments, the container is composed of a material that minimizes attachment of the tissue-derived epithelial stem cells and / or hydrogel to the surface of the container. Alternatively or additionally, the container can include a surface coating that minimizes attachment of the tissue-derived epithelial stem cells and / or hydrogel to the surface of the container.

[0162] In certain embodiments, the method can further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel -tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel -tissue- derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, fragmenting the filament-like structures result in structures that are at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% or at least about 90% shorter in length, width or both than the originally extruded hydrogel -tissue-derived epithelial stem cell mixture, e.g., filament-like structures.

[0163] The presently disclosed subject matter further provides methods for generating a suspension culture of tissue-derived epithelial organoids. In certain embodiments, the method can include introducing a mixture comprising a hydrogel and a tissue-derived epithelial stem cell into a medium to generate a suspended mixture. In certain embodiments, the mixture introduced into the medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells as described herein. In certain embodiments, the method can further include culturing the plurality of tissue-derived epithelial stem cells of the mixture in the medium to generate the tissue-derived epithelial organoids in suspension as described herein. In certain embodiments, the method can further include fragmenting the suspended mixture to generate fragmented structures comprising the tissue- derived epithelial organoids. In certain embodiments, the method for generating a suspension culture of tissue-derived epithelial organoids can include contacting a gastrointestinal stem cell, e.g., a plurality of tissue-derived epithelial stem cells, with a hydrogel to generate a hydrogel- tissue-derived epithelial stem cell mixture and depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate. In certain embodiments, the hydrogel-tissue- derived epithelial stem cell mixture is deposited onto the substrate as a droplet. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filament-like structure. In certain embodiments, the method can further include solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method can include suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the method includes culturing the suspended hydrogel- tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids. In certain embodiments, the method can further include dislodging the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the medium. In certain embodiments, the method can further include fragmenting the solidified hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids prior to or after suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the medium.

[0164] In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within a tissue fragment, an organoid fragment or a combination thereof. For example, but not by way of limitation, a tissue fragment and / or an organoid fragment that includes a tissue- derived epithelial stem cell or a plurality of tissue-derived epithelial stem cells can be used to generate a hydrogel-tissue-derived epithelial stem cell mixture. Alternatively or additionally, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from a tissue e.g., a tissue fragment), an organoid fragment (e.g., a tissue-derived epithelial organoid fragment) or a combination thereof. In certain embodiments, tissue-derived epithelial stem cells do not include pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)). In certain embodiments, the tissue-derived epithelial stem cells for use in the present disclosure can be obtained from in vitro cell cultures. In certain embodiments, the tissue fragment can be obtained from frozen samples or from fresh samples, e.g., frozen or fresh tissue samples and / or frozen or fresh tissue organoid fragments. In certain embodiments, the tissue fragment can be a fragment of a primary tissue. In certain embodiments, the tissue (e.g., the tissue fragment), e.g., of a subject, can be normal (e.g., non-cancerous and / or non-diseases). In certain embodiments, the tissue (e.g., the tissue fragment), e.g., of a subject, can be abnormal (e.g., cancerous and / or diseased). In certain embodiments, the tissue-derived epithelial stem cells can be isolated from a fragment of a primary tissue.

[0165] In certain embodiments, the primary tissue fragment can be a fragment of the lacrimal gland, the tonsils, the salivary gland, gastrointestinal tissue, the thyroid, the lung, the mammary gland, the liver, the bile duct, the stomach, the kidney, the pancreas, the endometrium, the fallopian tube, the cervix, the prostate, the bladder, the ovary, the taste bud or the placenta. In certain embodiments, tissue-derived epithelial stem cells can be isolated from a tissue (or fragment thereof) selected from the lacrimal gland, the tonsils, the salivary gland, gastrointestinal tissue, the thyroid, the lung, the mammary gland, the liver, the bile duct, the stomach, the kidney, the pancreas, the endometrium, the fallopian tube, the cervix, the prostate, the bladder, the ovary, the taste bud, the placenta and a combination thereof. In certain embodiments, the primary tissue fragment can be a fragment of the lacrimal gland. In certain embodiments, the primary tissue fragment can be a fragment of the tonsils. In certain embodiments, the primary tissue fragment can be a fragment of the salivary gland. In certain embodiments, the primary tissue fragment can be a fragment of gastrointestinal tissue. In certain embodiments, the primary tissue fragment can be a fragment of the thyroid. In certain embodiments, the primary tissue fragment can be a fragment of the lung. In certain embodiments, the primary tissue fragment can be a fragment of the mammary gland. In certain embodiments, the primary tissue fragment can be a fragment of the liver. In certain embodiments, the primary tissue fragment can be a fragment of the bile duct. In certain embodiments, the primary tissue fragment can be a fragment of the stomach. In certain embodiments, the primary tissue fragment can be a fragment of the kidney. In certain embodiments, the primary tissue fragment can be a fragment of the pancreas. In certain embodiments, the primary tissue fragment can be a fragment of the endometrium. In certain embodiments, the primary tissue fragment can be a fragment of the fallopian tube. In certain embodiments, the primary tissue fragment can be a fragment of the cervix. In certain embodiments, the primary tissue fragment can be a fragment of the prostate. In certain embodiments, the primary tissue fragment can be a fragment of the bladder. In certain embodiments, the primary tissue fragment can be a fragment of the ovary. In certain embodiments, the primary tissue fragment can be a taste bud. In certain embodiments, the primary tissue fragment can be a fragment of the placenta. In certain embodiments, the primary tissue fragment is selected from the group consisting of a fragment of lung tissue, a fragment of liver tissue, a fragment of gastrointestinal tissue, a fragment of mammary tissue, a fragment of pancreatic tissue and a combination thereof.

[0166] In certain embodiments, the primary tissue fragment can be a fragment of gastrointestinal tissue. In certain embodiments, the tissue fragment can be a fragment of the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine and / or rectum, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the oral mucosa, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the pharynx, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the esophagus, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the stomach, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the rectum, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the small intestine, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of the large intestine, e.g., of a subject. In certain embodiments, the tissue fragment can be a fragment of colon and / or ileum tissue, e.g., of a subject. For example, but not by way of limitation, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells can be isolated from colon and / or ileum tissue, e.g., of a subject. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells can be isolated from colon tissue (e.g., of a subject), e.g., to generate a colon organoid. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells can be isolated from ileum tissue (e.g., of a subject), e.g., to generate an ileum organoid. In certain embodiments, the tissue, e.g., of a subject, can be normal (z.e., non-cancerous). For example, but not by way of limitation, the colon and / or ileum tissue, e.g., of a subject, can be normal (z.e., non-cancerous). In certain embodiments, the tissue, e.g., of a subject, can be cancerous or diseased. For example, but not by way of limitation, the colon and / or ileum tissue, e.g., of a subject, can be cancerous and / or diseased colon and / or ileum tissue. In certain embodiments, the esophageal tissue, e.g., of a subject, can be cancerous and / or diseased esophageal tissue. In certain embodiments, the stomach tissue, e.g., of a subject, can be cancerous and / or diseased stomach tissue. In certain embodiments, the rectal tissue, e.g., of a subject, can be cancerous and / or diseased rectal tissue. In certain embodiments, the tissue-derived epithelial organoid can be a subject-derived gastrointestinal tumor organoid.

[0167] In certain embodiments, the primary tissue fragment is a fragment of lung tissue.

[0168] In certain embodiments, the primary tissue fragment is a fragment of liver tissue.

[0169] In certain embodiments, the primary tissue fragment is a fragment of mammary tissue.

[0170] In certain embodiments, the primary tissue fragment is a fragment of pancreatic tissue.

[0171] In certain embodiments, methods of the present disclosure produce tissue- derived epithelial organoids that are uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded within hydrogels attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by a method of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids because of the differences in nutrient availability as described in Example 1. For example, but not by way of limitation, tissue- derived epithelial organoids produced by a method of the present disclosure are more uniform in size over the width of the suspension culture droplet (e.g., the suspended hydrogel droplet) compared to reference tissue-derived epithelial organoids (e.g., tissue- derived epithelial organoids embedded within hydrogels attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoids are produced in hydrogel domes as disclosed in Example 1.

[0172] In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). For example, but not by way of limitation, the tissue-derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express markers at a higher level than reference tissue- derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue-derived epithelial organoids (e.g., a population of tissue-derived epithelial organoids) of the present disclosure express markers at a lower level than reference tissue-derived epithelial organoids (e.g, a population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell and / or proliferation marker, e.g., a gene associated with stem cell and / or proliferation. For example, but not by way of limitation, the stem cell and / or proliferation marker is MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUR0G3, NKX6.1, SM0C2, PDX1 and / or CD44. In certain embodiments, the marker is a differentiation marker, e.g., a gene associated with differentiation. For example, but not by way of limitation, the differentiation marker is Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (K14) and / or Smooth muscle Actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate. For example, but not by way of limitation, reference tissue-derived epithelial organoids are tissue-derived epithelial organoids that are produced in hydrogel domes as disclosed in Example 1.

[0173] In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a stem cell and / or proliferation marker at a higher level compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUR0G3, NKX6.1, SMOC2, PDX1 and / or CD44 and a combination thereof. In certain embodiments, the stem cell and / or proliferation markers are selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44 and a combination thereof. In certain embodiments, the stem cell and / or proliferation marker is MKI67. In certain embodiments, the stem cell and / or proliferation marker is ASCL2. In certain embodiments, the stem cell and / or proliferation marker is LGR5. In certain embodiments, the stem cell and / or proliferation marker is SOX9. In certain embodiments, the stem cell and / or proliferation marker is SMOC2. In certain embodiments, the stem cell and / or proliferation marker is CD44. In certain embodiments, the stem cell and / or proliferation marker is EpCAM. In certain embodiments, the stem cell and / or proliferation marker is CD49f. In certain embodiments, the stem cell and / or proliferation marker is CD133. In certain embodiments, the stem cell and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell and / or proliferation marker is NEUROG3. In certain embodiments, the stem cell and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell and / or proliferation marker is PDX1. In certain embodiments, the stem cell and / or proliferation marker is BMI1. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 60% greater, at least 70% greater, at least 80% greater, at least 90% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least 180% greater, at least 190% greater, at least 200% greater, at least 210% greater, at least 220% greater, at least 230% greater, at least 240% greater, at least 250% greater, at least 260% greater, at least 270% greater, at least 280% greater, at least 290% greater or at least 300% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 50% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 100% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 200% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a stem cell and / or proliferation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 300% greater than the expression level of a stem cell and / or proliferation marker in a population of reference tissue-derived epithelial organoids.

[0174] In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express a differentiation marker at a lower level compared to a population of reference tissue-derived epithelial organoids. Nonlimiting examples of differentiation markers include Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof. In certain embodiments, the differentiation markers are selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7 and a combination thereof. In certain embodiments, the differentiation marker is Keratin 20 (KRT20). In certain embodiments, the differentiation marker is FABP1. In certain embodiments, the differentiation marker is MUC2. In certain embodiments, the differentiation marker is MUC5B. In certain embodiments, the differentiation marker is TFF3. In certain embodiments, the differentiation marker is ALPI. In certain embodiments, the differentiation marker is SI. In certain embodiments, the differentiation marker is CEACAM7. In certain embodiments, the differentiation marker is keratin 19 (KRT19). In certain embodiments, the differentiation marker is Keratin 7 (KRT7). In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is SOX9. In certain embodiments, the differentiation marker is MUC 1. In certain embodiments, the differentiation marker is INS. In certain embodiments, the differentiation marker is GCG. In certain embodiments, the differentiation marker is AMY. In certain embodiments, the differentiation marker is ALB. In certain embodiments, the differentiation marker is CYP3A4. In certain embodiments, the differentiation marker is HNF4A. In certain embodiments, the differentiation marker is Cytokeratin 8 (K8). In certain embodiments, the differentiation marker is Cytokeratin 18 (KI 8). In certain embodiments, the differentiation marker is Cytokeratin 5 (K5). In certain embodiments, the differentiation marker is Cytokeratin 14 (K14). In certain embodiments, the differentiation marker is Smooth muscle Actin (SMA). In certain embodiments, the differentiation marker is MUC5AC. In certain embodiments, the differentiation marker is MUC6. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 100% less, at least 110% less, at least 120% less, at least 130% less, at least 140% less, at least 150% less, at least 160% less, at least 170% less, at least 180% less, at least 190% less, at least 200% less, at least 210% less, at least 220% less, at least 230% less, at least 240% less, at least 200% less, at least 250% less, at least 260% less, at least 270% less, at least 280% less, at least 290% less or at least 300% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 50% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 100% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 200% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of a differentiation marker in a population of tissue-derived epithelial organoids generated by methods of the present disclosure is at least 300% less than the expression level of a differentiation marker in a population of reference tissue-derived epithelial organoids.

[0175] In certain embodiments, a method for producing gastrointestinal organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel- tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue- derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the method can further include suspending the hydrogel-tissue- derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue- derived epithelial stem cell mixture. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 10 pL. In certain embodiments, the ratio of the volume of the hydrogel- tissue-derived epithelial stem cell mixture to the volume of medium is from about 1 :2 to about 1 : 15. In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue- derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which includes laminin, entactin, collagen IV and heparin sulfate proteoglycan. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets. In certain embodiments, the method can further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a medium, e.g., Colon Passage Media (Intesticult Organoid Growth Medium (OGM, StemCell Technologies cat. no. 06010) + 10 pM Y27632) or Ileum Media (OGM + 10 pM Y27632 + 2.5 pM CHIR99021) to generate colon or ileum organoids, respectively. In certain embodiments, the method can further include fragmenting the suspended hydrogel- tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue- derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed markers are MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0176] In certain embodiments, a method for producing lung organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue- derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the method can further include suspending the hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 10 pL. In certain embodiments, the ratio of the volume of the hydrogel -tissue- derived epithelial stem cell mixture to the volume of medium is from about 1:2 to about 1 : 15. In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. A nonlimiting example of ECM is MATRIGEL®. In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets. In certain embodiments, the method can further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel -tissue-derived epithelial stem cell mixture in a medium, e.g., SFFF medium containing 10 pM of ROCK inhibitor. In certain embodiments, the method can further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids).

[0177] In certain embodiments, a method for producing mammary organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel- tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue- derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the method can further include suspending the hydrogel-tissue- derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue- derived epithelial stem cell mixture. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 10 pL. In certain embodiments, the ratio of the volume of the hydrogel- tissue-derived epithelial stem cell mixture to the volume of medium is from about 1:2 to about 1 : 15. In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue- derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is CULTREX® growth factor reduced BME type 2 (Trevigen, 3533-010-02). In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets. In certain embodiments, the method can further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a medium, e.g., SFFF medium containing 10 pM of ROCK inhibitor. In certain embodiments, the method can further include fragmenting the suspended hydrogel- tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue- derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the differentially expressed markers are EpCAM, CD49f, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (K14), and / or Smooth muscle Actin (SMA).

[0178] In certain embodiments, a method for producing pancreatic organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel- tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue- derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. In certain embodiments, the method can further include suspending the hydrogel-tissue- derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue- derived epithelial stem cell mixture. For example, but not by way of limitation, the volume of the hydrogel-tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 10 pL. In certain embodiments, the ratio of the volume of the hydrogel- tissue-derived epithelial stem cell mixture to the volume of medium is from about 1:2 to about 1 : 15. In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue- derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of a BME is reduced growth factor BME 2-RGF (Basement Membrane Extract Type 2 3533-010- 02; AMSBIO, CULTREX®). In certain embodiments, the suspended hydrogel-tissue- derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets. In certain embodiments, the method can further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in optimized human pancreatic organoid expansion medium, e.g., including Basal medium supplemented with IX N2 and IX B27 (both from GIBCO), 1.25 mMN-Acetylcysteine (Sigma-Aldrich), 10% RSPO1 conditioned serum-free media, 10 nM [Leu15]-Gastrin I human (Sigma-Aldrich), 50 ng / mL EGF (Peprotech), 25 ng / mL Noggin (Peprotech), 100 ng / mL FGF10 (Peprotech), lO mM Nicotinamide (Sigma- Aldrich), 5 pM A83.01 (Tocris), 10 pMFSK (Tocris) and 3 pMPGE2 (Tocris) and supplemented with 10 pM Rho Kinase inhibitor (Y27632, Sigma-Aldrich) during the first 7 days. In certain embodiments, the method can further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel -tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel -tissue- derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoids are pancreatic organoids, and the differentially expressed markers are CD 133, LGR5, PDX1, SOX9, ALDH1A1, NEUR0G3, NKX6.1, (Keratin 19 (KRT19), MUC1, INS, GCG and / or AMY.

[0179] In certain embodiments, a method for producing liver organoids includes contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue- derived epithelial stem cell mixture. In certain embodiments, a plurality of tissue-derived epithelial stem cells can be combined with hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture. In certain embodiments, the plurality of tissue-derived epithelial stem cells includes about 3,000 to about 10,000 tissue-derived epithelial stem cells per well in a plate (e.g., a 48-well plate). In certain embodiments, the method can further include suspending the hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture. For example, but not by way of limitation, the volume of the hydrogel -tissue-derived epithelial stem cell mixture dispensed into the medium can be at least about 10 pL. In certain embodiments, the ratio of the volume of the hydrogel-tissue-derived epithelial stem cell mixture to the volume of medium is from about 1:2 to about 1 : 15. In certain embodiments, the hydrogel is solidified upon contact with the medium. For example, but not by way of limitation, the hydrogel of the hydrogel-tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures greater than about 37°C. In certain embodiments, the hydrogel can be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of basement membrane. A non-limiting example of an ECM is MATRIGEL® (BD Biosciences) or reduced growth factor BME 2 (Basement Membrane Extract, Type 2, Pathclear). In certain embodiments, the suspended hydrogel-tissue-derived epithelial stem cell mixture is dispensed into the medium with a certain geometric shape. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of a suspended hydrogel-tissue-derived epithelial stem cell mixture is a filament-like structure. In certain embodiments, the filament-like structure has a linear, snake or spiral shape. In certain embodiments, the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets. In certain embodiments, the method can further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in culture media, e.g., comprising AdDMEM / F12 (Invitrogen) supplemented with 1% N2 (GIBCO) and 1% B27 (GIBCO), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma), and the growth factors: 50 ng / ml EGF (Peprotech), 10% RSPO1 conditioned media (homemade), 100 ng / ml FGF 10 (Peprotech), 25 ng / ml HGF (Peprotech), 10 mM Nicotinamide (Sigma), 5 pM A83.01 (Tocris) and 10 pM FSK (Tocris), which, during the first 3 days after isolation, is supplemented with 25 ng / ml Noggin (Peprotech), 30% Wnt culture medium (as described in Barker et al. Cell Stem Cell 6:25-36 (2010)), and 10 pM (Y27632, Sigma Aldrich) or hES cell cloning recovery solution (Stemgent) to establish the culture. In certain embodiments, the medium is subsequently changed into a medium, e.g., without Noggin, Wnt, Y27632 and the hES cell cloning recovery solution. In certain embodiments, liver organoids are seeded and cultured in the culture medium described above supplemented with BMP7 (25 ng / ml) for 7 to 10 days. In certain embodiments, the medium is subsequently changed to the differentiation medium, e.g., which includes AdDMEM / F12 medium supplemented with 1% N2 and 1% B27 and containing EGF (50 ng / ml), gastrin (10 nM, Sigma), HGF (25 ng / ml), FGF 19 (100 ng / ml), A8301 (500 nM), DAPT (10 pM), BMP7 (25 ng / ml) and dexamethasone (30 pM) to generate hepatocyte organoids. In certain embodiments, the method can further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture can include shearing the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures, e.g., by pipetting the media containing suspended hydrogel-tissue-derived epithelial stem cell mixture up and down. In certain embodiments, fragmenting the suspending hydrogel-tissue-derived epithelial stem cell mixture results in structures that are shorter in length and / or width. For example, but not by way of limitation, fragmenting the filament-like structures result in structures that are shorter in length, width or both. In certain embodiments, the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells for use in the present disclosure is contained within an organoid fragment. In certain embodiments, methods of the present disclosure produce a population of tissue-derived epithelial organoids that express markers at different levels, e.g., higher or lower, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoids are liver organoids, and the differentially expressed markers are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0180] In certain embodiments, one or more steps of the disclosed methods can be performed using robotics and / or automated components. In certain embodiments, a method of the present disclosure can include the use of robotics and / or automated components to generate tissue-derived epithelial organoids. In certain embodiments, one or more steps of the disclosed methods can be performed using robotics and / or automated components to generate tissue-derived epithelial organoids. Non-limiting examples of robotics and / or automated components that can be used in the disclosed methods include automated liquid handlers (e.g., liquid handling robots), 3D printers, syringe pumps, electronic pipettes (e.g., with or without pipetting robots) or a combination thereof. A non-limiting example of an electronic pipettor (e.g., with a pipetting robot) is the Assist Plus from Integra Biosciences. In certain embodiments, one or more steps of the disclosed methods can be performed by automated liquid handlers (e.g., liquid handling robots).

[0181] In certain embodiments, passaging a tissue-derived epithelial organoid culture generated by a method of the present disclosure is performed by robotics and / or automated components. In certain embodiments, automated robotics can perform the methods described in “Organoid Maintenance” of Example 1 of the present disclosure, e.g, any one of the following steps: adding TrypLE Express, heating the organoid culture, triturating the organoid culture to dissociate cells, adding PBS, pelleting the cells, resuspending the cells in BME, cooling the culture, plating the cells, overlaying organoids with media, or a combination thereof. In certain embodiments, dissociating cells can be performed using robotics and / or automated components. In certain embodiments, performing media changes, e.g, during organoid maintenance or during the generation of tissue-derived epithelial organoids, can be performed using robotics and / or automated components.

[0182] In certain embodiments, the generation of BOBA and / or SOBA in cell culture media is performed by robotics and / or automated components. In certain embodiments, automated robotics can perform the methods described in “Suspended hydrogel BOBA culture” and / or “Suspended hydrogel SOBA fragment culture” of Example 1 of the present disclosure, e.g., any one of the following steps: heating media, dispensing organoid cell-BME solution into the warm media as droplets to produce BOBA, dispensing organoid cell-BME solution into the warm media in a linear, snake, or spiral motion in the X-Y plane to produce SOBA, triturating SOBA filament cultures to produce SOBA fragments, changing media, or a combination thereof. In certain embodiments, dispensing organoid cell-BME solution into the warm media to generate BOBA and / or SOBA can be performed using robotics and / or automated components, e.g., can be performed by a liquid handling robot. In certain embodiments, changing media can be performed using robotics and / or automated components, e.g., can be performed by a liquid handling robot. In certain embodiments, triturating SOBA filament cultures to produce SOBA fragments can be performed using robotics and / or automated components, e.g., can be performed by a liquid handling robot.

[0183] In certain embodiments, a high-throughput method, e.g., as described herein, can be performed using robotics and / or automated components. For example, but not by way of limitation, any one of the methods of use disclosed herein, e.g., as described in Section IV, can be performed, in part, using robotics and / or automated components. In certain embodiments, methods for screening agents, e.g., therapeutic agents, and for performing genomic screens using the disclosed tissue-derived epithelial organoids can be performed, in part, using robotics and / or automated components.

[0184] IV. METHODS OF USE

[0185] The present disclosure provides methods of using the disclosed organoids or compositions including such organoids. In certain embodiments, the tissue-derived epithelial organoids of the present disclosure can be used in screening assays. For example, but not by way of limitation, the present disclosure provides methods for screening agents, e.g., therapeutic agents, and for performing genomic screens using the disclosed tissue- derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids of the present disclosure can be used to generate organoid-based models.

[0186] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for identifying agents that have a therapeutic effect. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for identifying therapeutic agents that can be effective at preventing and / or treating a disease. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for identifying therapeutic agents that can be effective at ameliorating the symptoms of a disease.

[0187] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used investigating the biology and / or pathogenesis of a disease. For example, but not by way of limitation, the organoids or compositions thereof of the present disclosure can be contacted with an agent to investigate the biology and / or pathogenesis of a disease.

[0188] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for identifying agents, e.g., therapeutic agents, that can be toxic. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for identifying the concentrations at which an agent, e.g., therapeutic agent, can be toxic.

[0189] In certain embodiments, the method for identifying therapeutic agents that can be effective at preventing and / or treating a disease, for identifying therapeutic agents that can be effective at ameliorating the symptoms of a disease and / or for identifying therapeutic agents that can be toxic can include contacting a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid with a therapeutic agent. In certain embodiments, the method can include contacting a composition that includes a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid with a therapeutic agent. In certain embodiments, the tissue-derived epithelial organoid or the population of the tissue-derived epithelial organoid are embedded in hydrogel suspended in a medium as described herein.

[0190] In certain embodiments, the method for investigating the biology and / or pathogenesis of a disease can include contacting a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid with an agent. In certain embodiments, the method can include contacting a composition that includes a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid with an agent. In certain embodiments, the tissue-derived epithelial organoid or the population of the tissue-derived epithelial organoid are embedded in hydrogel suspended in a medium as described herein.

[0191] In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 minute to about 3 years, e.g., about 15 minutes to about 3 years, about 15 minutes to about 2.5 years, about 15 minutes to about 2 years, about 15 minutes to about 1.5 years, about 15 minutes to about 1 year, about 15 minutes to about 183 days, about 15 minutes to about 150 days, about 15 minutes to about 100 days, about 15 minutes to about 50 days, about 1 day to about 3 years, about 10 days to about 3 years, about 20 days to about 3 years, about 50 days to about 3 years, about 100 days to about 3 years, about 150 days to about 3 years, about 183 days to about 3 years, about 1 year to about 3 years, about 1.5 years to about 3 years, about 2 years to about 3 years or about 2.5 years to about 3 years. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue- derived epithelial organoids (or composition thereof) for about 1 minute to about 100 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue- derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 15 minutes to about 100 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 minute to about 150 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 15 minutes to about 150 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 minute to about 1 year. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 15 minutes to about 1 year. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 minute to about 2 years. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 15 minutes to about 2 years. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 minute to about 10 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 15 minutes to about 10 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 1 hour to about 10 days, about 12 hours to about 10 days, about 1 day to about 10 days, about 2 days to about 10 days, about 3 days to about 10 days, about 4 days to about 10 days, about 5 days to about 10 days, about 6 days to about 10 days, about 7 days to about 10 days, about 8 days to about 10 days, about 9 days to about 10 days, about 15 minutes to about 10 days, about 15 minutes to about 9 days, about 15 minutes to about 8 days, about 15 minutes to about 7 days, about 15 minutes to about 6 days, about 15 minutes to about 5 days, about 15 minutes to about 4 days, about 15 minutes to about 3 days, about 15 minutes to about 2 days, about 15 minutes to about 1 day, about 1 day to about 5 days, about 1 day to about 2 days, about 2 days to about 5 days or about 2 days to about 10 days. In certain embodiments, the agent, e.g., therapeutic agent, is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids (or composition thereof) for about 2 days to about 10 days.

[0192] In certain embodiments, the method can include contacting different populations of tissue-derived epithelial organoids (or composition thereof) with increasing concentrations of the agent, e.g., therapeutic agent, to allow dose-response studies.

[0193] In certain embodiments, the agent can be any agent of interest. In certain embodiments, the agent is a molecule that is known to affect the biology and / or pathogenesis of a disease. Non-limiting examples of agents that can be used in the disclosed methods include peptides, polypeptides, small molecules, cells, gene-editing systems or nucleic acids. In certain embodiments, such agents can be agents that affect cell signaling, nucleic acid expression, protein expression, cell growth, cell differentiation and / or cell survival.

[0194] In certain embodiments, the agent is a therapeutic agent. In certain embodiments, the therapeutic agent can be any therapeutic agent of interest. In certain embodiments, the therapeutic agent is obtained from a library of potential therapeutic agents. Non-limiting examples of therapeutic agents that can be analyzed and / or identified using the disclosed methods include peptide-based therapeutics, polypeptide-based therapeutics, small molecule therapeutics, cell-based therapeutics, gene-editing systems, nucleic acid-based therapeutics and combinations thereof.

[0195] In certain embodiments, the therapeutic agent is a peptide-based therapeutic. In certain embodiments, a peptide-based therapeutic includes peptides that have a molecular weight of about 5,000 Da or less. Non-limiting examples of peptide-based therapeutics include growth factors, anti-infective agents, anti-fungal agents, anti-bacterial agents, ligands for receptors and tyrosine kinase inhibitors. Additional non-limiting examples of peptide therapeutics are disclosed in Wang et al. (2022) Signal Transduction and Targeted Therapy 7:48 (e.g., Table 1 and Table 2), the contents of which is incorporated herein by reference in its entirety.

[0196] In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic. Non-limiting examples of polypeptide-based therapeutics include antibodybased therapeutics such as antibodies and antibody drug conjugates, hormones and enzymes. In certain embodiments, the antibody can be an agonist antibody or an antagonist antibody. In certain embodiments, the antibody can be an antibody fragment. Non-limiting examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv) and multispecific antibodies formed from antibody fragments.

[0197] In certain embodiments, the therapeutic agent is a small molecule therapeutic. For example, but not by way of limitation, the small molecule therapeutic is a compound that has a molecular weight less than about 1,000 Da. In certain embodiments, small molecule therapeutics include cell cycle regulators, kinase regulators (e.g., kinase inhibitors or activators), enzyme inhibitors, receptor regulators (e.g., receptor inhibitors or activators), anti-infective agents, anti-fungal agents, anti-bacterial agents, chemotherapeutics and anti-inflammatory agents.

[0198] In certain embodiments, the therapeutic agent is a cell-based therapeutic. Non-limiting examples of cell-based therapeutics include bacterial cells and immune cells. In certain embodiments, the immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells) and lymphocytes, e.g., B cells and T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells and helper T cells). In certain embodiments, the immune cells can be modified immune cells that have been genetically engineered to express a chimeric antigen receptor (CAR), e.g, CAR T cells and CAR NK cells, or a T cell receptor (TCR), e.g, a heterologous TCR.

[0199] In certain embodiments, the therapeutic agent is a gene-regulating system and / or a component of a gene-regulating system. In certain embodiments, the generegulating system and / or component of a gene-regulating system is a gene-editing system, CRISPRi, a gene expression promoting system, a gene repression promoting system, a nucleic acid-based therapeutic, a transcription factor and / or a regulator of post- transcriptional modifications.

[0200] In certain embodiments, the therapeutic agent is a gene-editing system. Nonlimiting examples of gene-editing systems include homing endonucleases or meganucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and CRISPR gene editing systems. In certain embodiments, the therapeutic agent is a CRISPR gene editing system, e.g., a CRISPR / Cas9 gene editing system.

[0201] In certain embodiments, the therapeutic agent is a nucleic acid-based therapeutic. Non-limiting examples of nucleic acid-based therapeutics include RNA-based therapeutics including siRNA, microRNAs, RNA aptamers, ribozymes, RNA decoys and RNAi. In certain embodiments, the nucleic acid-based therapeutic includes DNA-based therapeutics include anti-sense oligonucleotides (ASOs) and DNA aptamers.

[0202] In certain embodiments, the method can further include analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid). In certain embodiments, the method can further include analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid) that occurs in the presence of the agent. For example, but not by way of limitation, the method can further include analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids (or cells of the tissue-derived epithelial organoid) that is indicative of the effectiveness and / or toxicity of the therapeutic agent. In certain embodiments, the method includes analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids that is indicative of the effectiveness and / or toxicity of the therapeutic agent as compared to a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids that have not been treated with the therapeutic agent.

[0203] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used for performing a genomic screen. In certain embodiments, a genomic screen can be used for identifying gene editing systems for generating mutations. Nonlimiting examples of mutations include deletions, duplications, insertions and nucleotide substitutions. In certain embodiments, the method can include providing a tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid (or a composition thereof) and generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid. In certain embodiments, the mutation is generated using a gene-regulating system and / or a component of a gene-regulating system. In certain embodiments, the gene-regulating system and / or component of a gene-regulating system is a gene-editing system, CRISPRi, RNAi, a gene expression promoting system, a gene repression promoting system, a nucleic acid-based therapeutic, a transcription factor and / or a regulator of post-transcriptional modifications. In certain embodiments, the gene-editing system is a CRISPR system, e.g., a CRISPR / Cas9 gene editing system. In certain embodiments, the method can further include analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation. In certain embodiments, the method includes analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation as compared to a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids that do not have the mutation.

[0204] In certain embodiments, the present disclosure further provides methods for generating an epithelial cell model using the tissue-derived epithelial organoids of the present disclosure. In certain embodiments, the method can include providing the tissue- derived epithelial organoid or a population of the tissue-derived epithelial organoid (or a composition thereof), digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells and culturing the single cells in a medium to generate a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the medium is a differentiation medium. In certain embodiments, the medium is a stem cell promoting medium. In certain embodiments, the medium is a cell growth medium.

[0205] In certain embodiments, the cell monolayer can be used in any of the methods disclosed herein, e.g., used in a screening method disclosed herein. For example, but not by way of limitation, the cell monolayer can be used for screening therapeutic agents and for performing genomic screens. In certain embodiments, a monolayer of the present disclosure can be used to investigate the biology and / or pathogenesis of a disease.

[0206] In certain embodiments, a method for screening a therapeutic agent using a cell monolayer of the present disclosure can include contacting the cell monolayer and analyzing a change in the cell monolayer that is indicative of the effectiveness, disposition and / or toxicity of the therapeutic agent. In certain embodiments, the method includes analyzing a change in the cell monolayer that is indicative of the effectiveness and / or toxicity of the therapeutic agent as compared to a cell monolayer that has not been treated with the therapeutic agent.

[0207] In certain embodiments, a method for performing a genomic screen using a cell monolayer of the present disclosure can include providing the cell monolayer generated by a method described herein, generating a mutation in the genome of one or more cells of the cell monolayer and analyzing a change in the cell monolayer associated with the mutation. In certain embodiments, the method includes analyzing a change in the cell monolayer associated with the mutation as compared to a cell monolayer that does not have the mutation.

[0208] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in cell viability, cell proliferation, organoid size, cell morphology, organoid morphology, invasiveness into hydrogel, motility, differentiation status, mutational status, karyotype, chromosomal aberrations, nucleic acid expression levels, protein expression levels, nucleic acid modifications (e.g., methylation), post-translational modifications (e.g., phosphorylation, ubiquitination and / or glycosylation), activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity (e.g., enzymatic cleavage), chromatin accessibility, histone modifications and other epigenetic changes, physical properties of the organoid, including permeability of the gastrointestinal epithelial barrier, pH, oxygen tension and concentration of other metabolites in the lumen and in the basal membrane and secreted factors in the hydrogel and / or media, concentrations of cytokines and hormones, drug sensitivity, drug absorption and metabolism pharmacokinetics and pharmacodynamics, force measurements, measurements of interactions between biomolecules within the organoids, lumen and media / hydrogel and membrane potential.

[0209] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in cell viability.

[0210] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in cell proliferation.

[0211] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in organoid size.

[0212] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in mutational status.

[0213] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in RNA expression levels and / or protein expression levels.

[0214] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in RNA expression levels and / or protein expression levels of a stem cell and / or proliferation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, in the population of tissue-derived epithelial organoids or the cell monolayer can be a change in protein expression levels of a stem cell and / or proliferation marker. In certain embodiments, the change in the tissue- derived epithelial organoid, in the population of tissue-derived epithelial organoids or the cell monolayer can be a change in RNA expression levels of a stem cell and / or proliferation marker. For example, but not by way of limitation, the change can be a change in MKI67 expression, a change in EpCAM expression, a change in CD49f expression, a change in ASCL2 expression, a change in CD 133 expression, a change in LGR5 expression, a change in SOX9 expression, a change in ALDH1 Al expression, a change inNEUR0G3 expression, a change in NKX6.1 expression, a change in SM0C2 expression, a change in PDX1 expression, a change in BMI1 expression and / or a change in CD44 expression. In certain embodiments, the change can be a change in MKI67 expression. In certain embodiments, the change can be a change in ASCL2 expression. In certain embodiments, the change can be a change in LGR5 expression. In certain embodiments, the change can be a change in SOX9 expression. In certain embodiments, the change can be a change in SM0C2 expression. In certain embodiments, the change can be a change in CD44 expression. In certain embodiments, the change can be a change in EpCAM expression. In certain embodiments, the change can be a change in CD49f expression. In certain embodiments, the change can be a change in CD 133 expression. In certain embodiments, the change can be a change in ALDH1 Al expression. In certain embodiments, the change can be a change in NEUR0G3 expression. In certain embodiments, the change can be a change in NKX6.1 expression. In certain embodiments, the change can be a change in PDX1 expression. In certain embodiments, the change can be a change in BMI1 expression.

[0215] In certain embodiments, the change in the tissue-derived epithelial organoid (or cells thereof), in the population of tissue-derived epithelial organoids (or cells thereof) or the cell monolayer (or cells thereof) can be a change in RNA expression levels and / or protein expression levels of a differentiation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, in the population of tissue-derived epithelial organoids or the cell monolayer can be a change in RNA expression levels of a differentiation marker. In certain embodiments, the change in the tissue-derived epithelial organoid, in the population of tissue-derived epithelial organoids or the cell monolayer can be a change in protein expression levels of a differentiation marker. For example, but not by way of limitation, the change can be a change in Keratin 20 (KRT20) expression, a change in FABP1 expression, a change in MUC2 expression, a change in MUC5B expression, a change in MUC5AC expression, a change in MUC6 expression, a change in TFF3 expression, a change in ALPI expression, a change in SI expression, a change in CEACAM7 expression, a change in Keratin 19 (KRT19) expression, a change in Keratin 7 (KRT7) expression, a change in SOX9 expression, a change in MUC1 expression, a change in INS expression, a change in GCG expression, a change in AMY expression, a change in ALB expression, a change in CYP3 A4 expression, a change in HNF4A expression, a change in Cytokeratin 8 (K8) expression, a change in Cytokeratin 18 (KI 8) expression, a change in Cytokeratin 5 (K5) expression, a change in Cytokeratin 14 (K14) expression and / or a change in Smooth muscle Actin (SMA) expression. In certain embodiments, the change can be a change in KRT20 expression. In certain embodiments, the change can be a change in FABP1 expression. In certain embodiments, the change can be a change in MUC2 expression. In certain embodiments, the change can be a change in MUC5B expression. In certain embodiments, the change can be a change in TFF3 expression. In certain embodiments, the change can be a change in ALPI expression. In certain embodiments, the change can be a change in SI expression. In certain embodiments, the change can be a change in CEACAM7 expression. In certain embodiments, the change can be a change in Keratin 19 (KRT19) expression. In certain embodiments, the change can be a change in Keratin 7 (KRT7) expression. In certain embodiments, the change can be a change in SOX9 expression. In certain embodiments, the change can be a change in MUC1 expression. In certain embodiments, the change can be a change in INS expression. In certain embodiments, the change can be a change in GCG expression. In certain embodiments, the change can be a change in AMY expression. In certain embodiments, the change can be a change in ALB expression. In certain embodiments, the change can be a change in CYP3 A4 expression. In certain embodiments, the change can be a change in HNF4A expression. In certain embodiments, the change can be a change in Cytokeratin 8 (K8) expression. In certain embodiments, the change can be a change in Cytokeratin 18 (KI 8) expression. In certain embodiments, the change can be a change in Cytokeratin 5 (K5) expression. In certain embodiments, the change can be a change in Cytokeratin 14 (KI 4) expression. In certain embodiments, the change can be a change in Smooth muscle Actin (SMA) expression. In certain embodiments, the change can be a change in MUC5AC expression. In certain embodiments, the change can be a change in MUC6 expression.

[0216] In certain embodiments, to determine changes to the tissue-derived epithelial organoid, to the cells of the tissue-derived epithelial organoid and / or to the monolayer or the cells of the monolayer, the tissue-derived epithelial organoid cultures or monolayer cultures can be analyzed by flow cytometry, RNA and protein expression, cytokine and metabolite measurements in organoid and media, cell viability and proliferation assays, microscopy to monitor epithelial and immune cell motility, barrier function, migration, proliferation, subcellular localization of RNA, proteins and organelles, cell stiffness and other assays, tissue-derived epithelial organoids, cells of the tissue-derived epithelial organoid, cell monolayers and / or cells of the cell monolayer can be labeled with dyes or transfected with nucleic acids encoding fluorescent proteins and / or luciferase to enable visualization and quantitation of cells using imaging and bioluminescence assays.

[0217] In certain embodiments, the present disclosure provides methods for identifying a therapeutic agent that can be effective in treating a disease. In certain embodiments, the method can include (i) contacting (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of the tissue-derived epithelial organoid (or a composition thereof), (c) a cell monolayer derived from a tissue-derived epithelial organoid or (d) a population of tissue-derived epithelial organoids with a therapeutic agent and (ii) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue- derived epithelial organoids that is indicative of the effectiveness and / or toxicity of the therapeutic agent. In certain embodiments, the change is indicative of the effectiveness of the therapeutic agent. In certain embodiments, the change is indicative of the toxicity of the therapeutic agent. For example, but not by way of limitation, if the viability of the tissue- derived epithelial organoids or the cells of the tissue-derived epithelial organoid decreases in the presence of the therapeutic agent compared to the viability of tissue-derived epithelial organoids or cells of the tissue-derived epithelial organoid that were not treated with the therapeutic agent is an indication that the therapeutic agent is toxic.

[0218] In certain embodiments, the present disclosure provides methods for performing a genomic screen. In certain embodiments, the method can include (i) providing (a) a tissue-derived epithelial organoid (or a composition thereof), (b) a population of the tissue-derived epithelial organoid (or a composition thereof) or (c) a cell monolayer derived from a tissue-derived epithelial organoid, (ii) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid or the cell monolayer and (iii) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation. For example, but not by way of limitation, if a change is observed in the tissue-derived epithelial organoids or the cell monolayer that includes one or cells with a genomic mutation compared to reference cells or tissue-derived epithelial organoids, then this indicates that the change is a result of the genomic mutation.

[0219] V. SYSTEMS

[0220] The present disclosure further provides systems for use in the present disclosure. In certain embodiments, the present disclosure further provides systems for performing the presently disclosed methods. In certain embodiments, the present disclosure provides systems for generating a tissue-derived epithelial organoid, e.g., a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid or a cytotrophoblast organoid. In certain embodiments, the present disclosure provides systems for identifying therapeutic agents that can be effective at treating a disease. In certain embodiments, the present disclosure provides systems for performing a genomic screen. In certain embodiments, the present disclosure provides systems for generating an epithelial cell model.

[0221] In certain embodiments, systems of the present disclosure can include robotics and / or automated components capable of performing the presently disclosed methods. In certain embodiments, systems of the present disclosure include robotics and / or automated components capable of generating tissue-derived epithelial organoids. In certain embodiments, a system of the present disclosure includes one or more robotic and / or automated components for performing one or more steps of the disclosed methods for generating tissue-derived epithelial organoids. Non-limiting examples of robotics and / or automated components include automated liquid handlers (e.g., liquid handling robots), 3D printers, syringe pumps, or a combination thereof. In certain embodiments, a system of the present disclosure includes one or more automated liquid handlers.

[0222] In certain embodiments, the robotics and / or automated components are used to perform the presently disclosed methods and / or generate tissue-derived epithelial organoids for use in high throughput studies. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for performing a high-throughput method, e.g., as described herein. For example, but not by way of limitation, a system of the present disclosure can include one or more robotic and / or automated components can be used to perform any one of the methods of use disclosed herein. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for screening agents, e.g., therapeutic agents, and for performing genomic screens using the disclosed tissue-derived epithelial organoids.

[0223] In certain embodiments, the robotics and / or automated components are capable of passaging a tissue-derived epithelial organoid culture. In certain embodiments, automated robotics can perform the methods described in “Organoid Maintenance” of Example 1 of the present disclosure, e.g., any one of the following steps: adding TrypLE Express, heating the organoid culture, triturating the organoid culture to dissociate cells, adding PBS, pelleting the cells, resuspending the cells in BME, cooling the culture, plating the cells, overlaying organoids with media, or a combination thereof. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for dissociating cells. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for performing media changes, e.g., during organoid maintenance or during the generation of tissue-derived epithelial organoids.

[0224] In certain embodiments, the robotics and / or automated components are capable of generating BOBA and / or SOBA. In certain embodiments, automated robotics can perform the methods described in “Suspended hydrogel BOBA culture” and / or “Suspended hydrogel SOBA fragment culture” of Example 1 of the present disclosure, e.g., any one of the following steps: heating media, dispensing organoid cell-BME solution into the warm media as droplets to produce BOBA, dispensing organoid cell-BME solution into the warm media in a linear, snake, or spiral motion in the X-Y plane to produce SOBA, triturating SOBA filament cultures to produce SOBA fragments, changing media, or a combination thereof. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for dispensing organoid cell-BME solution into the warm media to generate BOBA and / or SOBA. In certain embodiments, a system of the present disclosure can include one or more liquid handling robots for dispensing organoid cell-BME solution into the warm media to generate BOBA and / or SOBA. In certain embodiments, a system of the present disclosure can include one or more robotic and / or automated components for performing media changes.

[0225] In certain embodiments, systems of the present disclosure can include a tissue-derived epithelial organoid, a population of tissue-derived epithelial organoids and / or a composition including a tissue-derived epithelial organoid, e.g., a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid or a cytotrophoblast organoid. In certain embodiments, the tissue-derived epithelial organoid is selected from the group consisting of a lung organoid, a gastrointestinal organoid, a liver organoid, a pancreatic organoid, a mammary organoid and a combination thereof. In certain embodiments, a system includes a tissue-derived epithelial organoid that is generated from one or more tissue-derived epithelial stem cells. In certain embodiments, the tissue-derived epithelial organoids are provided in a hydrogel. In certain embodiments, the tissue-derived epithelial organoids can be embedded in a hydrogel and suspended in a medium. Non-limiting examples of tissue-derived epithelial organoids (or compositions thereof) or methods of producing such tissue-derived epithelial organoids are disclosed in Sections II and III, respectively. In certain embodiments, the tissue-derived epithelial organoids can be provided in a container, e.g., a culture vessel. In certain embodiments, the tissue-derived epithelial organoids can be provided in a culture vessel, e.g., a flask and / or a multi -well plate.

[0226] In certain embodiments, systems of the present disclosure can further include instructions for using the tissue-derived epithelial organoids for determining whether a therapeutic agent to be tested will be effective in treating a disease. In certain embodiments, systems of the present disclosure can further include instructions for using the tissue-derived epithelial organoids for determining the effect of a genomic mutation. In certain embodiments, systems of the present disclosure can further include instructions for generating an epithelial cell model.

[0227] In certain non-limiting embodiments, systems of the present disclosure can further include one or more reagents and other components (e.g., dyes, antibodies, primers, probes and the like) to determine a change, e.g., change in the expression of a protein or nucleic acid, in a tissue-derived epithelial organoid, a cell of a tissue-derived epithelial organoid, a cell monolayer or a cell of a cell monolayer. Non-limiting examples of such changes are disclosed in Section III. In certain embodiments, a system of the present disclosure can include one or more robotics and / or automated components for analyzing a change in in a tissue-derived epithelial organoid, a cell of a tissue-derived epithelial organoid, a cell monolayer or a cell of a cell monolayer. VI. EXEMPLARY EMBODIMENTS

[0228] A. The presently disclosed subject matter provides a method of generating tissue-derived epithelial organoids, comprising: a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture; b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture; and c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids.

[0229] Al. The method of A, wherein a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture.

[0230] A2. The method of Al, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

[0231] A3. The method of Al or A2, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

[0232] A4. The method of A-A2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

[0233] A5. The method of any one of A-A4, wherein the hydrogel is solidified upon contact with the medium.

[0234] A6. The method of any one of A-A5, wherein suspending the hydrogel- tissue-derived epithelial stem cell mixture in the medium comprises submerging a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the medium.

[0235] A7. The method of any one of A-A6, wherein the temperature of the medium is from about 25°C to about 50°C.

[0236] A7-1. The method of any one of A-A7, wherein the temperature of the medium is from about 30°C to about 50°C.

[0237] A8. The method of any one of A-A7-1, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 25°C. A8-1. The method of any one of A-A8, wherein the temperature of the hydrogel-tissue-derived epithelial stem cell mixture is from about 2°C to about 20°C.

[0238] A9. The method of any one of A-A8-1, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

[0239] A10. The method of A9, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

[0240] Al l. The method of any one of A- A 10, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0241] A12. The method of any one of A-Al l, wherein the suspended hydrogel- tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

[0242] Al 3. The method of Al 2, wherein the suspended hydrogel -tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0243] A14. The method of any one of A-A13, wherein the hydrogel-tissue-derived epithelial stem cell mixture is suspended in the medium in droplets.

[0244] A15. The method of any one of A-A13, wherein the suspended hydrogel- tissue-derived epithelial stem cell mixture has a filament-like structure.

[0245] Al 6. The method of Al 5, wherein the filament-like structure has a linear, snake, or spiral shape.

[0246] Al 7. The method of any one of A- Al 6 further comprising fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0247] B. A method of generating a suspension culture of tissue-derived epithelial organoids, comprising: a) introducing a mixture comprising a hydrogel and a tissue-derived epithelial stem cell into a medium to generate a suspended mixture; and b) culturing the suspended mixture in the medium to generate the tissue- derived epithelial organoids in suspension.

[0248] Bl. The method of B, wherein the mixture introduced into the medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells.

[0249] B2. The method of Bl, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel. B3. The method of Bl or B2, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

[0250] B4. The method of any one of B-B2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

[0251] B5. The method of any one of B-B4, wherein the hydrogel is solidified upon contact with the medium.

[0252] B6. The method of any one of B-B5, wherein introducing a mixture in the medium comprises submerging a dispensing device containing the mixture in the medium and dispensing the mixture into the medium.

[0253] B7. The method of any one of B-B6, wherein the temperature of the medium is from about 25°C to about 50°C.

[0254] B7-1. The method of any one of B-B7, wherein the temperature of the medium is from about 30°C to about 50°C.

[0255] B8. The method of any one of B-B7-1, wherein the temperature of the mixture is from about 2°C to about 25°C.

[0256] B8-1. The method of any one of B-B8, wherein the temperature of the mixture is from about 2°C to about 20°C.

[0257] B9. The method of any one of B-B8-1, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

[0258] BIO. The method of B9, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

[0259] Bl l. The method of any one of B-B10, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0260] B12. The method of any one of B-Bl l, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

[0261] B13. The method of B12, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0262] B14. The method of any one of B-B13, wherein the mixture is introduced into the medium in droplets.

[0263] Bl 5. The method of any one of B-B3, wherein the mixture is introduced into the medium in filament-like structures.

[0264] Bl 6. The method of Bl 5, wherein the filament-like structures have a linear, snake, or spiral shape.

[0265] Bl 7. The method of any one of B-B16 further comprising fragmenting the suspended mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0266] Bl 8. The method of any one of A-B17, wherein the medium is present in a container selected from the group consisting of a petri dish, a multi-well plate, a conical tube, a reservoir, a culture bag, a bioreactor or a flask.

[0267] C. A method of generating a suspension culture of tissue-derived epithelial organoids, comprising: a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture; b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate; c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture; d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture; and e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids.

[0268] Cl. The method of C, wherein a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture.

[0269] C2. The method of Cl, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

[0270] C3. The method of Cl or C2, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

[0271] C4. The method of any one of C-C2, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

[0272] C5. The method of any one of C-C3 further comprising dislodging the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the medium.

[0273] C6. The method of any one of C-C4, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

[0274] C7. The method of C6, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

[0275] C8. The method of any one of C-C7, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

[0276] C9. The method of any one of C-C8, wherein the solidified hydrogel-tissue- derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

[0277] CIO. The method of C9, wherein the solidified hydrogel -tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0278] Cl 1. The method of any one of C-C10, wherein the hydrogel -tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet.

[0279] C12. The method of any one of C-C10, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filament-like structure.

[0280] C13. The method of Cl 2, wherein the filam ent-like structure has a linear, snake, or spiral shape.

[0281] Cl 4. The method of any one of C-C13 further comprising fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate fragmented structures comprising the tissue-derived epithelial organoids.

[0282] C15. The method of any one of C-C14, wherein the tissue-derived epithelial organoids have uniform morphology compared to reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0283] Cl 6. The method of Cl 5, wherein the tissue-derived epithelial organoids have uniform size.

[0284] Cl 7. The method of Cl 6, where the average diameter of the tissue-derived epithelial organoids is more uniform than the reference tissue-derived epithelial organoids.

[0285] Cl 8. The method of any one of A-C17, wherein a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0286] Cl 9. The method of Cl 8, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1 Al, NEUR0G3, NKX6.1, SM0C2, PDX1, CD44 and a combination thereof.

[0287] C20. The method of any one of A-C18, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0288] C21. The method of C20, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALP I, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

[0289] D. A tissue-derived epithelial organoid generated by the method of any one of A-C21.

[0290] DI. The tissue-derived epithelial organoid of D, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

[0291] E. A composition comprising a tissue-derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium.

[0292] El. The composition of E, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

[0293] E2. The composition of El, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0294] E3. The composition of any one of E-E2, wherein the hydrogel is a droplet. E4. The composition of any one of E-E2, wherein the hydrogel has a filament-like structure.

[0295] E5. The composition of E4, wherein the filament-like structure has a linear, snake, or spiral shape.

[0296] E6. The composition of any one of E-E5, wherein a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0297] E7. The composition of E6, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1 Al, NEUR0G3, NKX6.1, SM0C2, PDX1, CD44 and a combination thereof.

[0298] E8. The composition of any one of E-E5, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0299] E9. The composition of E8, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

[0300] F. A method for screening an agent, comprising: a) contacting the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of D or DI or the composition of any one of E-E9 with an agent; and b) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids that is indicative of the effectiveness and / or toxicity of the agent.

[0301] Fl. The method of F, wherein the agent is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 1 minute to about 3 years. Fl-1. The method of Fl, wherein the agent is contacted with the tissue- derived epithelial organoid or the population of tissue-derived epithelial organoids for about 15 minutes to about 3 years.

[0302] F2. The method of F, Fl or Fl-1, wherein the agent is a therapeutic agent.

[0303] F3. The method of F2, wherein the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof.

[0304] G. A method for performing a genomic screen, comprising: a) providing the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of D or DI or the composition of any one of E-E9; b) generating a mutation in the genome of one or more cells of the tissue-derived epithelial organoid; and c) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation.

[0305] Gl. The method of G, wherein the mutation is generated using a generegulating system.

[0306] G2. The method of Gl, wherein the gene-regulating system is a gene-editing system.

[0307] G3. The method of G2, wherein the gene-editing system is a CRISPR system.

[0308] G4. The method of any one of F-G2, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redux potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post- translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

[0309] H. A method for generating an epithelial cell model, comprising: a) providing the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of D; b) digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells; and c) culturing the single cells in a medium to generate a cell monolayer.

[0310] Hl. The method of H, wherein the single cells are cultured on a permeable cell culture insert.

[0311] H2. The method of H or Hl, wherein the medium is a differentiation medium. H3. The method of H or Hl, wherein the medium is a cell growth or stem cell promoting medium.

[0312] I. A method for screening an agent, comprising: a) contacting the cell monolayer generated by the method of any one of H-H3 with an agent; and b) analyzing a change in the cell monolayer that is indicative of the effectiveness, disposition, and / or toxicity of the agent.

[0313] 11. The method of I, wherein the agent is contacted with the cell monolayer for about 1 minute to about 3 years.

[0314] 11-1. The method of II, wherein the agent is contacted with the cell monolayer for about 15 minutes to about 3 years.

[0315] 12. The method of I, Il or 11-1, wherein the agent is a therapeutic agent.

[0316] 13. The method of 12, wherein the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof.

[0317] J. A method for performing a genomic screen, comprising: a) providing the cell monolayer generated by the method of any one of H-H3; b) generating a mutation in the genome of one or more cells of the cell monolayer; and c) analyzing a change in the cell monolayer associated with the mutation.

[0318] JI . The method of J, wherein the mutation is generated using a generegulating system.

[0319] J2. The method of JI, wherein the gene-regulating system is a gene-editing system.

[0320] J3. The method of J2, wherein the gene-editing system is a CRISPR system.

[0321] J4. The method of any one of J-J3, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, redux potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post- translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

[0322] JI . The method of any one of A-C21 and F-J4 or the composition of any one of E-E9, wherein the tissue fragment is a fragment from a tissue selected from the group consisting of the lacrimal gland, the tonsils, the salivary gland, gastrointestinal tissue, the thyroid, the lung, the mammary gland, the liver, the bile duct, the stomach, the kidney, the pancreas, the endometrium, the fallopian tube, the cervix, the prostate, the bladder, the taste bud, the ovary, the placenta and a combination thereof.

[0323] J5. The method of any one of A-C21 and F-J4 or the composition of any one of E-E9, wherein the tissue-derived epithelial stem cell is obtained from a fragment of an organoid selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

[0324] K. A system for culturing a tissue-derived epithelial organoid, comprising a tissue-derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium.

[0325] KI. The system of K, wherein the tissue-derived epithelial organoid is an intestinal organoid.

[0326] K2. The system of K or KI, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

[0327] K3. The system of any one of K-K2, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0328] K4. The system of any one of K-K3, wherein the hydrogel is a droplet.

[0329] K5. The system of any one of K-K3, wherein the hydrogel has a filamentlike structure.

[0330] K6. The system of K5, wherein the filament-like structure has a linear, snake, or spiral shape.

[0331] K7. The system of any one of K-K6, wherein a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0332] K8. The system of K7, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, S0X9, ALDH1 Al, NEUR0G3, NKX6.1, SM0C2, PDX1, CD44 and a combination thereof.

[0333] K9. The system of any one of K-K6, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

[0334] K10. The system of K9, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, MUC5AC, MUC6, TFF3, ALP I, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

[0335] KI 1. The system of any one of K-K10, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

[0336] K12. The system of any one of K-Kl l, further comprising robotics and / or automated components.

[0337] K13. The system of K12, wherein the robotics and / or automated components comprise liquid handling robots, 3D printers, syringe pumps, or a combination thereof.

[0338] L. The method of any one of A-C21 and F-J5, wherein one or more steps of the method is performed by robotics and / or automated components.

[0339] LI. The method of L, wherein the robotics and / or automated components comprise liquid handling robots, 3D printers, syringe pumps, or a combination thereof.

[0340] L2. The method of L or LI, wherein the robotics and / or automated components are liquid handling robots.

[0341] M. A method of producing the tissue-derived epithelial organoid of D-Dl or the composition of E-E9, wherein one or more steps of the method is performed by robotics and / or automated components.

[0342] ML The method of M, wherein the robotics and / or automated components comprise liquid handling robots, 3D printers, syringe pumps, or a combination thereof.

[0343] N. The method of any one of B-B18, wherein the introducing a mixture comprising a hydrogel and a tissue-derived epithelial stem cell into a medium to generate a suspended mixture is performed by robotics and / or automated components.

[0344] Nl. The method of any one of B-B18, wherein the culturing the suspended mixture in the medium to generate the tissue-derived epithelial organoids in suspension is performed by robotics and / or automated components.

[0345] N2. The method of N or Nl, wherein the robotics and / or automated components comprise liquid handling robots, 3D printers, syringe pumps, or a combination thereof.

[0346] N3. The method of N2, wherein the robotics and / or automated components comprises one or more liquid handling robots.

[0347] EXAMPLES

[0348] The presently disclosed subject matter will be better understood by reference to the following Examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0349] EXAMPLE 1: FORMATION OF INTESTINAL ORGANOIDS

[0350] This example provides a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries. This method streamlines the protocol, increases the scalability, enables kinetic sampling and improves culture uniformity without the need for specialized equipment or additional expertise. This method is compatible with multiple culture formats, and the organoids produced by this method can be used in downstream applications such as implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation as shown in Examples 2 and 3. The suspended BME hydrogel culture method allows intestinal organoids to be used more widely and at higher throughputs than previously possible.

[0351] Methods

[0352] Human Intestinal Organoid Derivation. Organoids were derived as previously described (Pleguezuelos-Manzano et al. (2020)) with some modifications. De- identified human colon and ileum tissue samples from deceased donors were procured by Donor Network West. Tissues were washed with Advanced DMEM / F12 media (ThermoFisher), cut into 5 cm x 5 cm segments, then the epithelium was scraped off the submucosa into the media and minced with a razor blade. The solution was pelleted at 450 x g for 5 min, then incubated in 2.5 pM EDTA in PBS without Mg2+or Ca2+at 37°C for 9 min (ileum) or 12 min (colon), with vortexing every 3-4 min until crypts were released. Crypts were pelleted at 450 x g for 5 min, washed in PBS, filtered through sterile gauze then a 100 pm cell strainer to remove debris, and pelleted at 450 x g for 5 min. Crypts were resuspended in CULTREX® Reduced Growth Factor Basement Membrane Matrix, Type II (BME, R&D Systems cat. no. 3533-010-02) on ice, plated in 50 pL domes in a 24-well plate, cured at 37°C for 15-30 minutes, then overlaid with 500 pL Colon Passage Media (Intesticult Organoid Growth Medium (OGM, StemCell Technologies cat. no. 06010) + 10 pM Y27632) or Ileum Media (OGM + 10 pM Y27632 + 2.5 pM CHIR99021). After the first 2-3 days in culture, media was changed every 2-3 days, or when media turned yellow, with plain OGM for colon cultures, or Ileum media for ileum cultures.

[0353] Organoid Maintenance. Organoid cultures were passaged every 1-2 weeks by digestion with TrypLE Express (ThermoFisher) at 37°C for 10 min then triturated with a P1000 pipette. The incubation was repeated up to 2 times if necessary to achieve a single cell suspension. TrypLE Express was inactivated by dilution with PBS, and cells were pelleted at 450 x g for 3 min. Cells were resuspended on ice in BME at 6 x 105cells / mL, plated in 24-well plates in 50 pL domes and cured at 37°C for 15-30 min. Domes were overlaid with Colon Passage Media or Ileum media for the first 2-3 days, and media was changed with plain OGM for colon or Ileum Media every 2-3 days. For colon organoid differentiation, cultures were washed with Advanced DMEM / F12 media, then overlaid with Intesticult Organoid Differentiation Media (ODM, StemCell Technologies cat. no. 100- 0214) + 5 pM DAPT for 5 days, with a media change every 2-3 days.

[0354] Suspended hydrogel BOBA culture. Single organoid cells in BME were prepared on ice as described above. Pre-warmed Colon Passage Media or Ileum Media was added to 6-well plates (5 mL / well), 100 cm petri dishes (15-30 mL / dish), or 50 mL conical tubes (30 mL / tube) and kept on a warm bead bath at 37°C. Ultra-low attachment (ULA) or standard tissue culture plates yielded similar results. FIG. 10 provides an exemplary schematic showing the generation of BOBA. To generate suspended BME droplets, or BOB As, the organoid cell-BME solution was dispensed directly into the warm media in 10 pL volumes using an electronic repeater pipette (Integra VIAFLO 300) with a wide-bore or cut pipette tip (opening about 2 mm) at a slow to moderate speed to avoid forming filaments or thin strands instead of droplets. The tip was submerged immediately beneath the liquid surface during dispense, then lifted after each dispense to ensure droplet separation. For larger format cultures, BOBAs were transferred into flasks by serological pipette or decanting. Media was changed every 2-3 days with plain OGM for colon or Ileum Media. In 6-well plate cultures, a sterile 70 pm cell strainer was placed in the well, the plate was tilted and 4 mL media was gently aspirated through the strainer. In flask cultures, the flask was tilted at an angle, BOBAs settled in a comer, then approximately 2 / 3 volume of spent media was replaced with a serological pipette.

[0355] Suspended hydrogel SOBA and SOBA fragment culture. Single organoid cells in BME were prepared on ice as described above. Pre-warmed Colon Passage Media was added to 6-well plates (5 mL / well), 100 cm petri dishes (15-30 mL / dish) and kept on a warm bead bath. FIG. 11 provides an exemplary schematic showing the generation of SOBA filaments. To generate suspended SOBA filaments, the cell-BME solution was gently aspirated into a syringe with a 15-gauge blunt tip needle, then extruded directly into the warm media while moving the submerged needle in a linear, snake, or spiral motion in the X-Y plane. The extrusion speed and motion in the X-Y plane can affect the filament length and / or width. To generate SOBA fragments, SOBA filament cultures were gently triturated twice using a 10 mL serological pipette or wide bore Pl 000 pipette tip. Additional media was added to bring the final BME-to-media ratio to 1 : 10. Media changes were performed as described above for BOBA cultures.

[0356] Brightfield microscopy and image analysis. Cultures were imaged by brightfield microscopy using a THUNDER DMi8 inverted light microscope (Leica) with a 2.5X, 4X, or 10X objective, and a DFC9000 GTC camera (Leica). Images were analyzed using Imaris Image Analysis software and the Imaris Batch software package (Oxford Instruments). For automated organoid diameter measurements, the Imaris Surfaces detection module was used with inverted brightfield images. Background and out-of-focus organoids were excluded using a background subtraction step (rolling ball, 19.5 pm diameter). Detected surfaces were then filtered based on four criteria: a software assigned quality metric (>3000 A.U.), length of the minor axis (>35 pm - excludes debris), oblong circularity (>0.2 - excludes shadows), and length of the major axis (between 35 pm and 600 pm, excludes false detection of overlapping organoids). Of the remaining surfaces (at least 40 per image analyzed), the diameter is reported as the longest side of the smallest object- oriented bounding box. This process was then run in batch across all analyzed images. Averages per image were calculated, and the mean of three total experiments for n = 3 replicates were plotted. Spatial organoid uniformity organoid diameter analysis was performed using FIJI (ImageJ). A horizontal rectangle (1.5 mm x 9 mm) ROI was set across the center of each image and divided into 1 mm sections in the X-axis. For each section, diameters were measured manually across the widest point of each organoid.

[0357] Immunofluorescence sample preparation and confocal microscopy. 24- well Dome cultures were fixed with 2% paraformaldehyde (PF A) in PBS. Domes were detached from plate using a spatula, and transferred into microcentrifuge tubes using a cut P1000 pipette tip. For BOBA cultures, 500 pL of culture was transferred to a microcentrifuge tube using a cut Pl 000 pipette tip, the media was removed and 2% PF A in PBS was added. Samples were incubated in fixative at RT for 15-30 min, then washed 3X in PBS. Samples were stained in microcentrifuge tubes with primary antibodies diluted in Blocking / Permeabilization Buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS) for at least 4 h at RT, then washed 3X in PBS. Primary antibodies used are as follows: u-Ki67 (Invitrogen cat. no. MA5-14520), U-MUC2 (Millipore cat. no. MABF1989), u- FABP1 (Novus cat. no. NBP-87695), and u-CHGA (Novus cat. no. NB120-15160). Samples were then incubated with secondary antibodies (donkey u-rabbit Alexa Fluor 488 (ThermoFisher cat. no. A-21206) or goat a-mouse Alexa Fluor 594 (ThermoFisher cat. no. A- 11032)), with DAPI, and Al exaFluor 660 Phalloidin diluted in Blocking / Permeabilization Buffer at RT for at least 2 h at RT. Images were collected on a Stellaris 8 Confocal Microscope (Leica) using a 40X objective and 3D-reconstructed using Imaris Image Analysis software (Oxford Instruments).

[0358] Transcriptomic analysis. For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to either BME domes or pelleted BOB As and stored at -80°C. RNA isolation was performed using the QiaCube Connect (Qiagen) and RNA was quantified using a Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PEI 50) and analysis was performed by Novogene. Reads were aligned using HISAT2 (Mortazavi et al 2008), differential gene expression analysis was performed using DESeq2 (Anders et al. 2014), and statistical significance calculated using the negative binomial distribution model with Benjamini -Hochberg FDR correction.

[0359] 96-well plate suspended BME organoid variability. Colon organoid SOBA fragments in a 225 cm2flask were collected after 9 d culture and gently triturated twice using a serological pipette to homogenize the sample without disrupting intact organoids. The organoid mixture was transferred to a reagent reservoir and then plated at 100 pL / well in a 96-well plate using a P200 multichannel pipette with wide-bore pipette tips. Dome cultures were prepared as described above and plated in 5 pL domes in the center of each well of a 96-well plate using a repeater pipette. Cultures were grown for 7 d before viability measurements were performed. All viability measurements were performed using the Cell Titer Gio 3D Assay kit (Promega), and luminescence was measured on an Ensight plate reader (Perkin Elmer).

[0360] Statistical Analysis. All statistical analyses were performed using Prism 9 software (Graphpad) unless otherwise stated. Statistical tests, n, and p-values are indicated in figure legends.

[0361] Results:

[0362] A suspended BME hydrogel method for human intestinal organoid culture. The conventional intestinal organoid culture method requires that a solution of organoid cells in cold ECM is deposited onto a plastic surface, cured in an incubator to form a hydrogel dome, then overlaid with growth medium (FIGs. 1A-1B) (Mahe et al. (2013); Pleguezuelos-Manzano et al. (2020); Sato et al. (2009); Sato et al. (2011)). To address the limitations of scaling this technique, a method was developed in which cold cell-ECM solutions are cured instantly as floating hydrogels suspended in warm media. A method using suspended droplets of BME, an Engelbreth-Holm-Swarm (EHS) cell-derived ECM equivalent to MATRIGEL®, has been shown and is termed BOBA (BME-embedded Organoid Bead Assembly) culture.

[0363] Intestinal organoid growth was first evaluated in the suspended BOBA culture method and compared to the conventional surface-attached BME dome method, referred to here as Dome culture. For the Dome culture, single cells from digested organoids were suspended in cold BME and plated as 50 pL domes in a 24-well plate, cured for 15-30 minutes in a 37°C incubator, then overlaid with growth media (Intesticult Organoid Growth Media). Media changes were performed individually for each well every 2-3 days. For the BOBA culture, the single cell-BME solution was deposited using an electronic repeater pipette with wide-bore tips as 10 pL droplets directly into pre-warmed media, where the hydrogel cured immediately into suspended BOBAs. The BOBAs could be generated in petri dishes, plates, or conical tubes, and were easily transferred to larger vessels like cell culture flasks via serological pipette or decanting (FIGs. 1A-1B). Media changes were performed for an entire flask by allowing BOBAs to settle, then replacing the top 75% volume of spent media with fresh media.

[0364] Human intestinal organoid cell-BME solutions were plated in parallel Dome or BOBA cultures and grown for 9 days in growth media. Organoid growth and size were similar in both methods as observed by brightfield microscopy (FIG. 1C) and quantified with organoid diameter measurements (FIG. ID). Organoid cell proliferation was also comparable, as determined by quantifying the abundance of cells expressing proliferation marker Ki-67 (FIGs. IE- IF). The BOB A method appeared to enable more organoid cell growth per cm2surface area, although statistical significance was observed only for small intestine (ileum) organoids (FIGs. 1G-1H). For colon organoids, Dome culture yielded a mean of 2.9 x 105viable cells per well or 1.5 x 105cells / cm2in a 24-well plate, while BOBA culture yielded a mean of 2.2 x 107viable cells or 2.9 x 105cells / per cm2in a 75 cm2flask (Figure 1G). For ileum organoids, Dome culture yielded a mean of 4.8 x 105viable cells per well or 2.6 x 105cells / cm2in a 24-well plate, while BOBA culture yielded a mean of 2.9 x 107viable cells or 3.9 x 105cells / per cm2in a 75 cm2flask (FIG. 1H). The number of viable cells per pL BME hydrogel was similar, indicating that the amount of growth given a fixed seeding density is comparable for both methods (FIG. 1G).

[0365] Organoid differentiation in BOBA culture. A key benefit of the intestinal organoid model is the ability to differentiate into the various intestinal epithelial cell types by altering the media composition, for example by withdrawing stem cell-promoting factors (Clevers (2016); Schutgens and Clevers (2019); Zachos et al. (2016)). Differentiation of organoids in Dome and BOBA cultures were compared. Organoids were grown in growth media for 7 days, then washed and transitioned to differentiation media (Intesticut Organoid Differentiation Medium with 5 pM DAPT) for 5 days. Brightfield microscopy showed that in both culture formats, proliferative organoids in growth media exhibited a cyst morphology with a large lumen (FIG. 2A) and differentiated organoids exhibited a dense spheroid morphology with elongated columnar cells and a small lumen (FIG. 2A).

[0366] Bulk RNA-seq analysis was performed for proliferative and differentiated organoids in Dome and BOBA culture. In both culture methods, differentiated organoids downregulated expression of stem cell and proliferation markers (MKI67, LGR5, SOX9, and CD44) and upregulated expression of differentiation markers for goblet cells (MUC2, MUC5B, TFF3) and enterocytes (KRT20, FABP1, ALPI, and CEACAM7) relative to proliferative organoids (FIG. 2B). Differentiated cell types were also observed by immunofluorescence (IF) confocal microscopy in both culture formats (FIG. 2C).

[0367] Characterization and optimization of BOBA culture conditions. Next, how varying culture parameters would impact organoid growth in the BOBA method was evaluated. Cultures were seeded in either 6-well plates (FIGs. 3 A-3B) or 25 cm2flasks (FIG. 3C), with various volumes of BME and a fixed cell seeding density of 6 x 105cells / mL of BME. All BOB As were generated in 10 pL droplets with 5 mL of growth media, and organoid growth was evaluated by quantifying organoid diameters and viable cell numbers after 9 days of culture (FIGs. 3B-3C). In 6-well plates, as the total BME volume per well increased from 0.5 mL to 2 mL, the organoid diameters decreased and the ratio of viable cells to volume BME decreased (although not statistically significant). The total number of viable cells and ratio of viable cells per cm2surface area was comparable for all conditions, despite a higher number of seeded cells in the higher BME volume conditions, indicating that there was less proliferation per cell seeded. Together, these data suggest that organoid growth is impaired when the BME volume exceeds a threshold for a fixed amount of media in a 6-well plate (FIG. 3B).

[0368] Interestingly, for BOBA cultures in 25 cm2flasks, organoids grew equally well in all tested conditions (FIG. 3C). As the total BME volume per flask increased from 0.5 mL to 2 mL, the organoid diameters and the ratio of viable cells to volume BME were similar. The total number of viable cells and ratio of viable cells per cm2surface area increased as the volume of BME in the culture appeared to increase (although not statistically significant due to experiment-to-experiment variability), suggesting that the threshold for BME volume to media is different for the 6-well plate and 25 cm2flasks. Both BME-to-media ratio and vessel type should be optimized for specific applications.

[0369] Organoid uniformity in BOBA culture. The conventional Dome method is known to cause organoid heterogeneity (Pleguezuelos-Manzano et al. (2020); Ringel et al. (2020)). Native ECM hydrogels limit gas and molecular diffusion, resulting in nutrient gradients and heterogeneous organoid growth (Colom et al. (2014) J Biomed Mater Res A 102, 2776-2784; Park et al. (2022); Shin et al. (2020)). It was observed that organoids grow more uniformly in BOBA culture than in Dome culture (FIG. 4). Colon organoid cultures were imaged by brightfield microscopy at the deepest point in each format - the bottom z- plane of Domes or the center z-plane of BOB As - and the average organoid diameter across a rectangular ROI in the center of each hydrogel was quantified (FIG. 4C). In agreement with previous reports (Park et al. (2022); Shin et al. (2020)), Dome culture organoids grew larger at the hydrogel edges and smaller at the core (FIG. 4B-4E). However, organoids in BOB As grew to comparable sizes across the hydrogel (FIG. 4B-E).

[0370] It was also observed that the organoids in the core of the dome culture often had the dense spheroid lumen-less morphology that often indicates differentiation (FIG. 4B). Supporting the hypothesis that there may be a subpopulation of differentiated organoids in Dome cultures, bulk RNA-seq analysis indicated that Dome culture organoids had lower expression of stem cell and proliferation markers and higher expression of enterocyte markers relative to their BOBA culture counterparts (FIG. 5).

[0371] Alternative geometries for suspended BME hydrogels. While the BOBA method provides significant upscaling advantages over the conventional Dome method, massive culture expansion of suspended BOBA hydrogel droplets without an automated liquid handler can still be a labor-intensive endeavor. To reduce time and labor required for suspended BME hydrogel culture, an alternate hydrogel geometry was devised, specifically a hydrogel filament. Extruded filaments have been employed in the bioprinting field to spatially control cell growth or to build layer-by-layer assemblies of 3D hydrogel structures, but typically rely on attachment to a surface (Kolesky et al. (2014) Adv. Mater. 26, 2966- 2966; Kolesky et al. (2016) Proc National Acad Sci 113, 3179-3184). To generate an organoid cell-containing hydrogel filament, termed SOBA (Syringe-extruded Organoid BME Assembly), a cold cell-BME solution was loaded into a syringe with a 15-gauge (1.37 mm inner diameter) blunt tip needle, then injected directly into warm medium while moving the tip across the X-Y plane (e.g., in a linear, snake, or spiral pattern). Filament fragments which more closely resemble the BOBA geometry were also generated, termed SOBA fragments, by gentle trituration of SOBA cultures with a wide bore Pl 000 tip or a 10 mL serological pipette.

[0372] Organoids grown in BOBA, SOBA, or SOBA fragment cultures for 9 days showed similar growth determined by brightfield microscopy (FIG. 6A), organoid diameter measurements (FIGs. 6B-6C) and viable cell counts (FIG. 6D). Relative to BOBA droplets, SOBA and SOBA fragment formats yield comparable organoid growth while enabling faster and less labor-intensive culture preparation. Compared to SOBA cultures, SOBA fragments are more evenly dispersed in the media, thus allowing a single culture to be more easily divided, sampled, or aliquoted.

[0373] Discussion:

[0374] This example describes the development of a suspended BME hydrogel culture method for human intestinal epithelial organoids that overcomes several challenges associated with the conventional surface-attached Dome hydrogel culture method. Compared to the Dome method, the BOBA, SOBA and SOBA filament methods simplify and expedite the protocol, enable compatibility with scalable culture vessels, allow kinetic culture sampling and improve organoid culture uniformity.

[0375] Several suspension culture protocols have been proposed for intestinal organoid and tumoroid scale, however in these methods organoids were cultured in liquid media containing low concentrations of solubilized MATRIGEL® (similar to BME) which does not form an intact hydrogel (Hirokawa et al. (2021) Commun Biology 4, 1067; Price et al. (2022) Sci Rep-Uk 12, 5571). This contrasts with the presently disclosed method which relies on the formation of fully cured, insoluble hydrogel. Furthermore, previous studies showed that while organoids and tumoroids can be grown in a 5% solution of soluble MATRIGEL®, concentrations above 5% resulted in reduced growth. Moreover, the intestinal organoids grown in 5% MATRIGEL® exhibited inverted epithelial polarity (Hirokawa et al. (2021)), which is consistent with previous reports of organoid polarity reversal in low ECM conditions (Co et al. (2019) Cell Reports 26, 2509-2520. e4).

[0376] The BOBA method provides several advantages over the conventional Dome method. First, it simplifies the protocol. Since the hydrogel is cured directly in the media, it eliminates the need for a separate curing incubation step, thus saving time and labor. Next, it reduces the amount of vessel surface area and technical precision required to plate domes. By relieving reliance on available surface area, the BOBA method takes advantage of all 3 dimensions of the culture vessel, resulting in increased hydrogel volume and organoid cells per culture. The BOBA method facilitates substantial culture scale up because it enables compatibility with flasks, which are available in larger sizes, are easier to handle, and enable rapid media changes. For example, in BOBA culture, 10 mL BME can be cultured in a single 225 cm2flask and media can be changed by simply replacing the supernatant with a serological pipette. However, using the Dome method, nine 24-well plates (200 wells of 50 pL domes) would be required for an equivalent culture and media would need to be changed individually for each well. This also reduces the amount of plastic consumed by over 70% (A 225 cm2flask contains 152.7 g of plastic and replaces 562.5 g of plastic in nine 24-well plates, data not shown). Further scale-up can be achieved using multi-layer flasks or “cell factories” that can accommodate several liters of culture volume. General guidelines for the disclosed suspended BME hydrogel culture setup for several vessel types is provided in Table 1. Culture vessel type has been reported to impact parameters like gas transfer (Allen et al. (2001) Am I Physiol-Lung C 281, L1021-L1027), and factors such as media formulation, hydrogel composition, and organoid line-specific growth rate can all impact organoid growth. Table 1

[0377] Seeding conditions for suspended BME hydrogel cultures.

[0378] The BOB A method also overcomes culture heterogeneity that occurs from hydrogel diffusion limitations that occur in Dome cultures (Park et al. (2022); Shin et al. (2020)). Several protocols have suggested plating smaller 10-15 pL hydrogel domes (Pleguezuelos-Manzano et al. (2020); Stewart et al. (2020) Methods Mol Biology 2121, 185-198), thereby reducing the diffusion path for molecular transport. However, plating smaller domes, even when plating multiple domes per well, reduces the total hydrogel volume per well. Another approach to overcome hydrogel diffusion restriction is to invert the plates during the hydrogel curing step such that gravity causes the cells to settle near the top surface of the dome, with few or no cells in the dome core (Pleguezuelos-Manzano et al. (2020)). This results in suboptimal use of expensive hydrogel volume, and ultimately fewer organoid cells that can be seeded per pL of hydrogel. Complex bioengineered approaches have been developed to increase hydrogel surface area to improve molecular transport (Park et al. (2022)), but these are difficult to scale and still rely on anchoring the hydrogel to a 2D surface. The BOBA method generates cultures that are more homogeneous without sacrificing the amount of hydrogel per well or number of cells per pL of hydrogel. Without being limited to a particular theory, the observed improvement in organoid culture uniformity can be explained by the BOBA hydrogels having (1) smaller diameters and thus shorter diffusion paths and (2) all outer surfaces exposed to the media enabling even molecular diffusion into the hydrogel.

[0379] Another documented challenge with organoid morphology heterogeneity in hydrogel domes is that organoids located near the plate bottom can attach to the plate surface, spread and flatten, and lose their 3D structure (Pleguezuelos-Manzano et al. (2020); Price et al. (2022)). Since the suspended hydrogel droplets are not in direct contact with the plate surface, organoid spreading and flattening does not occur.

[0380] In addition to BOBA hydrogel droplets, organoids can be grown as SOBA filaments and SOBA filament fragments. Organoids grow similarly in all 3 configurations, demonstrating the robustness of the suspension BME culture method. The SOBA method facilitates and expedites culture preparation since a large volume of cell-BME solution can be loaded into a single syringe to produce SOBA hydrogel filaments. About 10 mL of SOBAs can be generated in under a minute, whereas an equivalent Dome culture would require over 15 minutes to plate and additional 15-30 minutes to cure. Although the SOBAs are much longer in length than the BOBAs, heterogeneity in organoid growth (like that observed in Dome cultures) was not observed, likely because the SOBA diameters are smaller (typically less than 2 mm) and enable efficient nutrient transport from the media. SOBA fragments more closely resemble BOBAs in size and geometry, but are much faster to generate. Like the BOBAs, the SOBA fragments are evenly dispersed throughout the culture, which can be useful for kinetic sampling or dividing a culture for multiple applications or readouts.

[0381] Overall, the suspended hydrogel culture method enables massive organoid scale up, improves organoid culture uniformity, and saves labor, time and resources. These culture improvements make intestinal organoids more amenable to implementation for high- throughput applications like compound or genomic screens. The method can likely be extended to culture diseased intestinal organoids and tumoroids, intestinal organoids from other species, and organoids from different tissue types. Thus, BOBAs, SOBAs and SOBA filaments have the potential to advance and expand adoption of organoid technology.

[0382] EXAMPLE 2: USE OF ORGANOIDS IN SCREENING METHODS

[0383] This example discloses the use of the intestinal organoids generated by the method of Example 1 in a cytotoxicity assay.

[0384] Methods:

[0385] 96-well plate suspended BME organoid cytotoxicity assay. Two 96-well plates were seeded from homogenized colon organoid SOBA fragments in a 225 cm2flask at 90 pl per well. Compound stocks were diluted to 10X final concentration in Intesticult OGM and 10 pL was added to each well. An 8-dose dilution series with 5-fold dilutions starting at 100 pM was evaluated in technical quadruplicates. After 3 d treatment, viability measurements were performed using the Cell Titer Gio 3D Assay kit (Promega), and luminescence was measured on an Ensight plate reader (Perkin Elmer). 4PL fit curves were generated using Prism (GraphPad).

[0386] Results:

[0387] Application of suspended BME hydrogel organoids in a drug toxicity screening assay. Intestinal organoids generated in BOBA, SOBA or SOBA fragment formats can be used directly in downstream assays without additional organoid digestion or passaging steps. As a proof of concept, the implementation of these organoids was demonstrated in a drug toxicity screen. SOBA fragment-cultured organoids were grown in a 225 cm2flask (FIGs. 7A-7B), homogenized by trituration with a serological pipette, then transferred to 96-well plates (FIGs. 7A-7C). Well-to-well variability, measured by the Cell Titer Gio 3D ATP -based viability assay, was comparable for SOBA filament-grown organoids and Dome cultures plated in a 96-well plate (FIG. 7D).

[0388] For the drug toxicity screen, SOBA filament-grown organoids in the 96-well plates were treated with compounds known to cause intestinal toxicity (diacerin, sorafenib, SN-38, or docetaxel) or a DMSO vehicle control for 3 days. Viability was determined as a measure of ATP and dose response curves were generated (FIG. 7E). This is one example of how suspended BME hydrogel cultures can be used directly in a downstream application.

[0389] The utility of the suspended BME hydrogel-grown cultures is demonstrated here in two applications. First, SOBA filament cultures could be homogenized to generate 96-well plate cultures with low well-to-well organoid variability. As a proof of concept, a drug toxicity screen was performed; this method can be used in other medium-to-high throughput screens.

[0390] EXAMPLE 3: GENERATION OF ORGANOID-DERIVED MODELS

[0391] This example discloses the use of the intestinal organoids generated by the method of Example 1 in providing alternative organoid-derived models.

[0392] Methods:

[0393] Transwell monolayers. Colon organoid SOBA fragments in a 225 cm2flask were collected in a 50-mL conical tube and pelleted at 800 x g for 3 minutes. Supernatant was removed and organoids were digested to single cells by incubation in TrypLE Express in a 37°C water bath for 10 min, then triturated with a P1000 pipette. Incubation and trituration was repeated as necessary up to 2X. Cells were pelleted and washed with PBS before resuspension in either Monolayer Growth Media (Intesticult OGM + 10 pM Y- 27632) or Monolayer Differentiation Media (Intesticult ODM + 10 pM Y-27632). For each well of a 96-well PET Transwell plate (pore size 0.4 pm, Coming cat. no. 3450), 2.0 x 105cells in 100 pL of media was seeded in the apical chamber and 200 pL media was added to the basal chamber. Media in both chambers was changed every 2-3 days. Brightfield imaging was performed using a 10X objective and THUNDER microscope (Leica) with a DFC9000 GTC camera (Leica). Transepithelial electrical resistance (TEER) was determined by measuring resistance with a volt / ohm meter (EVOM3, WPI), then multiplying resistance by the Transwell surface area (0.143 cm2).

[0394] Results:

[0395] Suspended BME hydrogel cultures facilitate alternative organoid-derived models. In addition to using organoids directly in their suspended BME hydrogel formats, these organoids can facilitate generation of other organoid-derived models, especially those that require large cell inputs like monolayers and microphy si ologi cal system (MPS) devices. Suspended BME hydrogel organoids were used to generate colon epithelial Transwell monolayers and evaluated epithelial barrier function in two conditions. SOBA filament organoids cultured in a 225 cm2flask were digested to single cells, seeded at confluence on 96-well Transwell inserts, and cultured in monolayer growth or monolayer differentiation media for 7 days (FIG. 8A). The two conditions resulted in Transwell epithelial cultures with different morphologies: in growth media the resulting epithelium formed 3D structures that protruded from the monolayer surface, while in differentiation media these structures were absent and a “cobblestone” cell morphology typical of differentiated epithelial cells with mature tight junctions was visible (FIG. 8B). Consistent with this morphology, the epithelial barrier function, quantified by transepithelial electrical resistance (TEER) measurements, was higher for Transwell monolayers in differentiation media than in growth media by day 3 (FIG. 8C). This experiment is an example of how organoids grown using the BOBA, SOBA, or SOBA filament methods can enable the production of alternative intestinal organoid-derived in vitro models.

[0396] SOBA filament organoids could be used to produce organoid-derived Transwell monolayers, which provide the advantage of simultaneous apical and basolateral access, but are difficult to scale up because they require large cell numbers to seed. By enabling substantial organoid expansion, the suspended BME hydrogel culture method can facilitate development and implementation of complex intestinal organoid-derived models with higher tissue fidelity and experimental advantages. EXAMPLE 4: GENERATION OF LUNG ORGANOIDS

[0397] This example discloses the generation of lung organoids using the BME suspension method described herein.

[0398] Methods:

[0399] Human Lung A TH cell isolation and Organoid Derivation. De-identified human lung tissue from a deceased donor was procured by Donor Network West. Lung dissociation into single cells and organoid establishment was previously described (Konishi et al., 2022). Briefly, the tissue was washed with HBSS buffer and inflated with digestion buffer (Collagenase type I: 450 units / mL; Dispase: 5 units / mL; DNase I: 10 units / mL). After removal of the pleura and small airways, the remaining tissue was minced with a single edge blade, transferred to a canonical tube containing warm digestion buffer and incubated with agitation and vigorous mixing every 15 minutes for a total of 1 hour at 37°C. The solution was passed through a 100 pm cell strainer and pelleted at 450g for 10 min at 4°C. The cell pellet was resuspended in 5 mL of ACK buffer to lyse red blood cells and incubated for 3- 5 min at room temperature. The reaction was stopped by adding DMEM / F12+10%FBS. The cell suspension was subsequently filtered through a 40 pm cell strainer and pelleted at 450g for 5 min at 4°C. Cells were then labeled with CD45 magnetic MicroBeads (Miltenyi Biotech, cat. no. 130-045-801) and loaded onto a MACS® Column which was placed in the magnetic field of a MACS Separator (according to the manufacture protocol). The magnetically labeled CD45+ cells were retained within the column and discarded, and the unlabeled cells that passed through the column were collected. These cells were washed in Gentle MACS buffer, pelleted at 450g for 5 min at 4°C and incubated with DMEM+10%FBS containing 50 nM Lysotracker green (Invitrogen, cat. no. L7526) for 30 min at 37°C. The following antibodies were added to the cells for 30 min on ice: preconjugated HTII280+ Alexa 647 (Terrance Biotech; Invitrogen cat. no. A20186); EpcamPE- Cy7 (BioLegend, cat. no. 324222); CD45-Alexa700 (BioLegend, cat. no. 304024), CD31- Alexa594 (BioLegend, cat. no. 303126). The live / dead staining probe Sytox blue (Invitrogen) was also added. Cells were then pelleted, washed, resuspended in PBS+2%FBS and ATII cells were isolated by sorting for Sytox blue-, CD45-, CD31-, EpCAM+, HTII- 280+, lysotracker green+. ATII cells were resuspended in MATRIGEL® (Growth Factor Reduced, Phenol Red-Free; Corning, cat. no. 356231), plated in 50 pl domes in 6-well plates (60 x 104cells / mL, or 3000 cells / 50 pl of MATRIGEL®), curated at 37°C for 30 minutes and then overlaid with 3 ml of serum-free feeder-free (SFFF) medium (Konishi et al., 2022) containing 10 pM of the ROCK inhibitor Y27632 (Selleckchem, cat. no. SI 049) for 3 days. After the first 2-3 days in culture, SFFF medium without ROCK inhibitor was changed every 2-3 days.

[0400] Organoid Maintenance. Organoid cultures were passaged every 10-14 days. To passage, cultures were first incubated with Accutase (Cell Stem Cell Technologies, cat. no. 07922) for 15 min to soften the MATRIGEL®. The domes and organoids were then collected in a 15 ml tube and incubated with agitation for 15 min at 37°C. After centrifugation at 450g for 5 min at 4oC, cells were treated with TrypLE Express (Gibco, cat. no. 12604-021) for 10 min at 37°C. TrypLE Express was inactivated by dilution with PBS, and cells were pelleted at 450 x g for 5 min at 4°C. Cells were resuspended on ice in MATRIGEL® at 60 x 104cells / mL, plated in 6-well plates in 50 pL domes and cured at 37°C for 30 min. Domes were overlaid with SFFF medium containing 10 pM of ROCK inhibitor for the first 3 days, and SFFF medium without ROCK inhibitor was then added and changed every 2-3 days.

[0401] Suspended hydrogel BOBA culture. Single organoid cells in MATRIGEL® were prepared on ice as described above. Pre-warmed SFFF medium containing 10 pM of ROCK inhibitor was added to 6-well plates (4 mL / well) and kept on a warm bath at 37°C. To generate suspended BME droplets, or BOB As, the organoid cell-MATRIGEL® solution was dispensed directly into the warm media in 10 pL volumes using an electronic repeater pipette (reference) with a wide-bore or cut pipette tip (opening about 2 mm). The tip was submerged immediately beneath the liquid surface during dispense, then lifted after each dispense to ensure droplet separation. After 3 days, SFFF media was added and changed every 2-3 days. For media exchange, a sterile 70 pm cell strainer was placed in the well, the plate was tilted and 3 mL media was gently aspirated through the strainer.

[0402] Results:

[0403] As shown in FIG. 9, lung organoids were successfully generated using alveolar type II (ATII) stem cells isolated from lung tissue embedded in hydrogel suspended in media.

[0404] EXAMP...

Claims

WHAT IS CLAIMED IS:

1. A method of generating tissue-derived epithelial organoids, comprising:(a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture;(b) suspending the hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture; and(c) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids.

2. The method of claim 1, wherein a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture.

3. The method of claim 2, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

4. The method of claim 2 or 3, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

5. The method of any one of claims 1-3, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

6. The method of any one of claims 1-5, wherein the hydrogel is solidified upon contact with the medium.

7. The method of any one of claims 1-6, wherein suspending the hydrogel -tissue- derived epithelial stem cell mixture in the medium comprises submerging a dispensing device containing the hydrogel-tissue-derived epithelial stem cell mixture in the medium and dispensing the hydrogel-tissue-derived epithelial stem cell mixture into the medium.

8. The method of any one of claims 1-7, wherein the temperature of the medium is from about 25°C to about 50°C.

9. The method of any one of claims 1-8, wherein the temperature of the medium isfrom about 30°C to about 50°C.

10. The method of any one of claims 1-9, wherein the temperature of the hydrogel- tissue-derived epithelial stem cell mixture is from about 2°C to about 25°C.

11. The method of any one of claims 1-10, wherein the temperature of the hydrogel- tissue-derived epithelial stem cell mixture is from about 2°C to about 20°C.

12. The method of any one of claims 1-11, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

13. The method of claim 12, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

14. The method of any one of claims 1-13, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

15. The method of any one of claims 1-14, wherein the suspended hydrogel -tissue- derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

16. The method of claim 15, wherein the suspended hydrogel -tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

17. The method of any one of claims 1-16, wherein the hydrogel -tissue-derived epithelial stem cell mixture is suspended in the medium in droplets.

18. The method of any one of claims 1-16, wherein the suspended hydrogel -tissue- derived epithelial stem cell mixture has a filament-like structure.

19. The method of claim 18, wherein the filament-like structure has a linear, snake, or spiral shape.

20. The method of any one of claims 1-19 further comprising fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

21. A method of generating a suspension culture of tissue-derived epithelial organoids,comprising:(a) introducing a mixture comprising a hydrogel and a tissue-derived epithelial stem cell into a medium to generate a suspended mixture; and(b) culturing the suspended mixture in the medium to generate the tissue- derived epithelial organoids in suspension.

22. The method of claim 21, wherein the mixture introduced into the medium comprises the hydrogel and a plurality of tissue-derived epithelial stem cells.

23. The method of claim 22, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

24. The method of claim 22 or 23, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

25. The method of any one of claims 21-23, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

26. The method of any one of claims 21-25, wherein the hydrogel is solidified upon contact with the medium.

27. The method of any one of claims 21-26, wherein introducing the mixture in the medium comprises submerging a dispensing device containing the mixture in the medium and dispensing the mixture into the medium.

28. The method of any one of claims 21-27, wherein the temperature of the medium is from about 25°C to about 50°C.

29. The method of any one of claims 21-28, wherein the temperature of the medium is from about 30°C to about 50°C.

30. The method of any one of claims 21-29, wherein the temperature of the mixture is from about 2°C to about 25°C.

31. The method of any one of claims 21-30, wherein the temperature of the mixture is from about 2°C to about 20°C.

32. The method of any one of claims 21-31, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

33. The method of claim 32, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

34. The method of any one of claims 21-33, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

35. The method of any one of claims 21-34, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

36. The method of claim 35, wherein the suspended mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

37. The method of any one of claims 21-36, wherein the mixture is introduced into the medium in droplets.

38. The method of any one of claims 21-36, wherein the mixture is introduced into the medium in filament-like structures.

39. The method of claim 38, wherein the filament-like structures have a linear, snake, or spiral shape.

40. The method of any one of claims 21-39 further comprising fragmenting the suspended mixture to generate fragmented structures comprising the tissue-derived epithelial organoids.

41. The method of any one of claims 1-40, wherein the medium is present in a container selected from the group consisting of a petri dish, a multi-well plate, a conical tube, a reservoir, a culture bag, a bioreactor or a flask.

42. A method of generating a suspension culture of tissue-derived epithelial organoids, comprising:(a) contacting a tissue-derived epithelial stem cell with a hydrogel to generate a hydrogel-tissue-derived epithelial stem cell mixture;(b) depositing the hydrogel-tissue-derived epithelial stem cell mixture onto a substrate;(c) solidifying the hydrogel-tissue-derived epithelial stem cell mixture to generate a solidified hydrogel-tissue-derived epithelial stem cell mixture;(d) suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in a medium to generate a suspended hydrogel-tissue-derived epithelial stem cell mixture; and(e) culturing the suspended hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate tissue-derived epithelial organoids.

43. The method of claim 42, wherein a plurality of tissue-derived epithelial stem cells is contacted with the hydrogel to generate the hydrogel-tissue-derived epithelial stem cell mixture.

44. The method of claim 43, wherein the plurality of tissue-derived epithelial stem cells comprises from about 1 x 104tissue-derived epithelial stem cells / ml of hydrogel to about 1 x 107tissue-derived epithelial stem cells / ml of hydrogel.

45. The method of claim 43 or 44, wherein the plurality of tissue-derived epithelial stem cells is comprised within a tissue fragment, an organoid fragment, or a combination thereof.

46. The method of any one of claims 42-44, wherein the tissue-derived epithelial stem cell or the plurality of tissue-derived epithelial stem cells is isolated from primary epithelial tissue.

47. The method of any one of claims 32-46 further comprising dislodging the solidified hydrogel-tissue-derived epithelial stem cell mixture from the substrate prior to suspending the solidified hydrogel-tissue-derived epithelial stem cell mixture in the medium.

48. The method of any one of claims 42-47, wherein the hydrogel is selected from the group consisting of a synthetic hydrogel, a native hydrogel, and a combination thereof.

49. The method of claim 48, wherein the native hydrogel comprises a basement membrane extract (BME) or an extracellular matrix (ECM) component.

50. The method of any one of claims 42-49, wherein the hydrogel has a storage modulus G’ equal to or greater than the loss modulus G”.

51. The method of any one of claims 42-50, wherein the solidified hydrogel-tissue- derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

52. The method of claim 51, wherein the solidified hydrogel-tissue-derived epithelial stem cell mixture has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

53. The method of any one of claims 42-52, wherein the hydrogel -tissue-derived epithelial stem cell mixture is deposited onto the substrate as a droplet.

54. The method of any one of claims 42-52, wherein the hydrogel-tissue-derived epithelial stem cell mixture is deposited onto the substrate to have a filament-like structure.

55. The method of claim 54, wherein the filament-like structure has a linear, snake, or spiral shape.

56. The method of any one of claims 42-55 further comprising fragmenting the hydrogel-tissue-derived epithelial stem cell mixture in the medium to generate fragmented structures comprising the tissue-derived epithelial organoids.

57. The method of any one of claims 1-56, wherein the tissue-derived epithelial organoids have uniform morphology compared to reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

58. The method of claim 57, wherein the tissue-derived epithelial organoids have uniform size.

59. The method of claim 58, where the average diameter of the tissue-derived epithelial organoids is more uniform than the reference tissue-derived epithelial organoids.

60. The method of any one of claims 1-59, wherein a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

61. The method of claim 60, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44 and a combination thereof62. The method of any one of claims 1-61, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue- derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

63. The method of claim 62, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

64. A tissue-derived epithelial organoid generated by the method of any one of claims 1-63.

65. A composition comprising a tissue-derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium.

66. The composition of claim 65, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

67. The composition of claim 66, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

68. The composition of any one of claims 65-67, wherein the hydrogel is a droplet.

69. The composition of any one of claims 65-67, wherein the hydrogel has a filamentlike structure.

70. The composition of claim 69, wherein the filament-like structure has a linear, snake, or spiral shape.

71. The composition of any one of claims 65-70, wherein a stem cell and / or proliferationmarker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

72. The composition of claim 71, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, CD49f, ASCL2, CD 133, LGR5, SOX9, ALDH1A1, NEUR0G3, NKX6.1, SM0C2, PDX1, CD44 and a combination thereof.

73. The composition of any one of claims 65-72, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

74. The composition of claim 73, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

75. A method for screening an agent, comprising:(a) contacting the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of claim 64 or the composition of any one of claims 65-74 with an agent; and(b) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids that is indicative of the effectiveness, disposition, and / or toxicity of the agent.

76. The method of claim 75, wherein the agent is contacted with the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids for about 15 minutes to about 3 years.

77. The method of claim 75 or 76, wherein the agent is a therapeutic agent.

78. The method of claim 77, wherein the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof.

79. A method for performing a genomic screen, comprising:(a) providing the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of claim 64 or the composition of any one of claims 65-74;(b) generating a mutation in the genome of one or more cells of the tissue- derived epithelial organoid; and(c) analyzing a change in the tissue-derived epithelial organoid or in the population of tissue-derived epithelial organoids associated with the mutation.

80. The method of claim 79, wherein the mutation is generated using a gene-regulating system.

81. The method of claim 80, wherein the gene-regulating system is a gene-editing system.

82. The method of claim 81, wherein the gene-editing system is a CRISPR system.

83. The method of any one of claims 75-82, wherein the change is a change in a property selected from the group consisting of cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

84. A method for generating an epithelial cell model, comprising:(a) providing the tissue-derived epithelial organoid or a population of the tissue-derived epithelial organoid of claim 64;(b) digesting the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids into single cells; and(c) culturing the single cells in a medium to generate a cell monolayer.

85. The method of claim 84, wherein the single cells are cultured on a permeable cell culture insert.

86. The method of claim 84 or 85, wherein the medium is a differentiation medium.

87. The method of claim 84 or 85, wherein the medium is a cell growth or stem cell promoting medium.

88. A method for screening an agent, comprising:(a) contacting the cell monolayer generated by the method of any one of claims 84-87 with an agent; and(b) analyzing a change in the cell monolayer that is indicative of the effectiveness, disposition, and / or toxicity of the agent.

89. The method of claim 88, wherein the agent is contacted with the cell monolayer for about 15 minutes to about 3 years.

90. The method of claim 88 or 89, wherein the agent is a therapeutic agent.

91. The method of claim 90, wherein the therapeutic agent is a polypeptide-based therapeutic, a small molecule therapeutic, a cell therapeutic, a gene-editing system, a nucleic acid-based therapeutic or a combination thereof.

92. A method for performing a genomic screen, comprising:(a) providing the cell monolayer generated by the method of any one of claims 84-87;(b) generating a mutation in the genome of one or more cells of the cell monolayer; and(c) analyzing a change in the cell monolayer associated with the mutation.

93. The method of claim 92, wherein the mutation is generated using a gene-regulating system.

94. The method of claim 93, wherein the gene-regulating system is a gene-editing system.

95. The method of claim 94, wherein the gene-editing system is a CRISPR system.

96. The method of any one of claims 84-95, wherein the change is a change in a property selected from the group consisting of cell viability, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modifications, post-translational modifications, activation of a cell signaling pathway, repression of a cell signaling pathway, enzymatic activity, barrier integrity, and a combination thereof.

97. The method of any one of claims 1-63 and 75-96 or the composition of any one of claims 65-74, wherein the tissue fragment is a fragment from a tissue selected from the group consisting of the lacrimal gland, the tonsils, the salivary gland, gastrointestinal tissue, the thyroid, the lung, the mammary gland, the liver, the bile duct, the stomach, the kidney, the pancreas, the endometrium, the fallopian tube, the cervix, the prostate, the bladder, the taste bud, the ovary, the placenta and a combination thereof.

98. The method of any one of claims 1-63 and 75-96 or the composition of any one of claims 65-74, wherein the tissue-derived epithelial stem cell is obtained from a fragment of an organoid selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

99. A system for culturing a tissue-derived epithelial organoid, comprising a tissue- derived epithelial organoid and a medium, wherein the tissue-derived epithelial organoid is embedded within a hydrogel suspended in the medium.

100. The system of claim 99, wherein the tissue-derived epithelial organoid is an intestinal organoid.

101. The system of claim 99 or 100, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter greater than about 0.1 mm.

102. The system of any one of claims 99- 101, wherein the hydrogel has a geometric shape comprising a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

103. The system of any one of claims 99-102, wherein the hydrogel is a droplet.

104. The system of any one of claims 99-102, wherein the hydrogel has a filament-like structure.

105. The system of claim 104, wherein the filam ent-like structure has a linear, snake, or spiral shape.

106. The system of any one of claims 99-105, wherein a stem cell and / or proliferation marker is expressed in a population of the tissue-derived epithelial organoids at a higher level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

107. The system of claim 106, wherein the stem cell and / or proliferation marker is selected from the group consisting of MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUR0G3, NKX6.1, SMOC2, PDX1, CD44 and a combination thereof.

108. The system of any one of claims 99-107, wherein a differentiation marker is expressed in a population of the tissue-derived epithelial organoids at a lower level compared to a population of reference tissue-derived epithelial organoids, wherein the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded within hydrogels attached to a substrate.

109. The system of claim 108, wherein the differentiation marker is selected from the group consisting of Keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, Keratin 19 (KRT19), Keratin 7 (KRT7), SOX9, MUC1, INS, GCG, AMY, ALB, CYP3A4, HNF4A, Cytokeratin 8 (K8), Cytokeratin 18 (KI 8), Cytokeratin 5 (K5), Cytokeratin 14 (KI 4), Smooth muscle Actin (SMA) and a combination thereof.

110. The system of any one of claims 99-109, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopiantube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

111. The system of any one of claims 99-110 comprising one or more robotic and / or automated components for generating and / or culturing the tissue-derived epithelial organoid.

112. The system of claim 111, wherein the one or more robotic and / or automated components comprise a liquid handling robot.

113. A system for performing the method of any one of claims 1-63 and 75-98 comprising one or more robotic and / or automated components.

114. The system of claim 113, wherein the one or more robotic and / or automated components comprise a liquid handling robot.

115. The method of any one of claims 1-63 and 75-98, wherein one or more of the steps of the method are performed by one or more robotic and / or automated components.

116. The method of claim 115, wherein the one or more robotic and / or automated components comprise a liquid handling robot.

117. The tissue-derived epithelial organoid of claim 64, wherein the tissue-derived epithelial organoid is selected from the group consisting of a lacrimal gland organoid, a tonsil organoid, a salivary gland organoid, a gastrointestinal organoid, a thyroid organoid, a lung organoid, a mammary gland organoid, a liver organoid, a bile duct organoid, a gastric organoid, a kidney organoid, a pancreatic organoid, an endometrial organoid, a fallopian tube organoid, a cervix organoid, a prostate organoid, a bladder organoid, an ovarian organoid, a taste bud organoid, a cytotrophoblast organoid and a combination thereof.

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