Method for producing tissue-derived epithelial organoids and their use

Abstract

This disclosure provides a method for preparing tissue-derived epithelial organoids. In particular, this disclosure provides tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. This disclosure further provides a method for using such organoids.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of U.S. Provisional Application No. 63 / 508,132, filed on June 14, 2023, the content of which is incorporated herein by reference in its entirety.

[0002] Field The subject matter disclosed herein relates to tissue - derived epithelial organoids and methods of manufacturing and using such organoids.

Background Art

[0003] Background Tissue - derived epithelial organoids are three - dimensional (3D) multicellular spheroids that recapitulate tissue complexity and function in vivo and have emerged as physiologically relevant in vitro models of tissue. For example, intestinal organoids, also called "enteroids" or "colonoids", are derived from adult stem cells isolated from primary intestinal tissue, can be easily propagated, and can be 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 can recapitulate the 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 fundamental scientific discoveries and facilitating translational applications in many fields such as cancer biology, infectious diseases, and cystic fibrosis (Clevers (2016); Schutgens and Clevers (2019) Annu Rev Pathology Mech Dis 15, 1 - 24).

[0004] The challenge in implementing tissue-derived epithelial organoids in fields such as drug development is that scaling existing organoid culture technologies is difficult and requires cumbersome manual methods or the development of highly automated infrastructure (Louey et al. (2021) Slas Discov 26, 1138-1147). Existing technologies involve resuspending tissue-derived epithelial stem cells (either isolated from primary tissue or passaged from established organoid cultures) in a cold extracellular matrix (ECM) solution, most often CULTREX® Basement Membrane Extract (BME) or MATRIGEL® hydrogel. The ECM is deposited on the surface of a plate, then heated to solidify the ECM cell solution, creating a hydrogel dome attached to the surface, which is then overlaid with culture medium. The culture medium in each well is changed every few days to allow organoids to form over a period of 1-2 weeks (Mahe et al. Curr Protoc Mouse Biology 3,217-240, Pleguezuelos-Manzano et al. (2020) Curr Protoc Immunol 130,e106, Sato et al. (2009) Nature 459,262-265, Sato et al. (2011) Gastroenterology 141,1762-1772). This technique is limited by the available surface area for hydrogel dome formation, is time-consuming, labor-intensive, prone to user error, and difficult to scale up because the diffusion limits 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, 431-440.e8, Shin et al. (2020) iScience 23, 101372). Therefore, there is a need in the art for more efficient and high-throughput methods for generating tissue-derived epithelial organoids. [Overview of the project]

[0005] overview The subject matter disclosed herein is tissue-derived epithelial organoids and methods for producing such organoids. This disclosure further provides methods for using tissue-derived epithelial organoids and systems for carrying out the methods disclosed herein.

[0006] In certain embodiments, a method for generating tissue-derived epithelial organoids includes (a) contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and tissue-derived epithelial stem cells, (b) suspending the mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of the hydrogel and tissue-derived epithelial stem cells, and (c) culturing the suspended mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce tissue-derived epithelial organoids. In certain embodiments, a mixture of the hydrogel and tissue-derived epithelial stem cells is produced by contacting a plurality of tissue-derived epithelial stem cells with a hydrogel. In certain embodiments, the method further includes fragmenting the suspended mixture of the hydrogel and tissue-derived epithelial stem cells to produce a fragmented structure containing tissue-derived epithelial organoids.

[0007] In certain embodiments, the hydrogel solidifies upon contact with the culture medium. In certain embodiments, suspending the mixture of hydrogel and tissue-derived epithelial stem cells in the culture medium involves immersing a dispensing device containing the mixture of hydrogel and tissue-derived epithelial stem cells in the culture medium and dispensing the mixture of hydrogel and tissue-derived epithelial stem cells into the culture medium. In certain embodiments, the temperature of the culture medium is approximately 25°C to approximately 50°C. In certain embodiments, the temperature of the culture medium is approximately 30°C to approximately 50°C. In certain embodiments, the temperature of the culture medium is approximately 30°C to approximately 40°C. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is less than approximately 20°C. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 25°C. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 20°C. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 10°C.

[0008] This disclosure further provides a method for producing a turbid culture of tissue-derived epithelial organoids. In certain embodiments, the method comprises (a) introducing a mixture containing a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspension mixture, and (b) culturing the suspension mixture in the culture medium to produce turbid tissue-derived epithelial organoids. In certain embodiments, the mixture introduced into the culture medium comprises a hydrogel and a plurality of tissue-derived epithelial stem cells. In certain embodiments, the method comprises (a) introducing a mixture containing a hydrogel and a plurality of tissue-derived epithelial stem cells into a culture medium to produce a suspension mixture, and (b) culturing the mixture in the culture medium to produce turbid tissue-derived epithelial organoids. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. In certain embodiments, introducing the mixture into the culture medium comprises immersing a dispensing device containing the mixture into the culture medium to dispense the mixture into the culture medium. In certain embodiments, the temperature of the culture medium is approximately 25°C to approximately 50°C. In certain embodiments, the temperature of the culture medium is approximately 30°C to approximately 50°C. In certain embodiments, the temperature of the culture medium is approximately 25°C to approximately 40°C. In certain embodiments, the temperature of the culture medium is 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 about 2°C to about 25°C. In certain embodiments, the temperature of the mixture is about 2°C to about 20°C. In certain embodiments, the temperature of the mixture is about 2°C to about 10°C. In certain embodiments, the method further includes fragmenting the suspended mixture to generate fragmented structures containing tissue-derived epithelial organoids.

[0009] In a particular embodiment, a method for producing a turbid culture of tissue-derived epithelial organoids includes (a) contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and tissue-derived epithelial stem cells; (b) depositing the mixture of the hydrogel and tissue-derived epithelial stem cells onto a substrate; (c) solidifying the mixture of the hydrogel and tissue-derived epithelial stem cells to produce a solidified mixture of the hydrogel and tissue-derived epithelial stem cells; (d) suspending the solidified mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of the hydrogel and tissue-derived epithelial stem cells; and (e) culturing the suspended mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce tissue-derived epithelial organoids. In a particular embodiment, a mixture of the hydrogel and tissue-derived epithelial stem cells is produced by contacting a plurality of tissue-derived epithelial stem cells with a hydrogel. For example, but not limited to, the method includes (a) contacting a plurality of tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and tissue-derived epithelial stem cells; (b) depositing the mixture of the hydrogel and tissue-derived epithelial stem cells onto a substrate; (c) solidifying the mixture of the hydrogel and tissue-derived epithelial stem cells to produce a solidified mixture of the hydrogel and tissue-derived epithelial stem cells; (d) suspending the solidified mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of the hydrogel and tissue-derived epithelial stem cells; and (e) culturing the suspended mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a tissue-derived epithelial organoid. In certain embodiments, the method further includes removing the solidified mixture of the hydrogel and tissue-derived epithelial stem cells from the substrate before suspending the mixture in a culture medium.

[0010] In certain embodiments, a mixture of hydrogel and tissue-derived epithelial stem cells is deposited on a substrate as droplets. In certain embodiments, a mixture of hydrogel and tissue-derived epithelial stem cells is deposited on a substrate so as to have a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, the method further comprises fragmenting the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to generate a fragmented structure containing tissue-derived epithelial organoids.

[0011] In certain embodiments, the mixture of suspended hydrogel and tissue-derived epithelial stem cells, the suspension mixture, or the solidified hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the mixture of suspended hydrogel and tissue-derived epithelial stem cells, the suspension mixture, or the solidified hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1,000 mm, for example, about 0.1 mm to about 20 mm. In certain embodiments, the mixture of suspended hydrogel and tissue-derived epithelial stem cells, the suspension mixture, or the solidified hydrogel and tissue-derived epithelial stem cells is in a droplet. In certain embodiments, the mixture of suspended hydrogel and tissue-derived epithelial stem cells, the suspension mixture, or the solidified hydrogel and tissue-derived epithelial stem cells has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape.

[0012] In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells are contained within a tissue fragment, organoid fragment, or a combination thereof. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue. In certain embodiments, tissue-derived epithelial stem cells are obtained from tissue fragments selected from the group consisting of lacrimal glands, tonsils, salivary glands, gastrointestinal tissue, thyroid gland, lungs, mammary glands, liver, bile ducts, stomach, kidneys, pancreas, endometrium, fallopian tubes, cervix, prostate, bladder, ovaries, taste buds, placenta, and combinations thereof. Alternatively or additionally, tissue-derived epithelial stem cells may be obtained from fragments of organoids selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblast organoids, and combinations thereof.

[0013] In a particular embodiment, multiple tissue-derived epithelial stem cells are approximately 1 × 10⁶ 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7 Contains 1 ml of tissue-derived epithelial stem cell / hydrogel.

[0014] In certain embodiments, the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof. In certain embodiments, the natural hydrogel contains a basement membrane extract (BME) component 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 contains a BME component, an ECM component, or a polymer in w / v% greater than about 1 w / v%. In certain embodiments, the hydrogel has a storage modulus G' greater than or equal to a loss modulus G''.

[0015] In certain embodiments, the culture medium is contained within a container. In certain embodiments, the container is a petri dish, a multiwell plate, a conical tube, a reservoir, a culture bag, a bioreactor, or a flask.

[0016] This disclosure further provides tissue-derived epithelial organoids produced by the methods disclosed herein.

[0017] In certain embodiments, tissue-derived epithelial organoids produced by the method of this disclosure have a more uniform morphology compared to reference tissue-derived epithelial organoids. In certain embodiments, the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In certain embodiments, the tissue-derived epithelial organoids have a uniform size. In certain embodiments, the average diameter of the tissue-derived epithelial organoids is more uniform than that of the reference tissue-derived epithelial organoids.

[0018] In certain embodiments, stem cell markers and / or proliferation markers are expressed at higher levels in a population of tissue-derived epithelial organoids produced by the method of this disclosure compared to a population of reference tissue-derived epithelial organoids. In certain embodiments, differentiation markers are expressed at lower levels in a population of tissue-derived epithelial organoids produced by the method of this disclosure 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 in a hydrogel attached to a substrate. In certain embodiments, the stem cell markers and / or proliferation markers are selected from the group consisting of MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0019] The Disclosure further provides a composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter greater than about 0.1 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width and / or diameter of about 0.1 mm to about 1,000 mm, for example, about 0.1 mm to about 20 mm. In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine or spiral shape. In certain embodiments, stem cell markers and / or proliferation markers are expressed at a higher level in the population of tissue-derived epithelial organoids compared to the population of reference tissue-derived epithelial organoids. In certain embodiments, the stem cell marker 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 combinations thereof. In certain embodiments, the differentiation marker is expressed at lower levels in the tissue-derived epithelial organoid population compared to the reference tissue-derived epithelial organoid population. 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate.

[0020] This disclosure further provides a method for screening drugs, such as therapeutic agents. In certain embodiments, the method includes (a) contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids with a drug, such as a therapeutic agent, and (b) analyzing changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids that indicate the efficacy, pharmacokinetics and / or toxicity of the drug, such as a therapeutic agent. In certain embodiments, the drug, such as a therapeutic agent, is contacted with the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids for about 1 minute to about 3 years, for example, about 15 minutes to about 3 years. In certain embodiments, the drug is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In certain embodiments, the changes are those of properties selected from the group consisting of cell viability, cell metabolism, reduction potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression levels, nucleic acid expression levels, nucleic acid modifications, post-translational modifications, activation of cell signaling pathways, suppression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

[0021] This disclosure further provides methods for performing genome screening. In certain embodiments, the method includes (a) preparing a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) inducing a mutation in the genome of one or more cells of the tissue-derived epithelial organoids, and (c) analyzing the changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids associated with the mutation. In certain embodiments, the mutation is induced using a gene regulatory system. In certain embodiments, the gene regulatory 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, reduction potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

[0022] This disclosure further provides a method for generating an epithelial cell model. In certain embodiments, the method includes (a) preparing tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids or a composition of tissue-derived epithelial organoids, (b) digesting the tissue-derived epithelial organoids or population of tissue-derived epithelial organoids into single cells, and (c) culturing the single cells in a culture medium to produce a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the culture medium is a differentiation medium. In certain embodiments, the culture medium is a cell proliferation medium. In certain embodiments, the culture medium is a stem cell promoting medium.

[0023] This disclosure further provides a screening method using a cell monolayer produced by the method described herein. In certain embodiments, the screening method is a method for screening a drug, for example, a therapeutic agent. For example, but not limited to, the method may include (a) contacting a cell monolayer produced by the method described herein with a drug, for example, a therapeutic agent, and (b) analyzing changes in the cell monolayer that indicate the efficacy, pharmacokinetics, and / or toxicity of the drug, for example, a therapeutic agent. In certain embodiments, the drug, for example, a therapeutic agent, is contacted with the cell monolayer for about 1 minute to about 3 years, for example, for about 15 minutes to about 3 years. In certain embodiments, the drug is a therapeutic agent. In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell-based therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof. In certain embodiments, the screening method is genome screening. In certain embodiments, a method for carrying out genome screening may include (a) preparing a cell monolayer produced by the method described herein, (b) inducing a mutation in the genome of one or more cells in the cell monolayer, and (c) analyzing changes in the cell monolayer associated with the mutation. In certain embodiments, the mutation is induced using a gene regulatory system. In certain embodiments, the gene regulatory system is a gene editing system. In certain embodiments, the gene editing system is a CRISPR system. In certain embodiments, the changes are changes in properties selected from the group consisting of cell viability, cell metabolism, reduction potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression levels, nucleic acid expression levels, nucleic acid modifications, post-translational modifications, activation of cell signaling pathways, repression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

[0024] In certain embodiments, one or more steps of the methods of the present disclosure may be carried out using one or more robots and / or automated components. For example, but not limited to, one or more steps of the methods described herein for generating tissue-derived epithelial organoids, generating turbid cultures of tissue-derived epithelial organoids, screening drugs, performing genome screening, generating epithelial cell models, and / or screening methods using cell monolayers may be carried out using one or more robots and / or automated components. In certain embodiments, one or more robots and / or automated components are selected from the group consisting of liquid handling robots, 3D printers, syringe pumps, and combinations thereof. In certain embodiments, one or more robots and / or automated components include liquid handling robots.

[0025] This disclosure further provides a system for culturing tissue-derived epithelial organoids. In certain embodiments, the system comprises tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium. In certain embodiments, the hydrogel has a geometric shape having a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the hydrogel has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1,000 mm, for example, about 0.1 mm to about 20 mm. In certain embodiments, the hydrogel is a droplet. In certain embodiments, the hydrogel has a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, stem cell markers and / or proliferation markers are expressed at higher levels in the population of tissue-derived epithelial organoids compared to the population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate. In certain embodiments, the stem cell marker 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 combinations thereof. In certain embodiments, the differentiation marker is expressed at lower levels in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.In certain embodiments, the tissue-derived epithelial organoids are selected from the group consisting of lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblast organoids, and combinations thereof. In certain embodiments, the system includes one or more robots and / or automated components for generating and / or culturing the tissue-derived epithelial organoids. In certain embodiments, the one or more robots 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 robots and / or automated components include a liquid handling robot.

[0026] This disclosure further provides systems for carrying out the methods disclosed herein. For example, but not limited to, this disclosure provides systems for carrying out methods for generating tissue-derived epithelial organoids, methods for generating turbid cultures of tissue-derived epithelial organoids, methods for screening drugs, methods for performing genome screening, methods for generating epithelial cell models and / or screening methods using cell monolayers. In certain embodiments, the system includes one or more robots and / or automated components for generating and / or culturing tissue-derived epithelial organoids. In certain embodiments, the one or more robots 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 robots and / or automated components include liquid handling robots. [Brief explanation of the drawing]

[0027] Brief explanation of the drawing [Figure 1A-1H]Figures 1A to 1H show the scale-up of intestinal organoids in suspended BME hydrogel. (A) schematic diagram, (B) photograph, and (C) bright-field microscope image of human colon organoids in conventional dome culture (top) and suspended BOBA (BME-embedded organoid bead assembly) culture (bottom). The scale bar in the microscope image is 500 μm. (D) Organoid diameter in dome culture and BOBA culture in bright-field image. (E) Percentage of Ki67-positive cells in confocal image. The data shown are mean ± SD, Student's t-test, n=3, and 10 fields of view each. (F) 3D reconstructed confocal image of colon organoids in dome culture or BOBA culture. Ki67 is green, nuclei are blue, actin is white, and the scale bar is 20 μm. (G, H) Total viable cell count, cell count per 1 cm² surface area, or cell count per 1 μL BME for (G) colon organoids and (H) ileal organoids. All data presented are mean ± SD, Student's t-test *p ≤ 0.05, ****p ≤ 0.0001, n=3 experiments. [Figure 2A-2C] Figures 2A-2C show organoid differentiation in BOBA cultures. (A) Bright-field images of colon organoids in dome cultures (top) and BOBA cultures (bottom) show similar morphologies for proliferative and differentiated organoids. Scale bar is 100 μm. (B) Bulk RNA-seq shows that dome-cultured and BOBA-cultured colon organoids similarly downregulate stem and precursor markers and upregulate differentiation markers after transfer from growth medium to differentiation medium. (C) Markers for differentiated epithelial cell types (MUC2 in goblet cells, FABP1 in intestinal cells, and CHGA in enteroendocrine cells) are expressed in organoids cultured in both dome format (top) and BOBA format (bottom). Nuclei are blue, actin is white, scale bar is 10 μm. [Figure 3A-3C]Figures 3A to 3C show that BME volume and culture vessel can affect organoid growth in suspended BME hydrogel cultures. (A) Bright-field images of surface-attached domes (50 μL of BME in 0.5 mL of medium) in a 24-well plate or colon organoids (0.5, 1, or 2 mL of BME in 5 mL of medium) in BOBA cultures in wells of a 6-well plate. Scale bar is 200 μm. (B, C) Quantification of organoid diameter, total viable cell count, viable cell count per cm² surface area and viable cell count per μL of BME in (B) a 6-well plate or (C) a 25 cm² flask. Data shown are mean ± SD, one-way ANOVA multiple comparison study, n=3 experiments, *p≦0.05, **p≦0.01, ***p≦0.001, ****p≦0.0001. [Figure 4A-4E] Figures 4A to 4E show the uniformity of organoid size in BOBA cultures. (A) A schematic diagram illustrating the imaging strategy for uniformity analysis. (B, C) Bright-field images of colon organoids in the deepest plane in a 50 μl BME dome or a 10 μl suspended BOBA hydrogel. Scale bars are (B) 200 μm and (C) 1 mm. (D) Quantification of organoid diameter in the horizontal ROI of the dome or BOBA hydrogel at positions across the X-axis. Data shown are mean ± SD, with n=3 replicates in representative experiments from three experiments. (E) Quantification of mean organoid diameter at the edge or core of dome and BOBA cultures. All data shown are mean ± SD, two-way ANOVA, with n=3 replicates in representative experiments from three experiments. [Figure 5A-5B]Figures 5A and 5B show gene expression in organoids in dome cultures and BOBA cultures. (A, B) Bulk RNA-seq analysis shows differences in gene expression of (A) stem cells and growth markers and (B) intestinal cell markers between colon organoids in dome cultures or suspension BOBA cultures after 7 days of culture in growth medium. The data shown are mean ± SD for normalized counts measured by DESeq2, with n=3 replicates and statistical analysis using a negative binomial distribution model with BH-adjusted p-values, *padj ≤ 0.05, **padj ≤ 0.01. [Figures 6A-6D] Figures 6A–6D show that different suspension BME hydrogel culture formats exhibit comparable organoid proliferation. (A) Photographs and (B) Bright-Field Images of colon organoids in 6-well plate cultures in BOBA format, SOBA (Syringe Extrusion Organoid BME Assembly) format, or SOBA Fragment format. Alternative formats allow for rapid culture preparation for scale-up. Scale bar is 1 mm. (C) Quantification of organoid diameter and (D) Number of viable cells per well. Data shown are mean ± SD, one-way ANOVA multiple comparison test, n=3 experiments. [Figures 7A-7E]Figures 7A to 7E illustrate the application of suspended BME hydrogel organoid cultures in medium-throughput screening. (A) Schematic diagram of the experiment. SOBA fragment organoid cultures in a 225 cm² flask were triturated to obtain a homogeneous organoid suspension, which was then seeded into a 96-well plate. (B) Bright-field image of SOBA fragment cultures cultured in a 225 cm² flask. Scale bar is 1 mm. (C) Bright-field images of randomly selected wells across the 96-well plate show comparable inter-well organoid density. Scale bar is 1 mm. (D) Cell Titer Glo 3D (CTG) viability readouts show similar inter-well variability between dome cultures and suspended BME cultures in the 96-well plate. Data shown are mean ± SD. Student's t-test. (E) Representative dose-response viability curves (CTG assay) of suspended BME organoids treated with diaseline, sorafenib, SN-38, or docetaxel for 3 days. The data presented is mean ± standard deviation, with n=4. [Figures 8A-8C] Figures 8A-8C show the use of suspended BME hydrogel organoids to generate Transwell monolayers. (A) Schematic diagram of the experiment. SOBA fragment organoid cultures in a 225 cm² flask were digested into a single-cell suspension and then seeded confluence into 96-well BME-coated Transwell inserts. (B) Bright-field image of SOBA fragment organoid-derived Transwell monolayers 3 days after seeding. Transwells were established using monolayer growth medium or monolayer differentiation medium. Scale bar is 100 μm. (C) Transepithelial electrical resistance (TEER) of Transwell monolayers cultured in monolayer growth medium (black circles) or monolayer differentiation medium (white circles). Data shown are mean ± SD and n=6 wells. [Figure 9] Figure 9 shows an image of a lung AT2 organoid embedded in a hydrogel suspended in a culture medium within a BOBA culture. [Figure 10]Figure 10 shows a schematic diagram illustrating exemplary conditions for generating hydrogel droplets, which are referred to herein as basement membrane organoid bead aggregates (BOBAs). Organoids or dissociated organoid cells suspended in a low-temperature liquid hydrogel are dispensed as droplets into a culture medium at room temperature or higher, thereby allowing the hydrogel to harden immediately upon dispensing into the medium. [Figure 11] Figure 11 shows a schematic diagram illustrating exemplary conditions for generating hydrogel filaments, which are referred to herein as syringe-extruded organoid basement membrane assemblies (SOBAs). Organoids or dissociated organoid cells suspended in a low-temperature liquid hydrogel are extruded as filaments into a culture medium at room temperature or higher (by syringe, pipette, or any other dispensing device), allowing the hydrogel to harden immediately upon dispensing into the culture medium. [Modes for carrying out the invention]

[0028] Detailed explanation This disclosure provides tissue-derived epithelial organoids in turbid cultures and a method for producing such tissue-derived epithelial organoids. The method of this disclosure enables the production of large-scale organoid cultures without requiring cumbersome manual handling, specialized equipment, or automation. By growing organoid cells in a suspended hydrogel instead of a conventional surface-adhered hydrogel dome, the volume of the hydrogel and, consequently, the number of organoid cells that can be grown in the culture vessel can be significantly increased. Furthermore, because the method of this disclosure does not require the deposition of hydrogel on a two-dimensional (2D) surface, it is compatible with various culture vessels, such as culture flasks and culture bags, enabling further culture scale-up and resulting in high throughput.

[0029] The method disclosed herein allows for the use of suspension hydrogels of various geometric shapes and can reduce the actual culture preparation time. Furthermore, because the organoids are in a suspended state, they can be sampled, divided, or collected at various points in the culture, which is difficult with conventional organoid cultures immobilized on plates. As shown in Example 1, organoids in suspension BME hydrogels grow more uniformly than conventional surface-attached hydrogel domes, where limited molecular diffusion results in a nutrient gradient (Park et al. (2022), Shin et al. (2020)). The suspension hydrogel culture method disclosed herein provides a tissue-derived epithelial organoid model that is more suitable for high-throughput research, benefiting both basic science and translational fields.

[0030] For clarity, and without limitation, a detailed description of the subject matter disclosed herein is divided into the following subsections. I. Definition, II. Organoids and their compositions, III. Method for manufacturing organoids, IV. How to use, V. System, and VI. Exemplary Embodiments

[0031] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field of the subject matter of this disclosure. The following references provide general definitions of many of the terms used herein: 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). Where used herein, the following terms have the meanings set forth below unless otherwise specified.

[0032] As used herein, the use of the words “a” or “an” may mean “one” when used in combination with the term “comprising” in the claims and / or specification, but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.”

[0033] The terms “about” or “approximately” mean that a particular value is within an acceptable margin of error, as determined by those skilled in the art, and this depends to some extent on the method by which the value is measured or determined, i.e., on the limitations of the measurement system. For example, “about” may mean a standard deviation of 3 or more, according to convention in the art. Alternatively, “about” may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably up to 1% of a given value. Or, particularly with respect to biological systems or processes, the term may mean within one order of magnitude of the value, for example, within 5 times, or within 2 times.

[0034] The term "antibody" is used herein in its broadest sense and is not limited thereto, but encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0035] An "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments, though not limited to them, include Fv, Fab, Fab', and Fab'-SH, which are multispecific antibodies formed from F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and antibody fragments.

[0036] As used herein, the terms “culture medium” or “culture medium” refer to a liquid containing nutrients that surrounds and nourishes cells in a culture vessel (such as a culture flask and a multiwell plate). In certain embodiments, the culture medium may also contain growth factors or differentiation factors that induce desired changes in the cells.

[0037] As used herein, the terms “comprise(s) / include(s),” “having / has,” “can,” and “contain(s),” and their variations, are intended to be open-ended transitional phrases, terms, or words that do not preclude any additional acts or structures. This disclosure also considers other embodiments that “comprising,” “consisting of,” and “consisting essentially of” the embodiments or elements presented herein, whether expressly provided for.

[0038] As used herein, the term “contacting” cells with a compound, e.g., a therapeutic agent, means exposing cells to the compound, e.g., placing the compound in a position where it can be brought into contact with the cells. Contact can be achieved using any suitable method. For example, but not limited to, “contact” can be achieved by adding the compound to a container containing cells. Contact can also be achieved by adding the compound to a culture medium containing cells. In certain embodiments, “contact” means exposing cells, e.g., cells within tissue-derived epithelial organoids or cell monolayers, to a drug or compound. In certain embodiments, “contact” means exposing tissue-derived epithelial organoids or cell monolayers to the desired potential therapeutic agent or drug.

[0039] As used herein, the terms “derived from,” “established from,” or “differentiated from,” when made in reference to any cells disclosed herein, refer to cells obtained (e.g., isolated or purified) from parental cells in a cell line, tissue (such as dissociated tissue), or body fluid using any operation. Non-limiting examples of such operations include single-cell isolation, in vitro culture, treatment and / or mutagenesis using transfection with proteins, chemicals, radiation, viral infection, and nucleic acids, etc. In certain embodiments, derived cells may be selected from a mixed population in response to growth factors, cytokines, selected progression of cytokine therapy, adhesion, lack of adhesion, sorting procedures, or combinations thereof.

[0040] The terms "detection" or "the act of detecting" include any means of detection, including direct and indirect detection.

[0041] As used herein, the term "droplet," when used in relation to geometric shape, refers to a spherical or spherical shape.

[0042] As used herein, the terms “embedding” or “embedded” mean to cover or surround a cell at least partially. In certain embodiments, as used herein, the terms “embedding” or “embedded” mean to completely cover or surround a tissue-derived epithelial organoid with, for example, a hydrogel.

[0043] As used herein, the terms “expression” or “to express” refer to the transcription and / or translation of a nucleotide sequence.

[0044] The term “expression vector” is used to describe a nucleic acid molecule that is either linear or circular and can incorporate another nucleic acid sequence fragment of appropriate size. Such a nucleic acid fragment may contain additional segments that provide transcription of the gene encoded by the nucleic acid sequence fragment. Additional segments may include, but are not limited to, promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selection markers, and origins of replication, as are known in the art. Expression vectors are often derived from plasmids, cosmids, and viral vectors, and vectors are often recombinant molecules containing nucleic acid sequences from several sources.

[0045] As used herein, the term “gastrointestinal tract” refers to the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine, and rectum.

[0046] As used herein, the term “gastrointestinal stem cells” refers to stem cells of the gastrointestinal system.

[0047] As used herein, the terms “individual” or “subject” refer to vertebrates or invertebrates, such as humans or non-human animals, such as mammals. Mammals include, but are not limited to, humans, non-human primates, livestock, sports animals, rodents, and pets. Non-exclusive 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.

[0048] As used herein, the terms “intestinal” or “intestine” refer to the rectum, small intestine, and large intestine.

[0049] As used herein, the term "intestinal stem cells" refers to stem cells of the intestines.

[0050] As used herein, the term "in vitro" refers to an artificial environment and any process or reaction occurring within it. Examples of in vitro environments include, but are not limited to, cell cultures.

[0051] As used herein, the term "in vivo" refers to a natural environment (e.g., an animal or a cell) and processes or reactions that occur within that natural environment.

[0052] As used herein, the term “linear,” when used in reference to geometric shapes, refers to a shape similar to a line. In certain embodiments, a line may be a straight line, a curve, or a line of any shape.

[0053] As used herein, the term “isolated” with respect to cells, for example, gastrointestinal stem cells, refers to cells isolated from components of their natural environment.

[0054] As used herein, “marker” refers to a drug that enables direct or indirect detection. Examples of markers include, but are not limited to, fluorescent labels, dye labels, high-density electron labels, chemiluminescent labels, and radioactive labels. Non-exclusive 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 herein by reference). 32 P, 14 C, 125 I, 3 H and 131 I. Fluorescent substances (such as rare earth chelates or Lucifer Yellow and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferases (such as firefly luciferase and bacterial fluorescent plain enzymes) (U.S. Patent No. 4,737,456), fluorescein, 2,3-dihydrophthalazine diketone, and enzymes that produce detectable signals, such as horseradish peroxidase (HRP), alkaline phosphatase enzymes, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase (G6PD)), and heterocyclic oxidases (such as uricase and xanthine oxidase).

[0055] The terms “nucleic acid” or “polynucleotide” encompass all compounds and / or substances containing polymers of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Often, nucleic acid molecules are described by their base sequence, which represents the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented 5' to 3'. The term nucleic acid encompasses, for example, deoxyribonucleic acid (DNA), such as complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), such as messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acids described herein may contain naturally occurring or non-natural nucleotides. Examples of non-natural nucleotides include modified nucleotide bases having derivatized sugars or phosphate backbone links or chemically modified residues.

[0056] The term "operatably linked," when applied to nucleic acid sequences in an expression vector, indicates that the sequences are arranged to work together to achieve their intended purpose, i.e., that the promoter sequence enables the initiation of transcription, which proceeds through the linked coding sequence up to the termination signal.

[0057] As used herein, the term “organoid” refers to a three-dimensional cellular structure obtained by the proliferation of self-organizing stem cells, such as adult stem cells, that can differentiate into functional cell types. See Corro et al. (2020) Am.J.Physiol.Cell Physiol.319:C151–C165.

[0058] As used herein, the term “multiple” means a number greater than one. In certain embodiments, the term “multiple tissue-derived epithelial stem cells” means a number of tissue-derived epithelial stem cells greater than one. For example, multiple tissue-derived epithelial stem cells include, but are not limited to, at least two tissue-derived epithelial stem cells. In certain non-limiting embodiments, multiple tissue-derived epithelial stem cells may 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, or at least about 1,000,000,000 tissue-derived epithelial stem cells.

[0059] As used herein, the term “collection of tissue-derived epithelial organoids” refers to a group consisting of at least two tissue-derived epithelial organoids. In certain embodiments, “collection of tissue-derived epithelial organoids” refers to a group of tissue-derived epithelial organoids produced in the same manner. In certain non-limiting embodiments, the collection of tissue-derived epithelial organoids may 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, or at least about 1,000,000,000 tissue-derived epithelial organoids.

[0060] As used herein, the term “proliferation” refers to an increase in the number of cells.

[0061] As used herein, the term “promoter” means a region within a gene to which a transcription factor and / or RNA polymerase can bind to control the expression of the associated coding sequence. Promoters are generally, but not always, located in the 5' non-coding region of a gene upstream of the translation start codon. The promoter region of a gene may contain one or more consensus sequences that act as a recognizable binding site for the sequence-specific nucleic acid binding domain of a nucleic acid-binding protein. Nevertheless, such binding sites may also be located in regions outside the promoter, for example, within an intron or in an enhancer region located downstream of the coding sequence.

[0062] As used herein, the terms “solidify” or “solidified” refer to the hardening, thickening, polymerization, and / or increase in rigidity of a substance.

[0063] As used herein, the term “serpent” refers to a meandering shape when used in relation to a geometric shape.

[0064] As used herein, the term “spiral,” when used in reference to a geometric shape, refers to a continuous curved line that revolves around a central point or axis.

[0065] As used herein, the term “subset” refers to a smaller portion of a larger quantity of material.

[0066] As used herein, “tissue-derived epithelial stem cells” refers to epithelial stem cells obtained from 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)).

[0067] As used herein, “treatment” is an approach to obtain beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes, with respect to the purposes of this subject, include, but are not limited to, relief or improvement of one or more signs or symptoms, reduction of disease severity, stabilization (i.e., non-worsening) of the disease, prevention of disease, delay or slowing of disease progression, remission of a disease (e.g., cancer), and / or improvement or relief of the disease state. A reduction may be a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% in the likelihood of complications, severity of signs or symptoms, or progression to another grade. In certain embodiments, “treatment” may also mean inhibiting cancer growth or progression to a higher grade by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%.

[0068] II. Organoids and their compositions This disclosure provides tissue-derived epithelial organoids. In certain embodiments, the tissue-derived epithelial organoids are embedded in a substrate, such as an unbound hydrogel of a culture vessel. This disclosure further provides compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids are produced by methods disclosed herein, such as those disclosed in Section III.

[0069] In certain embodiments, the disclosure provides a composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel. In certain embodiments, the hydrogel is not attached to the surface of a substrate, such as the surface of a cell culture dish and / or a multiwell plate. In certain embodiments, the tissue-derived epithelial organoids embedded in the hydrogel are suspended in a culture medium.

[0070] In certain embodiments, the tissue-derived epithelial organoid is a lacrimal gland organoid, tonsil organoid, salivary gland organoid, gastrointestinal organoid, thyroid organoid, lung organoid, mammary gland organoid, liver organoid, bile duct organoid, stomach organoid, kidney organoid, pancreatic organoid, endometrial organoid, fallopian tube organoid, cervical organoid, prostate organoid, bladder organoid, ovarian organoid, taste bud organoid, or trophoblast 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 cervical 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 trophoblast organoid.In certain embodiments, the tissue-derived epithelial organoids are selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, mammary gland organoids, and combinations thereof.

[0071] 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.

[0072] In certain embodiments, the tissue-derived epithelial organoids are gastrointestinal organoids. In certain embodiments, the gastrointestinal organoids are intestinal organoids. For example, but not limited to, the tissue-derived epithelial organoids are colonic or ileal organoids. In certain embodiments, the tissue-derived epithelial organoids are colonic organoids. In certain embodiments, the tissue-derived epithelial organoids are ileal organoids. In certain embodiments, the tissue-derived epithelial organoids are rectal organoids. In certain embodiments, the tissue-derived epithelial organoids are esophageal organoids. In certain embodiments, the tissue-derived epithelial organoids are oral and maxillofacial organoids. In certain embodiments, the gastrointestinal organoids include goblet cells and enterocytes.

[0073] In certain embodiments, the tissue-derived epithelial organoid is a mammary gland organoid.

[0074] In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid. In certain embodiments, the pancreatic organoid includes tubular cells, as disclosed in Example 6, for example.

[0075] In certain embodiments, the tissue-derived epithelial organoid is a liver organoid. In certain embodiments, the liver organoid includes hepatocytes, as disclosed in Example 7, for example.

[0076] In certain embodiments, the compositions of the Disclosure may include one or more different types of tissue-derived epithelial organoids. For example, but not limited to, the compositions of the Disclosure may include colon organoids and ileal organoids. In certain embodiments, the compositions of the Disclosure may include liver organoids and bile duct organoids.

[0077] In certain embodiments, the composition of the present disclosure comprises about one or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium, for example, about two or more, about five or more, about ten 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 100,000,000 or more, or about 1,000,000,000 or more tissue-derived epithelial organoids. In certain embodiments, the composition of the present disclosure comprises about 50 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 100 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 1,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 5,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 10,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 100,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 500,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the present disclosure comprises about 1,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the disclosure comprises approximately 10,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. In certain embodiments, the composition of the disclosure comprises approximately 100,000,000 or more tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium.

[0078] In certain embodiments, the hydrogel is a three-dimensional (3D) scaffold. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 10°C. For example, but not limited to, the hydrogel is composed of a material that solidifies at temperatures above approximately 15°C, above approximately 20°C, above approximately 25°C, above approximately 30°C, above approximately 35°C, above approximately 40°C, above approximately 45°C, or above approximately 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 25°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 30°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 35°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 40°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 45°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures above approximately 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at temperatures between approximately 25°C and approximately 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature of approximately 25°C to approximately 40°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature of approximately 30°C to approximately 50°C. In certain embodiments, the hydrogel is composed of a material that solidifies at a temperature of approximately 30°C to approximately 40°C, for example, approximately 37°C.

[0079] In certain embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the hydrogel may be a mixture of a synthetic hydrogel and a natural hydrogel. In certain embodiments, the hydrogel does not contain chemically crosslinked proteins and / or polymers.

[0080] In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the natural hydrogel contains one or more naturally occurring components. For example, but not limited to, the natural hydrogel may contain one or more proteins, such as glycoproteins and / or polysaccharides. Non-limited 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 natural hydrogel may further contain one or more components such as polysaccharides, water, and / or elastin, but not limited to. In certain embodiments, the natural hydrogel of this disclosure contains laminin, entactin, and type IV collagen. In certain embodiments, the natural hydrogels of the Disclosure comprise laminin, entactin, type IV collagen, and heparin sulfate proteoglycans. In certain embodiments, the natural hydrogels comprise extracellular matrix (ECM) secreted by and / or derived from epithelial cells, endothelial cells, parietal endoderm-like cells, and / or connective tissue cells.

[0081] 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 which are incorporated herein by reference in their entirety.

[0082] In certain embodiments, the hydrogels of this disclosure do not contain alginate.

[0083] In certain embodiments, the hydrogel may 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 contains laminin, enterin, and type IV collagen. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. Non-limiting examples of BMEs include CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which contains laminin, enterin, type IV collagen, and heparin sulfate proteoglycan.

[0084] In certain embodiments, the hydrogel has a protein concentration greater than about 0.7 mg / ml, for example, a glycoprotein concentration. In certain embodiments, the hydrogel has a protein concentration greater than about 1 mg / ml, for example, a glycoprotein concentration. In certain embodiments, the hydrogel has a protein concentration greater than about 2 mg / ml, for example, a glycoprotein concentration. 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 of about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of approximately 5 mg / ml to approximately 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of approximately 6 mg / ml to approximately 10 mg / ml. In certain embodiments, the hydrogel has a protein concentration of 0.7 mg / ml or higher. In certain embodiments, the hydrogel has a protein concentration of 1 mg / ml or higher. In certain embodiments, the hydrogel has a protein concentration of 5 mg / ml or higher. In certain embodiments, the hydrogel has a protein concentration of 6 mg / ml or higher.

[0085] In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in large w / v% amounts exceeding approximately 1 w / v%. For example, but not limited to, the hydrogel contains approximately 1 w / v%, 1.5 w / v%, 2 w / v%, 2.5 w / v%, 3 w / v%, 3.5 w / v%, 4 w / v%, 4.5 w / v%, 5 w / v%, 5.5 w / v%, 6 w / v%, 6.5 w / v%, 7 w / v%, 7.5 w / v%, 8 w / v%, 8.5 w / v%, 9 w / v%, 9.5 w / v%, 10 w / v%, 10.5 w / v%, and 11 w / v%. The hydrogel contains BME components, ECM components, or polymers in w / v% of approximately 1% to approximately 10% w / v%, including w / v% of 11.5 w / v%, 12.5 w / v%, 13.5 w / v%, 14.5 w / v%, 15.5 w / v%, 16.5 w / v%, 17.5 w / v%, 18.5 w / v%, 19.5 w / v%, or approximately 20 w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% of approximately 1% to approximately 10% w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% of about 2 w / v% to about 10 w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% of about 5 w / v% to about 10 w / v%.

[0086] In certain embodiments, the hydrogel has a storage modulus G' that is greater than or equal to the loss modulus G''.

[0087] In certain embodiments, the hydrogel containing tissue-derived epithelial organoids suspended in a culture 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, although not limited to these, Hydrogels can exceed approximately 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, 12 mm, 12.5 mm, 13 mm, 13.5 mm, 14 mm, 14.5 mm, 15 mm, and 15.5 mm. , having a geometric shape with a length, width and / or diameter exceeding approximately 16 mm, 16.5 mm, 17.5 mm, 18 mm, 18.5 mm, 19 mm, 19.5 mm, 20 mm, 50 mm, 100 mm, 150 mm, 200 mm, 250 mm, 300 mm, 350 mm, 400 mm, 450 mm, 500 mm, 550 mm, 600 mm, 650 mm, 700 mm, 750 mm, 800 mm, 850 mm, 900 mm, 950 mm, or 1,000 mm.

[0088] 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, for example, 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. For example, although not limited to these, the diameters of the Hydrogel are approximately 0.1mm to 19mm, 0.1mm to 18mm, 0.1mm to 17mm, 0.1mm to 16mm, 0.1mm to 15mm, 0.1mm to 14mm, 0.1mm to 13mm, 0.1mm to 12mm, 0.1mm to 11mm, 0.1mm to 10mm, 0.1mm to 9mm, 0.1mm to 8mm, 0.1mm to 7mm, 0.1mm to 6mm, 0.1mm to 5mm, 0.1mm to 4mm, 0.1mm to 3mm, 0.1mm to 2mm, 0.1mm to 1mm, 0.5mm to 20mm, and 1mm to 2mm. It has a geometric shape with a length, width, and / or diameter of 0 mm, approximately 2 mm to 20 mm, approximately 3 mm to 20 mm, approximately 4 mm to 20 mm, approximately 5 mm to 20 mm, approximately 6 mm to 20 mm, approximately 7 mm to 20 mm, approximately 8 mm to 20 mm, approximately 9 mm to 20 mm, approximately 10 mm to 20 mm, approximately 11 mm to 20 mm, approximately 12 mm to 20 mm, approximately 13 mm to 20 mm, approximately 14 mm to 20 mm, approximately 15 mm to 20 mm, approximately 16 mm to 20 mm, approximately 17 mm to 20 mm, approximately 18 mm to 20 mm, approximately 19 mm to 20 mm, approximately 1 mm to 15 mm, approximately 1 mm to 10 mm, approximately 1 mm to 5 mm, or approximately 1 mm to 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.

[0089] In certain embodiments, the geometric shape of the hydrogel suspended in the culture medium is a droplet, as shown in Figure 6, for example. In a particular embodiment, the Hydrogel droplets are approximately 0.1 mm to approximately 20 mm in size, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, and approximately 0.5 mm. mm~20mm, 1mm~20mm, 2mm~20mm, 3mm~20mm, 4mm~20mm, 5mm~20mm, 6mm~20mm, 7mm~20mm, 8mm~20mm, 9mm~20mm, 10mm~20mm, 11mm~20mm, 12mm~20mm It has a diameter of 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 1 mm. In certain embodiments, the hydrogel droplets have a diameter of about 0.1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 20 mm. In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 10 mm. In certain embodiments, the hydrogel droplets have a diameter of about 1 mm to about 4 mm. In certain embodiments, each hydrogel droplet contains about one or more tissue-derived epithelial organoids, for example, about two or more, about five or more, about ten or more, about fifty 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.

[0090] In certain embodiments, the hydrogel has a filamentous structure, for example, as shown in Figure 6. In certain embodiments, the filamentous structure has a linear, snake-like, or helical shape. In certain embodiments, the filamentous structure has a linear shape. In certain embodiments, the filamentous structure has a snake-like shape. In certain embodiments, the filamentous structure has a helical shape. In certain embodiments, the filament-like structure has a length and / or width of approximately 0.1 mm to approximately 1000 mm, for example, approximately 0.1 mm to approximately 500 mm, approximately 0.1 mm to approximately 100 mm, approximately 0.1 mm to approximately 50 mm, approximately 0.1 mm to approximately 20 mm, approximately 0.1 mm to approximately 10 mm, approximately 1 mm to approximately 100 mm, approximately 20 mm to approximately 1000 mm, approximately 50 mm to approximately 1000 mm, approximately 100 mm to approximately 1000 mm, approximately 500 mm to approximately 1000 mm, approximately 1 mm to approximately 100 mm, or approximately 1 mm to approximately 50 mm. In a particular embodiment, the filament-like structure is approximately 0.1 mm to approximately 20 mm, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, and approximately 0.5 mm. Having a length and / or width of approximately 20mm, 1mm to 20mm, 2mm to 20mm, 3mm to 20mm, 4mm to 20mm, 5mm to 20mm, 6mm to 20mm, 7mm to 20mm, 8mm to 20mm, 9mm to 20mm, 10mm to 20mm, 11mm to 20mm, 12mm to 20mm, 13mm to 20mm, 14mm to 20mm, 15mm to 20mm, 16mm to 20mm, 17mm to 20mm, 18mm to 20mm, 19mm to 20mm, 1mm to 15mm, 1mm to 10mm, 1mm to 5mm, or 1mm to 4mm. In certain embodiments, the filament-like structure has a length of approximately 0.1 mm to approximately 1 mm.In certain embodiments, the filament-like structure has a length of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a width of approximately 0.1 mm to approximately 1 mm. In certain embodiments, the filament-like structure has a width of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 4 mm.

[0091] In a particular embodiment, the filament-like structure is approximately 0.1 mm to approximately 20 mm, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, approximately 0. 5mm~20mm, 1mm~20mm, 2mm~20mm, 3mm~20mm, 4mm~20mm, 5mm~20mm, 6mm~20mm, 7mm~20mm, 8mm~20mm, 9mm~20mm, 10mm~20mm, 11mm~20mm, 12mm~ It has a diameter of about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 5 mm. In certain embodiments, the filament-like structure has a diameter of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a diameter of approximately 1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a diameter of approximately 1 mm to approximately 5 mm.

[0092] In certain embodiments, each filament-like structure comprises about one or more tissue-derived epithelial organoids, for example, about two or more, about five or more, about ten 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.

[0093] In certain embodiments, the composition comprises any suitable cell culture medium. In certain embodiments, the cell culture medium contains components important for supporting the maintenance of cultured cells. In certain embodiments, the cell culture medium for use in the disclosure may be a nutrient solution containing standard cell culture components such as amino acids, vitamins, inorganic salts, carbon energy sources (e.g., glucose), and buffers, but is not limited to these. 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. Non-limiting examples of cell culture media, such as organoid growth media, are provided in the examples.

[0094] In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:1 or greater. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:1 to about 1:100. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) 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~approx. 1:100, approx. 1:50~approx. 1:100, approx. 1:55~approx. 1:100, approx. 1:60~approx. 1:100, approx. 1:65~approx. 1:100, approx. 1:70~approx. 1:100, approx. 1:75~approx. 1:100, approx. 1:80~approx. 1:100, approx. 1:85~approx. 1:100, approx. 1:90~approx. 1:100, approx. 1:95~approx. 1:100, 1:5~approx. 1:50, approx. 1:10~approx. 1:50, approx. 1:15~approx. 1:50, approx. 1:20~approx. 1:50, approx. 1:25~1:50, approx. 1:30~1:50, approx. 1:35~1:50, approx. 1:40~1:50, approx. 1:45~1:50, approx. 1:1~1:95, approx. 1:1~1:90, approx. 1:1~1:85, approx. 1:1~1:80, approx. 1:1~1:75, approx. 1:1~1:70, approx. 1:1~1:65, approx. 1:1~1:60, approx. 1:1~1:55, approx. 1:1~1:50, approx. 1:1~1: The ratios are approximately 1:45, 1:1 to 1:40, 1:1 to 1:35, 1:1 to 1:30, 1:1 to 1:35, 1:1 to 1:30, 1:1 to 1:25, 1:1 to 1:20, 1:1 to 1:15, 1:1 to 1:10, 1:1 to 1:5, 1:5 to 1:75, 1:5 to 1:60, 1:5 to 1:50, 1:1 to 1:40, 1:5 to 1:30, or 1:5 to 1:20. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:1 to 1:50. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:2 to approximately 1:50. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:1 to approximately 1:20.In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:1 to about 1:15. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:1. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:2. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:5. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:10, as shown in Example 8, for example. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:20. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is about 1:30. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:40. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:50. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:60. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:70. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:80. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:90. In certain embodiments, the ratio of the volume of hydrogel to the volume of culture medium in the composition (volume ratio) is approximately 1:100.

[0095] In certain embodiments, tissue-derived epithelial organoids are more uniform in size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate). In certain embodiments, tissue-derived epithelial organoids produced by the method of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids due to differences in nutrient availability, as described in Example 1. For example, but not limited to, tissue-derived epithelial organoids produced by the method of the present disclosure are more uniform in size across the width of a turbid culture droplet (e.g., a suspended hydrogel droplet) compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate). In certain embodiments, reference tissue-derived epithelial organoids are produced in a hydrogel dome as disclosed in Example 1.

[0096] In certain embodiments, the tissue-derived epithelial organoids of this disclosure (e.g., a population of tissue-derived epithelial organoids) express markers at different levels, e.g., higher or lower levels, than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). For example, but not limited to, the tissue-derived epithelial organoids of this disclosure (e.g., a population of tissue-derived epithelial organoids) express markers at higher levels 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 of this disclosure (e.g., a population of tissue-derived epithelial organoids) express markers at lower levels than reference tissue-derived epithelial organoids (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the markers are stem cell markers and / or proliferation markers, e.g., genes related to stem cells and / or proliferation, as shown in Figure 5A. For example, but not limited to, stem cell markers and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and / or CD44. In certain embodiments, the markers are differentiation markers, such as genes related to differentiation, as shown in Figure 5B. For example, but not limited to, 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate. For example, but not limited to, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid manufactured in a hydrogel dome as disclosed in Example 1.

[0097] In certain embodiments, the tissue-derived epithelial organoids are gastrointestinal organoids, and the markers differentially expressed in the gastrointestinal organoids of this disclosure are MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0098] In certain embodiments, the tissue-derived epithelial organoid is a mammary gland organoid, and the differentially expressed markers in the mammary gland organoid of this disclosure are EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA).

[0099] In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid, and the differentially expressed markers in the pancreatic organoid of this disclosure are CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, keratin 19 (KRT19), MUC1, INS, GCG, and / or AMY.

[0100] In certain embodiments, the tissue-derived epithelial organoid is a liver organoid, and the markers differentially expressed in the liver organoid of this disclosure are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0101] In certain embodiments, the stem cell marker and / or proliferation marker is expressed at a higher level by a population of tissue-derived epithelial organoids of the Disclosure (e.g., a population of tissue-derived epithelial organoids in the composition of the Disclosure) compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell markers and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof. In certain embodiments, the stem cell marker and / or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In certain embodiments, the stem cell marker and / or proliferation marker is MKI67. In certain embodiments, the stem cell marker and / or proliferation marker is ASCL2. In certain embodiments, the stem cell marker and / or proliferation marker is LGR5. In certain embodiments, the stem cell marker and / or proliferation marker is SOX9. In certain embodiments, the stem cell marker and / or proliferation marker is SMOC2. In certain embodiments, the stem cell marker and / or proliferation marker is CD44. In certain embodiments, the stem cell marker and / or proliferation marker is EpCAM. In certain embodiments, the stem cell marker and / or proliferation marker is CD49f. In certain embodiments, the stem cell marker and / or proliferation marker is CD133. In certain embodiments, the stem cell marker and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell marker and / or proliferation marker is NEUROG3. In certain embodiments, the stem cell marker and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell marker and / or proliferation marker is PDX1. In certain embodiments, the stem cell marker and / or proliferation marker is BMI1.In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, and at least 110% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. At least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher, or at least 300% higher. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 50% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 100% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 200% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 300% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids.

[0102] In certain embodiments, differentiation markers are expressed at lower levels by a population of tissue-derived epithelial organoids of the Disclosure (e.g., a population of tissue-derived epithelial organoids in the composition of the Disclosure) 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations 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 MUC1. 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 (K18).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 the differentiation marker in a population of epithelial organoids derived from the tissue of this disclosure is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, and at least 1% lower than the expression level of the differentiation marker in a population of epithelial organoids derived from reference tissue. 30% lower, at least 140% lower, at least 150% lower, at least 160% lower, at least 170% lower, at least 180% lower, at least 190% lower, at least 200% lower, at least 210% lower, at least 220% lower, at least 230% lower, at least 240% lower, at least 200% lower, at least 250% lower, at least 260% lower, at least 270% lower, at least 280% lower, at least 290% lower, or at least 300% lower. In certain embodiments, the expression level of the differentiation marker in a population of tissue-derived epithelial organoids of the Disclosure is at least 50% lower than the expression level of the differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of the differentiation marker in a population of tissue-derived epithelial organoids of the Disclosure is at least 100% lower than the expression level of the differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of differentiation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 200% lower than the expression levels of differentiation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of differentiation markers in a population of tissue-derived epithelial organoids of the Disclosure are at least 300% lower than the expression levels of differentiation markers in a population of reference tissue-derived epithelial organoids.

[0103] III. Method for Manufacturing Organoids This disclosure provides methods for producing tissue-derived epithelial organoids. In certain embodiments, this disclosure provides methods for producing tissue-derived epithelial organoids embedded in a hydrogel suspended in a culture medium. This disclosure further provides methods for producing turbid cultures of tissue-derived epithelial organoids. In certain embodiments, organoids can be produced using the methods described in Examples 1 and 4-8 and in Figures 10 and 11.

[0104] In certain embodiments, the Disclosure provides a method for producing lacrimal gland organoids, tonsil organoids, salivary gland organoids, gastrointestinal organoids, thyroid organoids, lung organoids, mammary gland organoids, liver organoids, bile duct organoids, stomach organoids, kidney organoids, pancreatic organoids, endometrial organoids, fallopian tube organoids, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblast organoids. In certain embodiments, the Disclosure provides a method for producing lacrimal gland organoids. In certain embodiments, the Disclosure provides a method for producing tonsil organoids. In certain embodiments, the Disclosure provides a method for producing salivary gland organoids. In certain embodiments, the Disclosure provides a method for producing gastrointestinal organoids. In certain embodiments, the Disclosure provides a method for producing thyroid organoids. In certain embodiments, the Disclosure provides a method for producing lung organoids. In certain embodiments, the Disclosure provides a method for producing mammary gland organoids. In certain embodiments, the Disclosure provides a method for producing liver organoids. In certain embodiments, the Disclosure provides a method for producing bile duct organoids. In certain embodiments, the Disclosure provides a method for producing gastric organoids. In certain embodiments, the Disclosure provides a method for producing kidney organoids. In certain embodiments, the Disclosure provides a method for producing pancreatic organoids. In certain embodiments, the Disclosure provides a method for producing endometrial organoids. In certain embodiments, the Disclosure provides a method for producing fallopian tube organoids. In certain embodiments, the Disclosure provides a method for producing cervical organoids. In certain embodiments, the Disclosure provides a method for producing prostate organoids. In certain embodiments, the Disclosure provides a method for producing bladder organoids. In certain embodiments, the Disclosure provides a method for producing ovarian organoids. In certain embodiments, the Disclosure provides a method for producing trophoblast organoids. In certain embodiments, the Disclosure provides a method for producing trophoblast organoids.

[0105] In certain embodiments, the present disclosure provides a method for producing tissue-derived epithelial organoids selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, mammary gland organoids, and combinations thereof.

[0106] In certain embodiments, the Disclosure provides a method for producing gastrointestinal organoids. In certain embodiments, the Disclosure provides a method for producing intestinal organoids. For example, but not limited to, intestinal organoids are colonic organoids or ileal organoids. In certain embodiments, intestinal organoids are colonic organoids. In certain embodiments, intestinal organoids are ileal organoids. In certain embodiments, the Disclosure provides a method for producing rectal organoids. In certain embodiments, the Disclosure provides a method for producing esophageal organoids. In certain embodiments, the Disclosure provides a method for producing oral organoids and maxillofacial organoids. In certain embodiments, the methods of the Disclosure can produce two or more different types of tissue-derived epithelial organoids, such as colonic organoids and ileal organoids.

[0107] In certain embodiments, the present disclosure provides a method for manufacturing lung organoids.

[0108] In certain embodiments, the present disclosure provides a method for producing liver organoids.

[0109] In certain embodiments, the present disclosure provides a method for manufacturing pancreatic organoids.

[0110] In certain embodiments, the present disclosure provides a method for producing mammary gland organoids.

[0111] In certain embodiments, a method for producing tissue-derived epithelial organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells.

[0112] In certain embodiments, the plurality of tissue-derived epithelial stem cells include at least two or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells include 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 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 1,000,000,000 or more tissue-derived epithelial stem cells. In certain embodiments, the plurality of tissue-derived epithelial stem cells include a hydrogel containing at least about 10 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 100 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 1,000 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 10,000 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 100,000 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 100,000 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 1,000,000 tissue-derived epithelial stem cells / ml, a hydrogel containing at least about 100,000,000 tissue-derived epithelial stem cells / ml, or a hydrogel containing at least about 1,000,000,000 tissue-derived epithelial stem cells / ml. In certain embodiments, the multiple tissue-derived epithelial stem cells comprise a hydrogel containing at least approximately 10,000 or more tissue-derived epithelial stem cells / ml.

[0113] In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises a hydrogel having from about 1×10 4 to about 1×10 10 tissue-derived epithelial stem cells / ml. For example, without limitation, the plurality of tissue-derived epithelial stem cells comprises a hydrogel having from about 1×10 4 to about 1×10 9 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×104 to about 1×10 8 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 4 to about 1×10 7 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 4 to about 1×10 6 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 4 to about 1×10 5 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 5 to about 1×10 10 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 6 to about 1×10 10 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 7 to about 1×10 10 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 8 to about 1×10 10 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 9 to about 1×10 10 tissue-derived epithelial stem cells / ml, a hydrogel having from about 1×10 5 to about 1×10 7 tissue-derived epithelial stem cells / ml, or a hydrogel having from about 1×10 5 to about 1×10 8 tissue-derived epithelial stem cells / ml. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises a hydrogel having from about 1×10 4 tissue-derived epithelial stem cells / ml to about 1×10 7 tissue-derived epithelial stem cells / ml. In certain embodiments, the plurality of tissue-derived epithelial stem cells comprises a hydrogel having from about 1×10 4Hydrogel containing individual tissue-derived epithelial stem cells / ml ~ approximately 1 x 10⁶ 6 Contains a hydrogel of individual tissue-derived epithelial stem cells / ml.

[0114] In certain embodiments, the method may further include suspending a mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of hydrogel and tissue-derived epithelial stem cells. In certain embodiments, suspending a mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium includes immersing a dispensing device containing the mixture of hydrogel and tissue-derived epithelial stem cells in the culture medium and dispensing the mixture into the culture medium. In certain embodiments, the dispensing of the mixture of hydrogel and tissue-derived epithelial stem cells into the culture medium can be repeated to produce a plurality of separated mixtures of hydrogel and tissue-derived epithelial stem cells suspended in the culture medium. In certain embodiments, the dispensing device may be any device that enables the delivery of the mixture. In certain embodiments, the delivery device dispenses the mixture in a precise and controlled manner. Non-limiting examples of the dispensing device include pipettes, droppers, and syringes. In certain embodiments, the dispensing device may be manually operated or automatically operated. For example, but not limited to, the dispensing device may be automatically operated. In certain embodiments, the dispensing device may be an automated liquid handler. In certain embodiments, the dispensing device may be a liquid handling robot.

[0115] In certain embodiments, the volume of the mixture of hydrogel and tissue-derived epithelial stem cells dispensed into the culture medium may be at least about 1 μl. For example, but not limited to, the volume of the mixture of hydrogel and tissue-derived epithelial stem cells dispensed into the culture medium may be at least about 5 μl, at least about 10 μl, at least about 50 μl, at least about 100 μl, at least about 500 μl, 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 10 L. In certain embodiments, the volume of the mixture of hydrogel and tissue-derived epithelial stem cells dispensed into the culture medium may be about 1 μl to about 1 L. In certain embodiments, the volume of the mixture of hydrogel and tissue-derived epithelial stem cells dispensed into the culture medium may be about 1 μl to about 1 ml. For example, though not limited to, the volume of the mixture of Hydro gel and tissue-derived epithelial stem cells dispensed into the culture medium is approximately 1 μl to 900 μl, 1 μl to 800 μl, 1 μl to 700 μl, 1 μl to 600 μl, 1 μl to 500 μl, 1 μl to 400 μl, 1 μl to 300 μl, 1 μl to 200 μl, 1 μl to 100 μl, 1 μl to 10 μl, 1 μl to 900 μl, and 10 μl to 1 m³. The volumes are approximately 100 μl to 1 ml, approximately 200 μl to 1 ml, approximately 300 μl to 1 ml, approximately 400 μl to 1 ml, approximately 500 μl to 1 ml, approximately 600 μl to 1 ml, approximately 700 μl to 1 ml, approximately 800 μl to 1 ml, approximately 900 μl to 1 ml, approximately 1 μl to 50 μl, approximately 5 μl to 20 μl, approximately 10 μl to 100 μl, approximately 10 μl to 500 μl, approximately 100 μl to 200 μl, or approximately 100 μl to 500 μl. In certain embodiments, the volume of the mixture of Hydro gel and tissue-derived epithelial stem cells dispensed into the culture medium may be approximately 1 μl to 100 μl.

[0116] In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 10°C. For example, but not limited to, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 15°C, approximately 20°C, approximately 25°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, or approximately 50°C. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 25°C. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 30°C. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 35°C. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 40°C. In certain embodiments, the hydrogel of the mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 45°C. In certain embodiments, the hydrogel of the mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures between approximately 25°C and approximately 50°C. In certain embodiments, the hydrogel of the mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures between approximately 30°C and approximately 50°C. In certain embodiments, the hydrogel of the mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures between approximately 25°C and approximately 40°C, for example, approximately 37°C. In certain embodiments, the hydrogel of the mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures between approximately 30°C and approximately 40°C, for example, approximately 37°C.

[0117] In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of about 25°C or lower before contact with the culture medium. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of about 20°C or lower before contact with the culture medium. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of about 15°C or lower before contact with the culture medium. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of about 10°C or lower before contact with the culture medium. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of about 5°C or lower before contact with the culture medium. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is at a temperature of approximately 25°C, approximately 24°C, approximately 23°C, approximately 22°C, approximately 21°C, approximately 20°C, approximately 19°C, approximately 18°C, approximately 17°C, approximately 16°C, approximately 15°C, approximately 14°C, approximately 13°C, approximately 12°C, approximately 11°C, approximately 10°C, approximately 9°C, approximately 8°C, approximately 7°C, approximately 6°C, approximately 5°C, approximately 4°C, approximately 3°C, or approximately 2°C before contact with the culture medium. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 25°C before contact with the culture medium. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 20°C before contact with the culture medium. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 15°C before contact with the culture medium. In certain embodiments, the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to 10°C before contact with the culture medium.

[0118] In certain embodiments, the hydrogel may contain any material to solidify. In certain embodiments, the hydrogel is a synthetic hydrogel, a natural hydrogel, or a combination thereof. In certain embodiments, the hydrogel is a synthetic hydrogel. In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the hydrogel may be a mixture of a synthetic hydrogel and a natural hydrogel. In certain embodiments, the hydrogel does not contain chemically crosslinked proteins and / or polymers.

[0119] In certain embodiments, the hydrogel is a natural hydrogel. In certain embodiments, the natural hydrogel contains one or more naturally occurring components. For example, but not limited to, the natural hydrogel may contain one or more proteins, such as glycoproteins and / or polysaccharides. Non-limited 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 natural hydrogel may further contain one or more components such as polysaccharides, water, and / or elastin, but not limited to. In certain embodiments, the natural hydrogel of this disclosure contains laminin, entactin, and type IV collagen. In certain embodiments, the natural hydrogels of the Disclosure comprise laminin, entactin, type IV collagen, and heparin sulfate proteoglycans. In certain embodiments, the natural hydrogels comprise extracellular matrix (ECM) secreted by and / or derived from epithelial cells, endothelial cells, parietal endoderm-like cells, and / or connective tissue cells.

[0120] 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 which are incorporated herein by reference in their entirety.

[0121] In certain embodiments, the hydrogels of this disclosure do not contain alginate.

[0122] In certain embodiments, the hydrogel may 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 contains laminin, enterin, and type IV collagen. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. Non-limiting examples of BMEs include CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which contains laminin, enterin, type IV collagen, and heparin sulfate proteoglycan.

[0123] In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 0.7 mg / ml, for example, a glycoprotein concentration. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 1 mg / ml, for example, a glycoprotein concentration. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 2 mg / ml, for example, a glycoprotein concentration. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 3 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 4 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 5 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration greater than approximately 6 mg / ml, greater than approximately 7 mg / ml, greater than approximately 8 mg / ml, greater than approximately 9 mg / ml, or greater than approximately 10 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of about 0.7 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of about 1 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of about 5 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of about 6 mg / ml to about 10 mg / ml. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of 0.7 mg / ml or more. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of 1 mg / ml or more. In certain embodiments, the hydrogel of a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of 5 mg / ml or more.In certain embodiments, the hydrogel in a mixture of hydrogel and tissue-derived epithelial stem cells has a protein concentration of 6 mg / ml or higher.

[0124] In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in large w / v% amounts exceeding about 1 w / v%. For example, but not limited to, the hydrogel may contain amounts exceeding about 1 w / v%, about 1.5 w / v%, about 2 w / v%, about 2.5 w / v%, about 3 w / v%, about 3.5 w / v%, about 4 w / v%, about 4.5 w / v%, about 5 w / v%, about 5.5 w / v%, about 6 w / v%, about 6.5 w / v%, about 7 w / v%, about 7.5 w / v%, about 8 w / v%, about 8.5 w / v%, about 9 w / v%, about 9.5 w / v%, or about 10 w / v%, about 10.5 w / v%, about 11 w / v% The hydrogel contains BME components, ECM components, or polymers in w / v% of more than v%, about 11.5 w / v%, about 12 w / v%, about 12.5 w / v%, about 13 w / v%, about 13.5 w / v%, about 14 w / v%, about 14.5 w / v%, about 15 w / v%, about 15.5 w / v%, about 16 w / v%, about 16.5 w / v%, about 17 w / v%, about 17.5 w / v%, about 18 w / v%, about 18.5 w / v%, about 19 w / v%, about 19.5 w / v%, or about 20 w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% from about 1 w / v% to about 10 w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% of about 2 w / v% to about 10 w / v%. In certain embodiments, the hydrogel contains BME components, ECM components, or polymers in w / v% of about 5 w / v% to about 10 w / v%.

[0125] In certain embodiments, the hydrogel has a storage modulus G' that is greater than or equal to the loss modulus G''.

[0126] In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium (volume ratio) is approximately 1:1 to approximately 1:50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium (volume ratio) is approximately 1:2 to approximately 1:50. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:5 to 1:50, 1:10 to 1:50, 1:15 to 1:50, 1:20 to 1:50, 1:25 to 1:50, 1:30 to 1:50, 1:35 to 1:50, 1:40 to 1:50, 1:45 to 1:50, 1:2 to 1:45, 1:2 to 1:40, 1:2 to 1:35, 1:2 to 1:30, 1:2 to 1:35, 1:2 to 1:30, 1:2 to 1:25, 1:2 to 1:20, 1:2 to 1:15, 1:2 to 1:10, or 1:2 to 1:5. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:2 to approximately 1:20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:2 to approximately 1:15. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:2. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:5. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:10, as shown in Example 8, for example. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:20. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:30. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:40. In certain embodiments, the ratio of the volume of the hydrogel to the volume of the culture medium is approximately 1:50.

[0127] In certain embodiments, a mixture of suspended hydrogel and tissue-derived epithelial stem cells is dispensed into a culture medium in a specific geometric shape. For example, but not limited to, the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter greater than about 0.1 mm. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a length greater than about 0.1 mm. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a width greater than about 0.1 mm. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a diameter greater than about 0.1 mm. For example, though not limited to, a mixture of suspended hydrogel and tissue-derived epithelial stem cells can be found in sizes of approximately 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, 6.5 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, 12 mm, 12.5 mm, 13 mm, 13.5 mm, 14 mm, 14.5 mm, 15 mm, and 1 It has a geometric shape with a length, width, and / or diameter exceeding 5.5 mm, approximately 16 mm, approximately 16.5 mm, approximately 17.5 mm, approximately 18 mm, approximately 18.5 mm, approximately 19 mm, approximately 19.5 mm, approximately 20 mm, approximately 50 mm, approximately 100 mm, approximately 150 mm, approximately 200 mm, approximately 250 mm, approximately 300 mm, approximately 350 mm, approximately 400 mm, approximately 450 mm, approximately 500 mm, approximately 550 mm, approximately 600 mm, approximately 650 mm, approximately 700 mm, approximately 750 mm, approximately 800 mm, approximately 850 mm, approximately 900 mm, approximately 950 mm, or approximately 1,000 mm.

[0128] In certain embodiments, the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1000 mm, for example, 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 a particular embodiment, the mixture of suspended Hydrogel and tissue-derived epithelial stem cells is approximately 0.1 mm to approximately 20 mm, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, and approximately 0.5 mm. It has a geometric shape with a length, width, and / or diameter of approximately m to 20 mm, approximately 1 mm to 20 mm, approximately 2 mm to 20 mm, approximately 3 mm to 20 mm, approximately 4 mm to 20 mm, approximately 5 mm to 20 mm, approximately 6 mm to 20 mm, approximately 7 mm to 20 mm, approximately 8 mm to 20 mm, approximately 9 mm to 20 mm, approximately 10 mm to 20 mm, approximately 11 mm to 20 mm, approximately 12 mm to 20 mm, approximately 13 mm to 20 mm, approximately 14 mm to 20 mm, approximately 15 mm to 20 mm, approximately 16 mm to 20 mm, approximately 17 mm to 20 mm, approximately 18 mm to 20 mm, approximately 19 mm to 20 mm, approximately 1 mm to 15 mm, approximately 1 mm to 10 mm, approximately 1 mm to 5 mm, or approximately 1 mm to 4 mm. In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1 mm.In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 4 mm. In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 1 mm to about 20 mm. In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 1 mm to about 10 mm. In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of about 1 mm to about 4 mm.

[0129] In certain embodiments, the geometric shape of the mixture of the suspended hydrogel and tissue-derived epithelial stem cells is spherical or spherical. In certain embodiments, the geometric shape of the mixture of the suspended hydrogel and tissue-derived epithelial stem cells is filamentous. In certain embodiments, the filamentous structure has a linear, serpentine, or helical shape. In certain embodiments, the filamentous structure has a linear shape. In certain embodiments, the filamentous structure has a serpentine shape. In certain embodiments, the filamentous structure has a helical shape.

[0130] In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplets (see, for example, Figure 10). In certain embodiments, the hydrogel droplets have a diameter greater than approximately 0.1 mm. In a particular embodiment, the Hydrogel droplets are approximately 0.1 mm to approximately 20 mm in size, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, and approximately 0.5 mm. mm~20mm, 1mm~20mm, 2mm~20mm, 3mm~20mm, 4mm~20mm, 5mm~20mm, 6mm~20mm, 7mm~20mm, 8mm~20mm, 9mm~20mm, 10mm~20mm, 11mm~20mm, 12mm~20mm It has a diameter of 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 0.1 mm to approximately 1 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 1 mm to approximately 20 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 1 mm to approximately 10 mm. In certain embodiments, the hydrogel droplets have a diameter of approximately 1 mm to approximately 4 mm.In certain embodiments, each hydrogel droplet contains approximately one or more tissue-derived epithelial stem cells, for example, approximately 5 or more, approximately 10 or more, approximately 50 or more, approximately 100 or more, approximately 500 or more, approximately 1,000 or more, approximately 5,000 or more, approximately 10,000 or more, approximately 100,000 or more, approximately 200,000 or more, approximately 300,000 or more, approximately 400,000 or more, approximately 500,000 or more, approximately 600,000 or more, approximately 700,000 or more, approximately 800,000 or more. , containing approximately 900,000 or more, approximately 1,000,000 or more, approximately 2,000,000 or more, approximately 3,000,000 or more, approximately 4,000,000 or more, approximately 5,000,000 or more, approximately 6,000,000 or more, approximately 7,000,000 or more, approximately 8,000,000 or more, approximately 9,000,000 or more, approximately 10,000,000 or more, approximately 100,000,000 or more, or approximately 1,000,000,000 or more tissue-derived epithelial stem cells.

[0131] In certain embodiments, the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a filamentous structure (see, for example, Figure 11). In certain embodiments, the filamentous structure has a length and / or width greater than about 0.1 mm. In certain embodiments, the filamentous structure has a length greater than about 0.1 mm. In certain embodiments, the filamentous structure has a width greater than about 0.1 mm. In certain embodiments, the filament-like structure has a length and / or width of approximately 0.1 mm to approximately 1000 mm, for example, approximately 0.1 mm to approximately 500 mm, approximately 0.1 mm to approximately 100 mm, approximately 0.1 mm to approximately 50 mm, approximately 0.1 mm to approximately 20 mm, approximately 0.1 mm to approximately 10 mm, approximately 1 mm to approximately 100 mm, approximately 20 mm to approximately 1000 mm, approximately 50 mm to approximately 1000 mm, approximately 100 mm to approximately 1000 mm, approximately 500 mm to approximately 1000 mm, approximately 1 mm to approximately 100 mm, or approximately 1 mm to approximately 50 mm. In a particular embodiment, the filament-like structure is approximately 0.1 mm to approximately 20 mm, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, and approximately 0.5 mm. Having a length and / or width of approximately 20mm, 1mm to 20mm, 2mm to 20mm, 3mm to 20mm, 4mm to 20mm, 5mm to 20mm, 6mm to 20mm, 7mm to 20mm, 8mm to 20mm, 9mm to 20mm, 10mm to 20mm, 11mm to 20mm, 12mm to 20mm, 13mm to 20mm, 14mm to 20mm, 15mm to 20mm, 16mm to 20mm, 17mm to 20mm, 18mm to 20mm, 19mm to 20mm, 1mm to 15mm, 1mm to 10mm, 1mm to 5mm, or 1mm to 4mm.In certain embodiments, the filament-like structure has a length of approximately 0.1 mm to approximately 1 mm. In certain embodiments, the filament-like structure has a length of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a length of approximately 1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a width of approximately 0.1 mm to approximately 1 mm. In certain embodiments, the filament-like structure has a width of approximately 0.1 mm to approximately 4 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a width of approximately 1 mm to approximately 4 mm.

[0132] In a particular embodiment, the filament-like structure is approximately 0.1 mm to approximately 20 mm, for example, approximately 0.1 mm to approximately 19 mm, approximately 0.1 mm to approximately 18 mm, approximately 0.1 mm to approximately 17 mm, approximately 0.1 mm to approximately 16 mm, approximately 0.1 mm to approximately 15 mm, approximately 0.1 mm to approximately 14 mm, approximately 0.1 mm to approximately 13 mm, approximately 0.1 mm to approximately 12 mm, approximately 0.1 mm to approximately 11 mm, approximately 0.1 mm to approximately 10 mm, approximately 0.1 mm to approximately 9 mm, approximately 0.1 mm to approximately 8 mm, approximately 0.1 mm to approximately 7 mm, approximately 0.1 mm to approximately 6 mm, approximately 0.1 mm to approximately 5 mm, approximately 0.1 mm to approximately 4 mm, approximately 0.1 mm to approximately 3 mm, approximately 0.1 mm to approximately 2 mm, approximately 0.1 mm to approximately 1 mm, approximately 0. 5mm~20mm, 1mm~20mm, 2mm~20mm, 3mm~20mm, 4mm~20mm, 5mm~20mm, 6mm~20mm, 7mm~20mm, 8mm~20mm, 9mm~20mm, 10mm~20mm, 11mm~20mm, 12mm~ It has a diameter of about 20 mm, about 13 mm to about 20 mm, about 14 mm to about 20 mm, about 15 mm to about 20 mm, about 16 mm to about 20 mm, about 17 mm to about 20 mm, about 18 mm to about 20 mm, about 19 mm to about 20 mm, about 1 mm to about 15 mm, about 1 mm to about 10 mm, about 1 mm to about 5 mm, or about 1 mm to about 4 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 10 mm. In certain embodiments, the filament-like structure has a diameter of approximately 0.1 mm to approximately 5 mm. In certain embodiments, the filament-like structure has a diameter of approximately 1 mm to approximately 20 mm. In certain embodiments, the filament-like structure has a diameter of approximately 1 mm to approximately 10 mm. In certain embodiments, as described in Example 8, the filament-like structure has a diameter of approximately 1 mm to approximately 5 mm.

[0133] In certain embodiments, each filament-like structure comprises approximately one or more tissue-derived epithelial stem cells, for example, approximately 5 or more, approximately 10 or more, approximately 50 or more, approximately 100 or more, approximately 500 or more, approximately 1,000 or more, approximately 5,000 or more, approximately 10,000 or more, approximately 100,000 or more, approximately 200,000 or more, approximately 300,000 or more, approximately 400,000 or more, approximately 500,000 or more, approximately 600,000 or more, approximately 700,000 or more, approximately 800,000 or more, approximately 900,000 Contains more than 1,000,000, 2,000,000, 3,000,000, 4,000,000, 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 tissue-derived epithelial stem cells.

[0134] In certain embodiments, the method may further include culturing tissue-derived epithelial stem cells from a mixture of a suspended hydrogel and tissue-derived epithelial stem cells in a culture medium to produce tissue-derived epithelial organoids. In certain embodiments, the cell culture medium contains components important for supporting the maintenance of tissue-derived epithelial stem cells and / or organoids. In certain embodiments, the cell culture medium for use in the disclosure may be a nutrient solution containing standard cell culture components such as amino acids, vitamins, inorganic salts, carbon energy sources (e.g., glucose) and buffers, but is not limited thereto. 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. Culture media known to support the growth of specific tissues and cell types are known in the art and can be used in connection with the subject matter disclosed herein. For example, but not limited to, examples of culture media that can be used in this 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 are incorporated herein by reference. Further non-limiting examples of culture media for use in this disclosure are provided in the examples, e.g., Example 8.

[0135] In certain embodiments, the culture medium resides in a container. Non-limiting examples of containers include petri dishes, multiwell plates, conical tubes, reservoirs, culture bags, bioreactors, or flasks. In certain embodiments, the conical tube is a 50 ml conical tube. In certain embodiments, the multiwell plate is a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, or 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 made of a material that minimizes the adhesion of tissue-derived epithelial stem cells and / or hydrogels to the surface of the container. Alternatively or additionally, the container may include a surface coating that minimizes the adhesion of tissue-derived epithelial stem cells and / or hydrogels to the surface of the container.

[0136] In certain embodiments, the method may further include fragmenting a mixture of suspended hydrogel and tissue-derived epithelial stem cells to generate fragmented structures containing tissue-derived epithelial organoids. In certain embodiments, fragmenting a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture by pipetting a culture medium containing the mixture of suspended hydrogel and tissue-derived epithelial stem cells up and down to generate fragmented structures. In certain embodiments, fragmenting a mixture of suspended hydrogel and tissue-derived epithelial stem cells yields structures that are shorter in length and / or width. For example, but not limited to, fragmenting filamentous structures yields structures that are shorter in length, width, or both. In certain embodiments, fragmentation of the filamentous structure yields a structure that is 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 initially extruded mixture of hydrogel and tissue-derived epithelial stem cells, for example, the filamentous structure.

[0137] The subject matter of this disclosure further provides a method for producing turbid cultures of tissue-derived epithelial organoids. In certain embodiments, the method may include introducing a mixture comprising a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspension mixture. In certain embodiments, the mixture introduced into the culture medium comprises a hydrogel and a plurality of tissue-derived epithelial stem cells as described herein. In certain embodiments, the method may further include culturing the plurality of tissue-derived epithelial stem cells of the mixture in a culture medium to produce a turbid tissue-derived epithelial organoid as described herein. In certain embodiments, the method may further include fragmenting the suspension mixture to produce fragmented structures comprising tissue-derived epithelial organoids.

[0138] In certain embodiments, a method for producing a turbid culture of tissue-derived epithelial organoids may include contacting gastrointestinal stem cells, for example, multiple tissue-derived epithelial stem cells, with a hydrogel to produce a mixture of the hydrogel and tissue-derived epithelial stem cells, and depositing the mixture of the hydrogel and tissue-derived epithelial stem cells onto a substrate. In certain embodiments, the mixture of the hydrogel and tissue-derived epithelial stem cells is deposited on the substrate as droplets. In certain embodiments, the mixture of the hydrogel and tissue-derived epithelial stem cells is deposited on the substrate so as to have a filamentous structure. In certain embodiments, the method may further include solidifying the mixture of the hydrogel and tissue-derived epithelial stem cells to produce a solidified mixture of the hydrogel and tissue-derived epithelial stem cells. In certain embodiments, the method may include suspending the solidified mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of the hydrogel and tissue-derived epithelial stem cells. In certain embodiments, the method includes culturing the suspended mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce tissue-derived epithelial organoids. In certain embodiments, the method may further include removing the mixture of solidified hydrogel and tissue-derived epithelial stem cells from the substrate before suspending the mixture in a culture medium. In certain embodiments, the method may further include fragmenting the mixture of solidified hydrogel and tissue-derived epithelial stem cells before or after suspending the mixture in a culture medium to generate fragmented structures containing tissue-derived epithelial organoids.

[0139] In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in this disclosure are contained within tissue fragments, organoid fragments, or combinations thereof. For example, but not limited to, tissue fragments and / or organoid fragments containing tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells can be used to produce a mixture of hydrogel and tissue-derived epithelial stem cells. Alternatively or additionally, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells are isolated from tissue (e.g., tissue fragments), organoid fragments (e.g., tissue-derived epithelial organoid fragments), or combinations 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, tissue-derived epithelial stem cells for use in this disclosure can be obtained from in vitro cell cultures. In certain embodiments, tissue fragments can be obtained from frozen or fresh samples, e.g., frozen or fresh tissue samples and / or frozen or fresh tissue organoid fragments. In certain embodiments, tissue fragments may be fragments of primary tissue. In certain embodiments, the tissue of interest (e.g., a tissue fragment) may be normal (e.g., non-cancerous and / or non-disease-related). In certain embodiments, the tissue of interest (e.g., a tissue fragment) may be abnormal (e.g., cancerous and / or disease-related). In certain embodiments, tissue-derived epithelial stem cells can be isolated from a fragment of primary tissue.

[0140] In certain embodiments, the primary tissue fragment may be a fragment of the lacrimal gland, tonsils, salivary glands, gastrointestinal tissue, thyroid gland, lungs, mammary glands, liver, bile ducts, stomach, kidneys, pancreas, endometrium, fallopian tubes, cervix, prostate gland, bladder, ovaries, taste buds, or placenta. In certain embodiments, tissue-derived epithelial stem cells may be isolated from tissues (or fragments thereof) selected from the lacrimal gland, tonsils, salivary glands, gastrointestinal tissue, thyroid gland, lungs, mammary glands, liver, bile ducts, stomach, kidneys, pancreas, endometrium, fallopian tubes, cervix, prostate gland, bladder, ovaries, taste buds, placenta, and combinations thereof. In certain embodiments, the primary tissue fragment may be a fragment of the lacrimal gland. In certain embodiments, the primary tissue fragment may be a fragment of the tonsils. In certain embodiments, the primary tissue fragment may be a fragment of the salivary gland. In certain embodiments, the primary tissue fragment may be a fragment of gastrointestinal tissue. In certain embodiments, the primary tissue fragment may be a fragment of the thyroid gland. In certain embodiments, the primary tissue fragment may be a fragment of the lungs. In certain embodiments, the primary tissue fragment may be a fragment of the mammary gland. In certain embodiments, the primary tissue fragment may be a fragment of the liver. In certain embodiments, the primary tissue fragment may be a fragment of the bile duct. In certain embodiments, the primary tissue fragment may be a fragment of the stomach. In certain embodiments, the primary tissue fragment may be a fragment of the kidney. In certain embodiments, the primary tissue fragment may be a fragment of the pancreas. In certain embodiments, the primary tissue fragment may be a fragment of the endometrium. In certain embodiments, the primary tissue fragment may be a fragment of the fallopian tube. In certain embodiments, the primary tissue fragment may be a fragment of the cervix. In certain embodiments, the primary tissue fragment may be a fragment of the prostate gland. In certain embodiments, the primary tissue fragment may be a fragment of the bladder. In certain embodiments, the primary tissue fragment may be a fragment of the ovary. In certain embodiments, the primary tissue fragment may be a taste bud. In certain embodiments, the primary tissue fragment may be a fragment of the placenta. In certain embodiments, the primary tissue fragment is selected from the group consisting of lung tissue fragments, liver tissue fragments, gastrointestinal tissue fragments, mammary tissue fragments, pancreatic tissue fragments, and combinations thereof.

[0141] In certain embodiments, the primary tissue fragment may be a fragment of gastrointestinal tissue. In certain embodiments, the tissue fragment may be, for example, a fragment of the oral mucosa, pharynx (throat), esophagus, stomach, small intestine, large intestine, and / or rectum of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the oral mucosa of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the pharynx of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the esophagus of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the stomach of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the rectum of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the small intestine of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the large intestine of the subject. In certain embodiments, the tissue fragment may be, for example, a fragment of the colon and / or ileum tissue of the subject. For example, but not limited to, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells may be isolated from, for example, the colon and / or ileum tissue of the subject. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells may be isolated from (e.g., subject) colon tissue to generate colon organoids, for example. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells may be isolated from (e.g., subject) ileal tissue to generate ileal organoids, for example. In certain embodiments, for example, subject tissue may be normal (i.e., non-cancerous). For example, but not limited to, subject colon and / or ileal tissue may be normal (i.e., non-cancerous). In certain embodiments, for example, subject tissue may be cancerous or pathological. For example, but not limited to, subject colon and / or ileal tissue may be cancerous and / or pathological colon and / or ileal tissue. In certain embodiments, for example, subject esophageal tissue may be cancerous and / or pathological esophageal tissue. In certain embodiments, for example, subject gastric tissue may be cancerous and / or pathological gastric tissue. In certain embodiments, for example, subject rectal tissue may be cancerous and / or pathological rectal tissue. In certain embodiments, the tissue-derived epithelial organoid may be a gastrointestinal tumor organoid derived from the subject.

[0142] In certain embodiments, the primary tissue fragment is a fragment of lung tissue.

[0143] In certain embodiments, the primary tissue fragment is a fragment of liver tissue.

[0144] In certain embodiments, the primary tissue fragment is a fragment of mammary gland tissue.

[0145] In certain embodiments, the primary tissue fragment is a fragment of pancreatic tissue.

[0146] In certain embodiments, the method of the present disclosure produces tissue-derived epithelial organoids with more uniform size compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate). In certain embodiments, the tissue-derived epithelial organoids produced by the method of the present disclosure are more uniform in size compared to reference tissue-derived epithelial organoids due to differences in nutrient availability, as described in Example 1. For example, but not limited to, the tissue-derived epithelial organoids produced by the method of the present disclosure are more uniform in size across the width of a turbid culture droplet (e.g., a suspended hydrogel droplet) compared to reference tissue-derived epithelial organoids (e.g., tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate). In certain embodiments, the reference tissue-derived epithelial organoids are produced in a hydrogel dome as disclosed in Example 1.

[0147] In certain embodiments, the method of the present disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). For example, but not limited to, the tissue-derived epithelial organoids of the present disclosure (e.g., a population of tissue-derived epithelial organoids) express a marker at a higher level than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). Alternatively or additionally, in certain embodiments, the tissue-derived epithelial organoids of the present disclosure (e.g., a population of tissue-derived epithelial organoids) express a marker at a lower level than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the marker is a stem cell marker and / or proliferation marker, e.g., a gene associated with stem cells and / or proliferation. For example, but not limited to, stem cell markers and / or proliferation markers include 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, such as a gene associated with differentiation. For example, but not limited to, 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA). In certain embodiments, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate. For example, but not limited to, the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid manufactured in a hydrogel dome as disclosed in Example 1.

[0148] In certain embodiments, the method of the present disclosure produces a population of tissue-derived epithelial organoids that express stem cell markers and / or proliferation markers at higher levels compared to a population of reference tissue-derived epithelial organoids. Non-limiting examples of stem cell markers and / or proliferation markers include MKI67, EpCAM, BMI1, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, and / or CD44, and combinations thereof. In certain embodiments, the stem cell marker and / or proliferation marker is selected from the group consisting of MKI67, ASCL2, LGR5, SOX9, SMOC2, CD44, and combinations thereof. In certain embodiments, the stem cell marker and / or proliferation marker is MKI67. In certain embodiments, the stem cell marker and / or proliferation marker is ASCL2. In certain embodiments, the stem cell marker and / or proliferation marker is LGR5. In certain embodiments, the stem cell marker and / or proliferation marker is SOX9. In certain embodiments, the stem cell marker and / or proliferation marker is SMOC2. In certain embodiments, the stem cell marker and / or proliferation marker is CD44. In certain embodiments, the stem cell marker and / or proliferation marker is EpCAM. In certain embodiments, the stem cell marker and / or proliferation marker is CD49f. In certain embodiments, the stem cell marker and / or proliferation marker is CD133. In certain embodiments, the stem cell marker and / or proliferation marker is ALDH1A1. In certain embodiments, the stem cell marker and / or proliferation marker is NEUROG3. In certain embodiments, the stem cell marker and / or proliferation marker is NKX6.1. In certain embodiments, the stem cell marker and / or proliferation marker is PDX1. In certain embodiments, the stem cell marker and / or proliferation marker is BMI1.In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids produced by the method of the present disclosure are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, and at least 1% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. 10% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, at least 200% higher, at least 210% higher, at least 220% higher, at least 230% higher, at least 240% higher, at least 250% higher, at least 260% higher, at least 270% higher, at least 280% higher, at least 290% higher, or at least 300% higher. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids produced by the method of the present disclosure are at least 50% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids produced by the method of this disclosure are at least 100% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids produced by the method of this disclosure are at least 200% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of stem cell markers and / or proliferation markers in a population of tissue-derived epithelial organoids produced by the method of this disclosure are at least 300% higher than the expression levels of stem cell markers and / or proliferation markers in a population of reference tissue-derived epithelial organoids.

[0149] In certain embodiments, the method of the present disclosure produces a population of tissue-derived epithelial organoids that express differentiation markers at lower levels 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof. In certain embodiments, the differentiation marker is selected from the group consisting of keratin 20 (KRT20), FABP1, MUC2, MUC5B, TFF3, ALPI, SI, CEACAM7, and combinations 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 MUC1. 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 (K18).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 the differentiation marker in a population of tissue-derived epithelial organoids produced by the method of the present disclosure is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, At least 130% lower, at least 140% lower, at least 150% lower, at least 160% lower, at least 170% lower, at least 180% lower, at least 190% lower, at least 200% lower, at least 210% lower, at least 220% lower, at least 230% lower, at least 240% lower, at least 200% lower, at least 250% lower, at least 260% lower, at least 270% lower, at least 280% lower, at least 290% lower, or at least 300% lower. In certain embodiments, the expression level of the differentiation marker in a population of tissue-derived epithelial organoids produced by the method of the Disclosure is at least 50% lower than the expression level of the differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression level of the differentiation marker in a population of tissue-derived epithelial organoids produced by the method of the Disclosure is at least 100% lower than the expression level of the differentiation marker in a population of reference tissue-derived epithelial organoids. In certain embodiments, the expression levels of differentiation markers in a population of tissue-derived epithelial organoids produced by the method of the present disclosure are at least 200% lower than the expression levels of differentiation markers in a population of reference tissue-derived epithelial organoids.In certain embodiments, the expression levels of differentiation markers in a population of tissue-derived epithelial organoids produced by the method of the present disclosure are at least 300% lower than the expression levels of differentiation markers in a population of reference tissue-derived epithelial organoids.

[0150] In certain embodiments, a method for producing gastrointestinal organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells are approximately 1 × 10⁻⁶ 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7The method contains 1 ml of tissue-derived epithelial stem cells / hydrogel. In certain embodiments, the method may further include suspending the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of hydrogel and tissue-derived epithelial stem cells. For example, but not limited to, the volume of the hydrogel and tissue-derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In certain embodiments, the ratio of the volume of the hydrogel and tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel in the hydrogel and tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures above about 37°C. In certain embodiments, the hydrogel may be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is CULTREX® Basement Membrane Extract Type 2 (R&D Systems), which contains laminin, entactin, type IV collagen, and heparin sulfate proteoglycan. In certain embodiments, a mixture of suspended hydrogel and tissue-derived epithelial stem cells is dispensed into a culture medium in a specific geometric shape. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is spherical or spherical-like. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape.In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form. In certain embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the suspended hydrogel-tissue-derived epithelial stem cell mixture in a culture medium, for example, colonic transit medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies catalog no. 06010) + 10 μM Y27632) or ileal medium (OGM + 10 μM Y27632 + 2.5 μM CHIR99021) to produce colonic organoids or ileal organoids, respectively. In certain embodiments, the method may further include fragmenting the suspended hydrogel-tissue-derived epithelial stem cell mixture to produce fragmented structures containing tissue-derived epithelial organoids. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture by pipetting a culture medium containing the mixture of suspended hydrogel and tissue-derived epithelial stem cells up and down to generate fragmented structures. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells yields structures that are shorter in length and / or width. For example, but not limited to, fragmentation of filamentous structures yields structures that are shorter in length, width, or both. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in the Disclosure are contained within an organoid fragment. In certain embodiments, the method of the Disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, differentially expressed markers include MKI67, LGR5, SOX9, CD44, MUC2, MUC5B, TFF3, KRT20, FABP1, ALPI, and / or CEACAM7.

[0151] In certain embodiments, a method for producing lung organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells are approximately 1 × 10⁻⁶ 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7The method contains 1 ml of tissue-derived epithelial stem cells / hydrogel. In certain embodiments, the method may further include suspending the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of hydrogel and tissue-derived epithelial stem cells. For example, but not limited to, the volume of the hydrogel and tissue-derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In certain embodiments, the ratio of the volume of the hydrogel and tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel in the hydrogel and tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures above about 37°C. In certain embodiments, the hydrogel may be a commercially available ECM. A non-limiting example of an ECM is MATRIGEL®. In certain embodiments, the suspended hydrogel and tissue-derived epithelial stem cell mixture is dispensed into the culture medium in a specific geometric shape. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is spherical or spherical-like. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is filamentous. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form. In certain embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the mixture of suspended hydrogel and tissue-derived epithelial stem cells in a culture medium, for example, SFFF medium containing 10 μM of ROCK inhibitor. In certain embodiments, the method may further include fragmenting the mixture of suspended hydrogel and tissue-derived epithelial stem cells to produce fragmented structures containing tissue-derived epithelial organoids. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture of suspended hydrogel and tissue-derived epithelial stem cells by pipetting a culture medium containing the mixture up and down to generate fragmented structures.In certain embodiments, a shorter structure in length and / or width is obtained by fragmenting a mixture of a suspended hydrogel and tissue-derived epithelial stem cells. For example, but not limited to, a shorter structure in length, width, or both is obtained by fragmenting a filamentous structure. In certain embodiments, the tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in the Disclosure are contained within the organoid fragment. In certain embodiments, the method of the Disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids).

[0152] In certain embodiments, a method for producing mammary gland organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells are approximately 1 × 10⁶ 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7The method contains 1 ml of tissue-derived epithelial stem cells / hydrogel. In certain embodiments, the method may further include suspending the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of hydrogel and tissue-derived epithelial stem cells. For example, but not limited to, the volume of the hydrogel and tissue-derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In certain embodiments, the ratio of the volume of the hydrogel and tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel in the hydrogel and tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures above about 37°C. In certain embodiments, the hydrogel may be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is CULTREX® growth factor reduced BME type 2 (Trevigen, 3533-010-02). In certain embodiments, a mixture of suspended hydrogel and tissue-derived epithelial stem cells is dispensed into a culture medium in a specific geometric shape. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is spherical or spherical-like. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, the mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form. In certain embodiments, the method may further include culturing the tissue-derived epithelial stem cells of the mixture of suspended hydrogel and tissue-derived epithelial stem cells in a culture medium, for example, SFFF medium containing 10 μM of ROCK inhibitor. In certain embodiments, the method may further include fragmenting a mixture of a suspended hydrogel and tissue-derived epithelial stem cells to generate fragmented structures containing tissue-derived epithelial organoids.In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture by pipetting a culture medium containing the mixture of suspended hydrogel and tissue-derived epithelial stem cells up and down to generate fragmented structures. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells yields structures that are shorter in length and / or width. For example, but not limited to, fragmentation of filamentous structures yields structures that are shorter in length, width, or both. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in the Disclosure are contained within an organoid fragment. In certain embodiments, the method of the Disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, differentially expressed markers include EpCAM, CD49f, cytokeratin 8 (K8), cytokeratin 18 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), and / or smooth muscle actin (SMA).

[0153] In certain embodiments, a method for producing pancreatic organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells are approximately 1 × 10⁻⁶ 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7The method contains 1 ml of tissue-derived epithelial stem cells / hydrogel. In certain embodiments, the method may further include suspending the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of hydrogel and tissue-derived epithelial stem cells. For example, but not limited to, the volume of the hydrogel and tissue-derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In certain embodiments, the ratio of the volume of the hydrogel and tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel in the hydrogel and tissue-derived epithelial stem cell mixture is composed of a material that solidifies at temperatures above about 37°C. In certain embodiments, the hydrogel may be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. A non-limiting example of BME is the low growth factor BME 2-RGF (basement membrane extract type 2 3533-010-02, AMSBIO, CULTREX®). In certain embodiments, a mixture of suspended hydrogel and tissue-derived epithelial stem cells is dispensed into a culture medium in a specific geometric shape. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is spherical or spherical-like. In certain embodiments, the geometric shape of the mixture of suspended hydrogel and tissue-derived epithelial stem cells is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape. In certain embodiments, a mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form. In certain embodiments, the tissue-derived epithelial stem cells of the suspended hydrogel and tissue-derived epithelial stem cell mixture are added to an optimized human pancreatic organoid growth medium, for example, a basal medium containing 1X N2 and 1X B27 (both from GIBCO), 1.25 mM N-acetylcysteine ​​(Sigma-Aldrich), 10% RSPO1-prepared serum-free medium, and 10 nM [Leu] 15The method involves supplementing the culture medium with ]-Gastrin I human (Sigma-Aldrich), 50 ng / mL EGF (Peprotech), 25 ng / mL Noggin (Peprotech), 100 ng / mL FGF10 (Peprotech), 10 mM nicotinamide (Sigma-Aldrich), 5 μM A83.01 (Tocris), 10 μM FSK (Tocris), and 3 μM PGE2 (Tocris), and culturing in a medium supplemented with 10 μM Rho kinase inhibitor (Y27632, Sigma-Aldrich) for the first 7 days. In certain embodiments, the method may further include fragmenting a mixture of a suspended hydrogel and tissue-derived epithelial stem cells to generate fragmented structures containing tissue-derived epithelial organoids. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture by pipetting a culture medium containing the mixture of suspended hydrogel and tissue-derived epithelial stem cells up and down to generate fragmented structures. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells yields structures that are shorter in length and / or width. For example, but not limited to, fragmentation of filamentous structures yields structures that are shorter in length, width, or both. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in the Disclosure are contained within an organoid fragment. In certain embodiments, the method of the Disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoid is a pancreatic organoid, and the differentially expressed markers are CD133, LGR5, PDX1, SOX9, ALDH1A1, NEUROG3, NKX6.1, keratin 19 (KRT19), MUC1, INS, GCG, and / or AMY.

[0154] In certain embodiments, a method for producing liver organoids includes contacting tissue-derived epithelial stem cells with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, multiple tissue-derived epithelial stem cells can be combined with the hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells. In certain embodiments, the multiple tissue-derived epithelial stem cells comprise about 3,000 to about 10,000 tissue-derived epithelial stem cells per well of a plate (e.g., a 48-well plate). In certain embodiments, the method may further include suspending the mixture of the hydrogel and the tissue-derived epithelial stem cells in a culture medium to produce a suspended mixture of the hydrogel and the tissue-derived epithelial stem cells. For example, but not limited to, the volume of the hydrogel and tissue-derived epithelial stem cell mixture dispensed into the culture medium may be at least about 10 μL. In certain embodiments, the ratio of the volume of the hydrogel and tissue-derived epithelial stem cell mixture to the volume of the culture medium is about 1:2 to about 1:15. In certain embodiments, the hydrogel solidifies upon contact with the culture medium. For example, but not limited to, the hydrogel in a mixture of hydrogel and tissue-derived epithelial stem cells is composed of a material that solidifies at temperatures above approximately 37°C. In certain embodiments, the hydrogel may be a commercially available ECM. In certain embodiments, the ECM is a basement membrane extract (BME), which is a soluble form of the basement membrane. Non-limiting examples of ECMs include MATRIGEL® (BD Biosciences) or reduced growth factor BME 2 (basement membrane extract, type 2, Pathclear). In certain embodiments, the suspended hydrogel and tissue-derived epithelial stem cell mixture is dispensed into a culture medium in a specific geometric shape. In certain embodiments, the geometric shape of the suspended hydrogel and tissue-derived epithelial stem cell mixture is spherical or spherical-like. In certain embodiments, the geometric shape of the suspended hydrogel and tissue-derived epithelial stem cell mixture is a filamentous structure. In certain embodiments, the filamentous structure has a linear, serpentine, or spiral shape.In certain embodiments, a mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form. In certain embodiments, the method involves adding the tissue-derived epithelial stem cells from the suspended hydrogel and tissue-derived epithelial stem cell mixture to, for example, 1% N2 (GIBCO) and 1% B27 (GIBCO), 1.25 mM N-acetylcysteine ​​(Sigma), 10 nM gastrin (Sigma), and growth factors such as 50 ng / ml EGF (Peprotech) and 10% RSPO1 The prepared medium (in-house prepared) comprises AdDMEM / F12 (Invitrogen) supplemented with 100 ng / ml FGF10 (Peprotech), 25 ng / ml HGF (Peprotech), 10 mM nicotinamide (Sigma), 5 μM A83.01 (Tocris), and 10 μM FSK (Tocris), and may further include culturing in a culture medium 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 μM (Y27632, Sigma Aldrich) or hES cell cloning recovery solution (Stemgent) for the first three days after isolation to establish the medium. In certain embodiments, this medium is then replaced with a medium that does not contain, for example, Noggin, Wnt, Y27632, and hES cell cloning recovery solution. In certain embodiments, liver organoids are seeded and cultured for 7-10 days in the culture medium supplemented with BMP7 (25 ng / ml). In certain embodiments, this medium is then replaced with a differentiation medium, such as AdDMEM / F12 medium supplemented with 1% N2 and 1% B27, and containing EGF (50 ng / ml), gastrin (10 nM, Sigma), HGF (25 ng / ml), FGF19 (100 ng / ml), A8301 (500 nM), DAPT (10 μM), BMP7 (25 ng / ml), and dexamethasone (30 μM), in order to generate hepatocyte organoids. In certain embodiments, the method may further include fragmenting a mixture of suspended hydrogel and tissue-derived epithelial stem cells to generate fragmented structures containing tissue-derived epithelial organoids.In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells may include, for example, shearing the mixture by pipetting a culture medium containing the mixture of suspended hydrogel and tissue-derived epithelial stem cells up and down to generate fragmented structures. In certain embodiments, fragmentation of a mixture of suspended hydrogel and tissue-derived epithelial stem cells yields structures that are shorter in length and / or width. For example, but not limited to, fragmentation of filamentous structures yields structures that are shorter in length, width, or both. In certain embodiments, tissue-derived epithelial stem cells or a plurality of tissue-derived epithelial stem cells for use in the Disclosure are contained within an organoid fragment. In certain embodiments, the method of the Disclosure produces a population of tissue-derived epithelial organoids that express a marker at a different level, e.g., a higher or lower level, than a reference tissue-derived epithelial organoid (e.g., a population of reference tissue-derived epithelial organoids). In certain embodiments, the tissue-derived epithelial organoid is a liver organoid, and the differentially expressed markers are LGR5, ALB, CYP3A4, HNF4A, KRT19, KRT7, and / or SOX9.

[0155] In certain embodiments, one or more steps of the method of the disclosure may be carried out using a robot and / or automated component. In certain embodiments, the method of the disclosure may include the use of a robot and / or automated component for generating tissue-derived epithelial organoids. In certain embodiments, one or more steps of the method of the disclosure may be carried out using a robot and / or automated component to generate tissue-derived epithelial organoids. Non-limiting examples of robots and / or automated components that can be used in the method of the disclosure include automated liquid handlers (e.g., liquid handling robots), 3D printers, syringe pumps, electronic pipettes (e.g., with or without pipetting robots) or combinations thereof. Non-limiting examples of electronic pipettes (e.g., with pipetting robots) include the Assist Plus from Integra Biosciences. In certain embodiments, one or more steps of the method of the disclosure may be carried out by an automated liquid handler (e.g., a liquid handling robot).

[0156] In certain embodiments, the subculturing of tissue-derived epithelial organoid cultures produced by the method of the present disclosure is carried out by a robot and / or automated component. In certain embodiments, the automated robot can carry out any one of the methods described in "Organoid Maintenance" of Example 1 of the present disclosure, for example, the steps of: adding TrypLE Express; heating the organoid culture; triturating the organoid culture to dissociate cells; adding PBS; pelletizing cells; resuspending cells in BME; cooling the culture; seeding cells; overlaying organoids with culture medium; or a combination thereof. In certain embodiments, cell dissociation can be carried out using a robot and / or automated component. In certain embodiments, for example, changing the culture medium during organoid maintenance or during the generation of tissue-derived epithelial organoids can be carried out using a robot and / or automated component.

[0157] In certain embodiments, the production of BOBA and / or SOBA in cell culture medium is carried out by a robot and / or automated component. In certain embodiments, the automated robot can perform any one of the following steps: heating the medium; dispensing the organoid cell-BME solution as droplets into the warm medium to produce BOBA; dispensing the organoid cell-BME solution into the warm medium in a linear, serpentine, or helical motion in the XY plane to produce SOBA; tritulate the SOBA filament culture to produce SOBA fragments; changing the medium; or a combination thereof. In certain embodiments, dispensing the organoid cell-BME solution into the warm medium to produce BOBA and / or SOBA can be carried out using a robot and / or automated component, for example, by a liquid handling robot. In certain embodiments, changing the medium can be carried out using a robot and / or automated component, for example, by a liquid handling robot. In certain embodiments, the trituration of SOBA filament cultures to produce SOBA fragments can be carried out using robotic and / or automated components, for example, by a liquid handling robot.

[0158] In certain embodiments, high-throughput methods, such as those described herein, can be carried out using robots and / or automated components. For example, but not limited to, any one of the uses disclosed herein, such as those described in Section IV, can be carried out in part using robots and / or automated components. In certain embodiments, methods for screening drugs, such as therapeutic agents, and methods for performing genome screening using tissue-derived epithelial organoids of the Disclosure can be carried out in part using robots and / or automated components.

[0159] IV.How to use This disclosure provides methods for using the organoids of this disclosure or compositions comprising such organoids. In certain embodiments, the tissue-derived epithelial organoids of this disclosure can be used in screening assays. For example, but not limited to, this disclosure provides methods for screening drugs, such as therapeutic agents, and methods for performing genome screening using the tissue-derived epithelial organoids of this disclosure. In certain embodiments, organoid-based models can be generated using the tissue-derived epithelial organoids of this disclosure.

[0160] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to identify therapeutic agents having therapeutic effects. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to identify therapeutic agents that may be effective in preventing and / or treating diseases. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to identify therapeutic agents that may be effective in improving the symptoms of diseases.

[0161] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to investigate the biology and / or pathogenesis of diseases. For example, but not limited to, the organoids or compositions thereof of the present disclosure can be brought into contact with a drug to investigate the biology and / or pathogenesis of diseases.

[0162] In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to identify potentially toxic drugs, such as therapeutic agents. In certain embodiments, the organoids or compositions thereof of the present disclosure can be used to identify concentrations at which drugs, such as therapeutic agents, may be toxic.

[0163] In certain embodiments, a method for identifying a therapeutic agent that may be effective in preventing and / or treating a disease, a method for identifying a therapeutic agent that may be effective in improving the symptoms of a disease, and / or a therapeutic agent that may be toxic may include contacting a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the method may include contacting a composition containing a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids with a therapeutic agent. In certain embodiments, the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium as described herein.

[0164] In certain embodiments, a method for investigating the biology and / or pathogenesis of a disease may include contacting a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids with a drug. In certain embodiments, the method may include contacting a composition containing a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids with a drug. In certain embodiments, the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids is embedded in a hydrogel suspended in a culture medium as described herein.

[0165] In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for a period of about 1 minute to about 3 years, for example, 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, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 100 days. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 100 days. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 150 days. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 150 days. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 1 year. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 1 year. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 2 years. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 2 years. In certain embodiments, a drug, such as a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 1 minute to about 10 days.In certain embodiments, a drug, for example, a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a composition thereof) for about 15 minutes to about 10 days. In certain embodiments, a drug, for example, a therapeutic agent, is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids (or a 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 Contact for several days, approximately 9 to 10 days, approximately 15 minutes to 10 days, approximately 15 minutes to 9 days, approximately 15 minutes to 8 days, approximately 15 minutes to 7 days, approximately 15 minutes to 6 days, approximately 15 minutes to 5 days, approximately 15 minutes to 4 days, approximately 15 minutes to 3 days, approximately 15 minutes to 2 days, approximately 15 minutes to 1 day, approximately 1 day to 5 days, approximately 1 day to 2 days, approximately 2 days to 5 days, or approximately 2 days to 10 days. In certain embodiments, a drug, such as a therapeutic agent, is contacted with tissue-derived epithelial organoids or a group (or composition thereof) of tissue-derived epithelial organoids for approximately 2 to 10 days.

[0166] In certain embodiments, the method may involve contacting different populations (or compositions thereof) of tissue-derived epithelial organoids with increasing concentrations of a drug, such as a therapeutic agent, to enable dose-response testing.

[0167] In certain embodiments, the agent may be any agent for any purpose. In certain embodiments, the agent may be a molecule known to affect the biology and / or pathogenesis of a disease. Non-limiting examples of agents that may be used in the methods of this disclosure include peptides, polypeptides, small molecules, cells, gene editing systems, or nucleic acids. In certain embodiments, such agents may be agents that affect cell signaling, nucleic acid expression, protein expression, cell proliferation, cell differentiation, and / or cell survival.

[0168] In certain embodiments, the drug is a therapeutic agent. In certain embodiments, the therapeutic agent may 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 methods of this disclosure include peptide-based therapeutic agents, polypeptide-based therapeutic agents, small molecule therapeutic agents, cell-based therapeutic agents, gene editing systems, nucleic acid-based therapeutic agents, and combinations thereof.

[0169] In certain embodiments, the therapeutic agent is a peptide-based therapeutic agent. In certain embodiments, the peptide-based therapeutic agent comprises a peptide having a molecular weight of approximately 5,000 Da or less. Non-limiting examples of peptide-based therapeutic agents include growth factors, anti-infective agents, antifungal agents, antimicrobial agents, ligands for receptors, and tyrosine kinase inhibitors. Additional non-limiting examples of peptide therapeutic agents are disclosed in Wang et al. (2022) Signal Transduction and Targeted Therapy 7:48 (e.g., Tables 1 and 2), the contents of which are incorporated herein by reference in their entirety.

[0170] In certain embodiments, the therapeutic agent is a polypeptide-based therapeutic agent. Non-limiting examples of polypeptide-based therapeutic agents include antibody-based therapeutic agents such as antibodies and antibody-drug conjugates, hormones, and enzymes. In certain embodiments, the antibody may be an agonist antibody or an antagonist antibody. In certain embodiments, the antibody may 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.

[0171] In certain embodiments, the therapeutic agent is a small molecule therapeutic agent. For example, but not limited to, a small molecule therapeutic agent is a compound having a molecular weight of less than approximately 1,000 Da. In certain embodiments, small molecule therapeutic agents 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, antifungal agents, antibacterial agents, chemotherapeutic agents, and anti-inflammatory agents.

[0172] In certain embodiments, the therapeutic agent is a cell-based therapeutic agent. Non-limiting examples of cell-based therapeutic agents include bacterial cells and immune cells. In certain embodiments, non-limiting examples of immune cells include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells (NK cells), and lymphocytes, such as 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 may be chimeric antigen receptors (CARs), such as CAR T cells and CAR NK cells, or modified immune cells genetically engineered to express T cell receptors (TCRs), such as heterologous TCRs.

[0173] In certain embodiments, the therapeutic agent is a gene regulatory system and / or a component of a gene regulatory system. In certain embodiments, the gene regulatory system and / or a component of a gene regulatory system is a gene editing system, CRISPRi, a gene expression promoter, a gene repressor promoter, a nucleic acid-based therapeutic agent, a transcription factor and / or a post-transcriptional modification regulator.

[0174] In certain embodiments, the therapeutic agent is a gene editing system. Non-limiting examples of gene editing systems include homing endonucleases or meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR gene editing systems. In certain embodiments, the therapeutic agent is a CRISPR gene editing system, such as a CRISPR / Cas9 gene editing system.

[0175] In certain embodiments, the therapeutic agent is a nucleic acid-based therapeutic agent. Non-limiting examples of nucleic acid-based therapeutic agents include RNA-based therapeutic agents such as siRNA, microRNA, RNA aptamers, ribozymes, RNA decoys, and RNAi. In certain embodiments, nucleic acid-based therapeutic agents include DNA-based therapeutic agents, including antisense oligonucleotides (ASOs) and DNA aptamers.

[0176] In certain embodiments, the method may further include analyzing changes in tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids). In certain embodiments, the method may further include analyzing changes in tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids) that occur in the presence of a drug. For example, but not limited to, the method may further include analyzing changes in tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids (or cells of tissue-derived epithelial organoids) that indicate the efficacy and / or toxicity of a therapeutic agent. In certain embodiments, the method includes analyzing changes in tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids that indicate the efficacy and / or toxicity of a therapeutic agent compared to tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids that have not been treated with the therapeutic agent.

[0177] In certain embodiments, the organoids or compositions thereof of this disclosure can be used to perform genome screening. In certain embodiments, genome screening may be used to identify gene editing systems that induce mutations. Non-limiting examples of mutations include deletions, duplications, insertions, and nucleotide substitutions. In certain embodiments, the method may include preparing tissue-derived epithelial organoids or a population (or composition thereof) of tissue-derived epithelial organoids, and inducing mutations in the genome of one or more cells of the tissue-derived epithelial organoids. In certain embodiments, the mutations are produced using gene regulatory systems and / or components of gene regulatory systems. In certain embodiments, gene regulatory systems and / or components of gene regulatory systems are gene editing systems, CRISPRi, RNAi, gene expression promoters, gene repressor promoters, nucleic acid-based therapeutics, transcription factors, and / or post-transcriptional modification regulators. In certain embodiments, the gene editing system is a CRISPR system, e.g., a CRISPR / Cas9 gene editing system. In certain embodiments, the method may further include analyzing changes in tissue-derived epithelial organoids or a population of tissue-derived epithelial organoids associated with the mutations. In certain embodiments, the method includes a step of analyzing changes in a population of tissue-derived epithelial organoids associated with mutations, compared to a population of tissue-derived epithelial organoids that do not have mutations, or a population of tissue-derived epithelial organoids.

[0178] In certain embodiments, the Disclosure further provides a method for generating an epithelial cell model using the tissue-derived epithelial organoids of the Disclosure. In certain embodiments, the Method may include preparing a tissue-derived epithelial organoid or a population (or composition thereof) of tissue-derived epithelial organoids, digesting the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids into single cells, and culturing the single cells in a culture medium to produce a cell monolayer. In certain embodiments, the single cells are cultured on a permeable cell culture insert. In certain embodiments, the culture medium is a differentiation medium. In certain embodiments, the culture medium is a stem cell promoting medium. In certain embodiments, the culture medium is a cell proliferation medium.

[0179] In certain embodiments, the cell monolayer can be used in any of the methods disclosed herein, for example, in the screening methods disclosed herein. For example, but not limited to, the cell monolayer can be used to perform therapeutic drug screening and genomic screening. In certain embodiments, the monolayer of this disclosure can be used to investigate the biology and / or pathogenesis of disease.

[0180] In certain embodiments, a method for screening therapeutic agents using cell monolayers of the present disclosure may include contacting cell monolayers and analyzing changes in the cell monolayers that indicate the efficacy, pharmacokinetics, and / or toxicity of the therapeutic agent. In certain embodiments, the method includes the step of analyzing changes in cell monolayers that indicate the efficacy and / or toxicity of the therapeutic agent compared to cell monolayers that have not been treated with the therapeutic agent.

[0181] In certain embodiments, a method for performing genome screening using a cell monolayer of the present disclosure may include preparing a cell monolayer produced by the method described herein, inducing mutations in the genome of one or more cells in the cell monolayer, and analyzing the changes in the cell monolayer associated with the mutations. In certain embodiments, the method includes analyzing the changes in the cell monolayer associated with the mutations compared to a cell monolayer without mutations.

[0182] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells) or a population of cell monolayers (or their cells) include changes in cell viability, changes in cell proliferation, changes in organoid size, changes in cell morphology, changes in organoid shape, changes in invasiveness to hydrogels, changes in motility, changes in differentiation state, changes in mutation state, changes in karyotype, changes in chromosomal aberrations, changes in nucleic acid expression levels, changes in protein expression levels, changes in nucleic acid modifications (e.g., methylation), changes in post-translational modifications (e.g., phosphorylation, ubiquitination, and / or glycosylation), changes in activation of cell signaling pathways, These changes may include alterations in the suppression of cellular signaling pathways, changes in enzyme activity (e.g., enzymatic cleavage), changes in chromatin accessibility, histone modifications and other epigenetic changes, changes in the physical properties of organoids, such as changes in the permeability of the gastrointestinal epithelial barrier, changes in the pH of other metabolites in the lumen and basement membrane, changes in oxygen tension and concentration, as well as changes in secreted factors in the hydrogel and / or culture medium, changes in cytokine and hormone concentrations, changes in drug sensitivity, changes in the pharmacokinetics and pharmacodynamics of drug absorption and metabolism, changes in force measurements within organoids, lumens and culture medium / hydrogel, changes in measurements of biomolecular interactions, and changes in membrane potential.

[0183] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), a population of tissue-derived epithelial organoids (or their cells), or a cell monolayer (or their cells) may be changes in cell viability.

[0184] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), a population of tissue-derived epithelial organoids (or their cells), or a cell monolayer (or their cells) may be changes in cell proliferation.

[0185] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), a population of tissue-derived epithelial organoids (or their cells), or a cell monolayer (or their cells) may be changes in the size of the organoids.

[0186] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), tissue-derived epithelial organoids (or their cells), or a population of cell monolayers (or their cells) may be changes in the mutation state.

[0187] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), a population of tissue-derived epithelial organoids (or their cells), or a cell monolayer (or their cells) may be changes in RNA expression levels and / or protein expression levels.

[0188] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells) in a population or cell monolayer (or their cells) may be changes in the RNA expression levels and / or protein expression levels of stem cell markers and / or proliferation markers. In certain embodiments, changes in tissue-derived epithelial organoids in a population or cell monolayer may be changes in the protein expression levels of stem cell markers and / or proliferation markers. In certain embodiments, changes in tissue-derived epithelial organoids in a population or cell monolayer may be changes in the RNA expression levels of stem cell markers and / or proliferation markers. For example, but not limited to, changes may include changes in MKI67 expression, EpCAM expression, CD49f expression, ASCL2 expression, CD133 expression, LGR5 expression, SOX9 expression, ALDH1A1 expression, NEUROG3 expression, NKX6.1 expression, SMOC2 expression, PDX1 expression, BMI1 expression, and / or CD44 expression. In certain embodiments, the change may be a change in MKI67 expression. In certain embodiments, the change may be a change in ASCL2 expression. In certain embodiments, the change may be a change in LGR5 expression. In certain embodiments, the change may be a change in SOX9 expression. In certain embodiments, the change may be a change in SMOC2 expression. In certain embodiments, the change may be a change in CD44 expression. In certain embodiments, the change may be a change in EpCAM expression. In certain embodiments, the change may be a change in CD49f expression. In certain embodiments, the change may be a change in CD133 expression. In certain embodiments, the change may be a change in ALDH1A1 expression. In certain embodiments, the change may be a change in NEUROG3 expression. In certain embodiments, the change may be a change in NKX6.1 expression. In certain embodiments, the change may be a change in PDX1 expression. In certain embodiments, the change may be a change in BMI1 expression.

[0189] In certain embodiments, changes in tissue-derived epithelial organoids (or their cells), a population of tissue-derived epithelial organoids (or their cells), or a cell monolayer (or their cells) may be changes in the RNA expression levels and / or protein expression levels of differentiation markers. In certain embodiments, changes in tissue-derived epithelial organoids in a population of tissue-derived epithelial organoids or a cell monolayer may be changes in the RNA expression levels of differentiation markers. In certain embodiments, changes in tissue-derived epithelial organoids in a population of tissue-derived epithelial organoids or a cell monolayer may be changes in the protein expression levels of differentiation markers. For example, but not limited to, changes may include changes in keratin 20 (KRT20) expression, FABP1 expression, MUC2 expression, MUC5B expression, MUC5AC expression, MUC6 expression, TFF3 expression, ALPI expression, SI expression, CEACAM7 expression, keratin 19 (KRT19) expression, keratin 7 (KRT7) expression, SOX9 expression, MUC1 expression, INS expression, GCG expression, AMY expression, ALB expression, CYP3A4 expression, HNF4A expression, cytokeratin 8 (K8) expression, cytokeratin 18 (K18) expression, cytokeratin 5 (K5) expression, cytokeratin 14 (K14) expression, and / or smooth muscle actin (SMA) expression. In certain embodiments, the change may be a change in KRT20 expression. In certain embodiments, the change may be a change in FABP1 expression. In certain embodiments, the change may be a change in MUC2 expression. In certain embodiments, the change may be a change in MUC5B expression. In certain embodiments, the change may be a change in TFF3 expression. In certain embodiments, the change may be a change in ALPI expression. In certain embodiments, the change may be a change in SI expression. In certain embodiments, the change may be a change in CEACAM7 expression. In certain embodiments, the change may be a change in keratin 19 (KRT19) expression. In certain embodiments, the change may be a change in keratin 7 (KRT7) expression. In certain embodiments, the change may be a change in SOX9 expression. In certain embodiments, the change may be a change in MUC1 expression.In certain embodiments, the change may be a change in INS expression. In certain embodiments, the change may be a change in GCG expression. In certain embodiments, the change may be a change in AMY expression. In certain embodiments, the change may be a change in ALB expression. In certain embodiments, the change may be a change in CYP3A4 expression. In certain embodiments, the change may be a change in HNF4A expression. In certain embodiments, the change may be a change in cytokeratin 8 (K8) expression. In certain embodiments, the change may be a change in cytokeratin 18 (K18) expression. In certain embodiments, the change may be a change in cytokeratin 5 (K5) expression. In certain embodiments, the change may be a change in cytokeratin 14 (K14) expression. In certain embodiments, the change may be a change in smooth muscle actin (SMA) expression. In certain embodiments, the change may be a change in MUC5AC expression. In certain embodiments, the change may be a change in MUC6 expression.

[0190] In certain embodiments, tissue-derived epithelial organoid cultures or monolayer cultures can be analyzed by flow cytometry, RNA and protein expression in organoids and culture media, cytokine and metabolite measurement, cell viability and proliferation assays, microscopy to monitor epithelial and immune cell motility, barrier function, migration, proliferation, intracellular localization of RNA, proteins and organelles, and cell stiffness, as well as other assays, in order to investigate the transformation of tissue-derived epithelial organoids, the cells of tissue-derived epithelial organoids, cell monolayers, and / or cell monolayers.

[0191] In certain embodiments, the present disclosure provides a method for identifying therapeutic agents that may be effective in treating a disease. In certain embodiments, the method may include (i) contacting (a) tissue-derived epithelial organoids (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), (c) a cell monolayer derived from tissue-derived epithelial organoids, or (d) a population of tissue-derived epithelial organoids with a therapeutic agent, and (ii) analyzing changes in the tissue-derived epithelial organoids or population of tissue-derived epithelial organoids that indicate the efficacy and / or toxicity of the therapeutic agent. In certain embodiments, the changes indicate the efficacy of the therapeutic agent. In certain embodiments, the changes indicate the toxicity of the therapeutic agent. For example, but not limited to, if the viability of tissue-derived epithelial organoids or cells of tissue-derived epithelial organoids decreases in the presence of the therapeutic agent compared to the viability of tissue-derived epithelial organoids or cells of tissue-derived epithelial organoids that were not treated with the therapeutic agent, this is an indicator that the therapeutic agent is toxic.

[0192] In certain embodiments, the Disclosure provides a method for performing genome screening. In certain embodiments, the method may include (i) (a) preparing a tissue-derived epithelial organoid (or a composition thereof), (b) a population of tissue-derived epithelial organoids (or a composition thereof), or (c) a cell monolayer derived from a tissue-derived epithelial organoid; (ii) inducing a mutation in the genome of one or more cells in the tissue-derived epithelial organoid or cell monolayer; and (iii) analyzing the changes in the tissue-derived epithelial organoid or population of tissue-derived epithelial organoids associated with the mutation. For example, but not limited to, if changes are observed in a cell monolayer containing a tissue-derived epithelial organoid or one or more cells having a genomic mutation compared to a reference cell or tissue-derived epithelial organoid, this indicates that the changes are a result of a genomic mutation.

[0193] V. System This disclosure further provides systems for use in the present disclosure. In certain embodiments, this disclosure further provides systems for carrying out the methods of the present disclosure. In certain embodiments, this disclosure provides systems for generating tissue-derived epithelial organoids, such as 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblast organoids. In certain embodiments, this disclosure provides systems for identifying therapeutic agents that may be effective in treating diseases. In certain embodiments, this disclosure provides systems for performing genome screening. In certain embodiments, this disclosure provides systems for generating epithelial cell models.

[0194] In certain embodiments, the system of the Disclosure may include a robot and / or automated component capable of carrying out the method of the Disclosure. In certain embodiments, the system of the Disclosure includes a robot and / or automated component capable of producing tissue-derived epithelial organoids. In certain embodiments, the system of the Disclosure includes one or more robots and / or automated components for carrying out one or more steps of the method of the Disclosure for producing tissue-derived epithelial organoids. Non-limiting examples of robots and / or automated components include automated liquid handlers (e.g., liquid handling robots), 3D printers, syringe pumps, or combinations thereof. In certain embodiments, the system of the Disclosure includes one or more automated liquid handlers.

[0195] In certain embodiments, robots and / or automated components are used to carry out the methods disclosed herein and / or to generate tissue-derived epithelial organoids for use in high-throughput experiments. In certain embodiments, the system of the disclosure may include one or more robots and / or automated components for carrying out high-throughput methods, as described herein, for example. For example, but not limited to, the system of the disclosure may include one or more robots and / or automated components that can be used to carry out any one of the methods of use disclosed herein. In certain embodiments, the system of the disclosure may include one or more robots and / or automated components for screening drugs, for example therapeutic agents, and for performing genome screening using the tissue-derived epithelial organoids of the disclosure.

[0196] In certain embodiments, a robot and / or automated component can subculture a tissue-derived epithelial organoid culture. In certain embodiments, the automated robot can perform any one of the methods described in "Organoid Maintenance" of Example 1 of the Disclosure, for example, the following steps: adding TrypLE Express; heating the organoid culture; triturating the organoid culture to dissociate cells; adding PBS; pelletizing cells; resuspending cells in BME; cooling the culture; seeding cells; overlaying organoids with culture medium; or any combination thereof. In certain embodiments, the system of the Disclosure may include one or more robots and / or automated components for dissociating cells. In certain embodiments, the system of the Disclosure may include one or more robots and / or automated components for performing culture medium changes, for example, during organoid maintenance or during the generation of tissue-derived epithelial organoids.

[0197] In certain embodiments, a robot and / or automated component can produce BOBA and / or SOBA. In certain embodiments, an automated robot can perform any one of the following steps: heating a culture medium; dispensing the organoid cell-BME solution as droplets into a warm culture medium to produce BOBA; dispensing the organoid cell-BME solution into a warm culture medium in a linear, serpentine, or helical motion in the XY plane to produce SOBA; triturating a SOBA filament culture to produce SOBA fragments; changing the culture medium; or a combination thereof. In certain embodiments, the system of the disclosure may include one or more robots and / or automated components for dispensing the organoid cell-BME solution into a warm culture medium to produce BOBA and / or SOBA. In certain embodiments, the system of the Disclosure may include one or more liquid handling robots for dispensing organoid cell-BME solution into a warming medium to generate BOBA and / or SOBA. In certain embodiments, the system of the Disclosure may include one or more robots and / or automated components for performing medium changes.

[0198] In certain embodiments, the system of the present disclosure may include tissue-derived epithelial organoids, a population of tissue-derived epithelial organoids and / or tissue-derived epithelial organoids, such as 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, or trophoblast organoids. In certain embodiments, the tissue-derived epithelial organoids are selected from the group consisting of lung organoids, gastrointestinal organoids, liver organoids, pancreatic organoids, mammary gland organoids and combinations thereof. In certain embodiments, the system includes tissue-derived epithelial organoids 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, tissue-derived epithelial organoids may be embedded in a hydrogel and suspended in a culture medium. Non-limiting examples of tissue-derived epithelial organoids (or compositions thereof) or methods for producing such tissue-derived epithelial organoids are disclosed in Sections II and III, respectively. In certain embodiments, tissue-derived epithelial organoids may be provided in a container, such as a culture vessel. In certain embodiments, tissue-derived epithelial organoids may be provided in a culture vessel, such as a flask and / or a multiwell plate.

[0199] In certain embodiments, the system of the disclosure may further include instructions for using tissue-derived epithelial organoids to determine whether a therapeutic agent under test is effective in treating a disease. In certain embodiments, the system of the disclosure may further include instructions for using tissue-derived epithelial organoids to investigate the effects of genomic mutations. In certain embodiments, the system of the disclosure may further include instructions for generating epithelial cell models.

[0200] In certain non-limiting embodiments, the system of the Disclosure may further include one or more reagents and other components (e.g., dyes, antibodies, primers, probes, etc.) for examining changes in tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, or cells of cell monolayers, such as changes in protein or nucleic acid expression. Non-limiting examples of such changes are disclosed in Section III. In certain embodiments, the system of the Disclosure may include one or more robots and / or automated components for analyzing changes in tissue-derived epithelial organoids, cells of tissue-derived epithelial organoids, cell monolayers, or cells of cell monolayers.

[0201] VI. Exemplary Embodiments A. The subject matter of this disclosure is a method for generating tissue-derived epithelial organoids, a) To produce a mixture of tissue-derived epithelial stem cells and tissue-derived epithelial stem cells by contacting tissue-derived epithelial stem cells with a hydrogel. b) Suspending a mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a mixture of suspended hydrogel and tissue-derived epithelial stem cells, and c) A method is provided which includes culturing a mixture of a suspended hydrogel and tissue-derived epithelial stem cells in a culture medium to produce tissue-derived epithelial organoids.

[0202] A1. The method according to A, wherein multiple tissue-derived epithelial stem cells are brought into contact with a hydrogel to produce a mixture of the hydrogel and the tissue-derived epithelial stem cells.

[0203] A2. Multiple tissue-derived epithelial stem cells, approximately 1 × 10 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7 The method according to A1, comprising 1 ml of tissue-derived epithelial stem cell / hydrogel.

[0204] A3. The method according to A1 or A2, wherein multiple tissue-derived epithelial stem cells are contained in a tissue fragment, organoid fragment, or a combination thereof.

[0205] A4. The method described in A to A2, wherein tissue-derived epithelial stem cells or multiple tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

[0206] A5. A method according to any one of A to A4, wherein the hydrogel solidifies upon contact with the culture medium.

[0207] A6. The method according to any one of A to A5, wherein suspending the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium is performed by immersing a dispensing device containing the mixture of hydrogel and tissue-derived epithelial stem cells in a culture medium and dispensing the mixture of hydrogel and tissue-derived epithelial stem cells into the culture medium.

[0208] A7. The method according to any one of A to A6, wherein the temperature of the culture medium is approximately 25°C to approximately 50°C.

[0209] A7-1. The method according to any one of A to A7, wherein the culture medium temperature is approximately 30°C to approximately 50°C.

[0210] A8. The method according to any one of A to A7-1, wherein the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 25°C.

[0211] A8-1. The method according to any one of A to A8, wherein the temperature of the mixture of hydrogel and tissue-derived epithelial stem cells is approximately 2°C to approximately 20°C.

[0212] A9. The method according to any one of A to A8-1, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

[0213] A10. The method according to A9, wherein the natural hydrogel contains a basement membrane extract (BME) component or an extracellular matrix (ECM) component.

[0214] A11. The method according to any one of A to A10, wherein the hydrogel has a storage modulus G' greater than or equal to the loss modulus G''.

[0215] A12. The method according to any one of A to A11, wherein the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width and / or diameter greater than approximately 0.1 mm.

[0216] A13. The method according to A12, wherein the mixture of suspended hydrogel and tissue-derived epithelial stem cells has a geometric shape having a length, width, and / or diameter of approximately 0.1 mm to approximately 1,000 mm.

[0217] A14. The method according to any one of A to A13, wherein a mixture of hydrogel and tissue-derived epithelial stem cells is suspended in a culture medium in droplet form.

[0218] A15. The method according to any one of A to A13, wherein the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a filamentous structure.

[0219] A16. The method according to A15, wherein the filamentous structure has a linear, serpentine, or spiral shape.

[0220] A17. The method according to any one of A to A16, further comprising fragmenting a mixture of a suspended hydrogel and tissue-derived epithelial stem cells to produce a fragmented structure containing tissue-derived epithelial organoids.

[0221] B. A method for producing turbid cultures of tissue-derived epithelial organoids, a) Introducing a mixture containing a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspension mixture, and b) A method comprising culturing a suspension mixture in a culture medium to produce turbid tissue-derived epithelial organoids.

[0222] B1. The method according to B, wherein the mixture introduced into the culture medium comprises a hydrogel and multiple tissue-derived epithelial stem cells.

[0223] B2. Multiple tissue-derived epithelial stem cells, approximately 1 × 10 4 Individual tissue-derived epithelial stem cells / hydrogel 1 ml ~ approximately 1 x 10 7The method according to B1, comprising 1 ml of tissue-derived epithelial stem cells / hydrogel.

[0224] B3. The method according to B1 or B2, wherein multiple tissue-derived epithelial stem cells are contained in a tissue fragment, organoid fragment, or a combination thereof.

[0225] B4. The method according to any one of B to B2, wherein tissue-derived epithelial stem cells or multiple tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

[0226] B5. A method according to any one of B to B4, wherein the hydrogel solidifies upon contact with the culture medium.

[0227] B6. The method according to any one of B to B5, wherein introducing the mixture into the culture medium involves immersing a dispensing device containing the mixture into the culture medium and dispensing the mixture into the culture medium.

[0228] B7. The method according to any one of B to B6, wherein the temperature of the culture medium is approximately 25°C to approximately 50°C.

[0229] B7-1. The method according to any one of B to B7, wherein the temperature of the culture medium is approximately 30°C to approximately 50°C.

[0230] B8. The method according to any one of B to B7-1, wherein the temperature of the mixture is approximately 2°C to approximately 25°C.

[0231] B8-1. The method according to any one of B to B8, wherein the temperature of the mixture is approximately 2°C to approximately 20°C.

[0232] B9. The method according to any one of B to B8-1, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

[0233] B10. The method according to B9, wherein the natural hydrogel contains a basement membrane extract (BME) component or an extracellular matrix (ECM) component.

[0234] B11. The method according to any one of B to B10, wherein the hydrogel has a storage modulus G' greater than or equal to the loss modulus G''.

[0235] B12. The method according to any one of B to B11, wherein the suspended mixture has a geometric shape having a length, width and / or diameter greater than approximately 0.1 mm.

[0236] B13. The method according to B12, wherein the suspended mixture has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0237] B14. The method according to any one of B to B13, wherein the mixture is introduced into the culture medium in droplet form.

[0238] B15. The method according to any one of B to B3, wherein the mixture is introduced into the culture medium in a filamentous structure.

[0239] B16. The method according to B15, wherein the filamentous structure has a linear, serpentine, or spiral shape.

[0240] B17. The method according to any one of B to B16, further comprising fragmenting a suspension mixture to produce a fragmented structure containing tissue-derived epithelial organoids.

[0241] B18. The method according to any one of A to B17, wherein the culture medium is contained in a container selected from the group consisting of a petri dish, a multiwell plate, a conical tube, a reservoir, a culture bag, a bioreactor, or a flask.

[0242] C. A method for producing a turbid culture of tissue-derived epithelial organoids, a) To produce a mixture of tissue-derived epithelial stem cells and tissue-derived epithelial stem cells by contacting tissue-derived epithelial stem cells with a hydrogel. b) Depositing a mixture of hydrogel and tissue-derived epithelial stem cells onto a substrate. c) Solidifying a mixture of a hydrogel and tissue-derived epithelial stem cells to produce a solidified mixture of a hydrogel and tissue-derived epithelial stem cells; d) Suspending the solidified mixture of a hydrogel and tissue-derived epithelial stem cells in a medium to produce a suspended mixture of a hydrogel and tissue-derived epithelial stem cells, and e) A method comprising culturing the suspended mixture of a hydrogel and tissue-derived epithelial stem cells in a medium to produce tissue-derived epithelial organoids.

[0243] C1. The method according to C, wherein a plurality of tissue-derived epithelial stem cells are contacted with a hydrogel to produce a mixture of a hydrogel and tissue-derived epithelial stem cells.

[0244] C2. The method according to C1, wherein the plurality of tissue-derived epithelial stem cells contain from about 1×10 4 tissue-derived epithelial stem cells / ml of hydrogel to about 1×10 7 tissue-derived epithelial stem cells / ml of hydrogel.

[0245] C3. The method according to C1 or C2, wherein the plurality of tissue-derived epithelial stem cells are contained in tissue fragments, organoid fragments or a combination thereof.

[0246] C4. The method according to any one of C to C2, wherein the tissue-derived epithelial stem cells or the plurality of tissue-derived epithelial stem cells are isolated from primary epithelial tissue.

[0247] C5. The method according to any one of C to C3, further comprising removing the solidified mixture of a hydrogel and tissue-derived epithelial stem cells from a substrate before suspending the solidified mixture of a hydrogel and tissue-derived epithelial stem cells in a medium.

[0248] C6. The method according to any one of C to C4, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

[0249] The method according to C6, wherein the natural hydrogel comprises a basement membrane extract (BME) component or an extracellular matrix (ECM) component.

[0250] C8. The method according to any one of C1 to C7, wherein the hydrogel has a storage modulus G' that is greater than or equal to the loss modulus G".

[0251] C9. The method according to any one of C1 to C8, wherein the mixture of the solidified hydrogel and the tissue-derived epithelial stem cells has a geometric shape with a length, width, and / or diameter greater than about 0.1 mm.

[0252] C10. The method according to C9, wherein the mixture of the solidified hydrogel and the tissue-derived epithelial stem cells has a geometric shape with a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0253] C11. The method according to any one of C1 to C10, wherein the mixture of the hydrogel and the tissue-derived epithelial stem cells is deposited on a substrate as droplets.

[0254] C12. The method according to any one of C1 to C10, wherein the mixture of the hydrogel and the tissue-derived epithelial stem cells is deposited on a substrate and has a filament-like structure.

[0255] C13. The method according to C12, wherein the filament-like structure has a linear, serpentine, or helical shape.

[0256] C14. The method according to any one of C1 to C13, further comprising fragmenting the mixture of the hydrogel and the tissue-derived epithelial stem cells in a medium to generate a fragmented structure comprising tissue-derived epithelial organoids.

[0257] C15. The method according to any one of C1 to C14, wherein the tissue-derived epithelial organoids have a uniform morphology compared to reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

[0258] C16. The method according to C15, wherein the tissue-derived epithelial organoids have a uniform size.

[0259] C17. The method according to C16, wherein the average diameter of the tissue-derived epithelial organoids is more uniform than that of the reference tissue-derived epithelial organoids.

[0260] C18. The method according to any one of A to C17, wherein a stem cell marker and / or proliferation marker is expressed at a higher level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

[0261] C19. The method according to C18, wherein the stem cell marker 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 combinations thereof.

[0262] C20. The method according to any one of A to C18, wherein the differentiation marker is expressed at a lower level in the tissue-derived epithelial organoid population compared to the reference tissue-derived epithelial organoid population, and the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate.

[0263] C21. The method according to C20, 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0264] A tissue-derived epithelial organoid produced by any one of the methods described in DA-C21.

[0265] D1. The tissue-derived epithelial organoid described in D, wherein the tissue-derived epithelial organoid is selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblast organoids, and combinations thereof.

[0266] E. A composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium.

[0267] E1. The composition according to E, wherein the hydrogel has a geometric shape having a length, width and / or diameter greater than about 0.1 mm.

[0268] E2. The composition according to E1, wherein the hydrogel has a geometric shape having a length, width, and / or diameter of about 0.1 mm to about 1,000 mm.

[0269] E3. One of the compositions E to E2, wherein the hydrogel is a droplet.

[0270] E4. A composition from any one of E to E2, wherein the hydrogel has a filamentous structure.

[0271] E5. The composition according to E4, wherein the filamentous structure has a linear, serpentine, or spiral shape.

[0272] E6. A composition according to any one of E1 to E5, wherein a stem cell marker and / or a proliferation marker is expressed at a higher level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

[0273] E7. A composition according to E6, wherein the stem cell marker and / or the 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 combinations thereof.

[0274] E8. A composition according to any one of E1 to E5, wherein a differentiation marker is expressed at a lower level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

[0275] E9. A composition according to 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0276] F. A method for screening a drug, comprising: a) contacting a tissue-derived epithelial organoid or a population of tissue-derived epithelial organoids according to D or D1, or a composition according to any one of E1 to E9 with the drug, and b) The method comprising analyzing changes in a population of tissue-derived epithelial organoids or tissue-derived epithelial organoids that exhibit efficacy and / or toxicity of a drug.

[0277] F1. The method according to F, wherein the drug is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids for approximately 1 minute to approximately 3 years.

[0278] F1-1. The method according to F1, wherein the drug is brought into contact with tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids for approximately 15 minutes to approximately 3 years.

[0279] F2. The method according to F, F1, or F1-1, wherein the drug is a therapeutic agent.

[0280] F3. The method according to F2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

[0281] G. A method for performing genome screening, a) Prepare tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids described in D or D1, or one of the compositions described in E to E9. b) to induce mutations in the genome of one or more cells of tissue-derived epithelial organoids, and c) The method comprising analyzing changes in tissue-derived epithelial organoids or populations of tissue-derived epithelial organoids related to the mutation.

[0282] G1. The method described in G, which involves inducing mutations using a gene regulatory system.

[0283] G2. The method described in G1, in which the gene regulatory system is a gene editing system.

[0284] G3. The gene editing system used is the CRISPR system, as described in G2.

[0285] G4. The method according to any one of F-G2, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, reduction potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, suppression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

[0286] H. A method for generating an epithelial cell model, a) Prepare the tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids described in D. b) Digesting tissue-derived epithelial organoids or a group of tissue-derived epithelial organoids into single cells, and c) A method comprising culturing a single cell in a culture medium to produce a cell monolayer.

[0287] H1. The method according to H, wherein a single cell is cultured on a permeable cell culture insert.

[0288] The method described in H or H1, wherein the medium is a differentiation medium.

[0289] H3. The method according to H or H1, wherein the culture medium is a cell proliferation medium or a stem cell promoting medium.

[0290] I. A method for screening drugs, a) Contacting a cell monolayer produced by any one of the methods described in H~H3 with a drug, and b) The method comprising analyzing changes in a cellular monolayer that indicate the efficacy, pharmacokinetics, and / or toxicity of a drug.

[0291] I1. The method described in I, in which the drug is brought into contact with a cell monolayer for approximately 1 minute to approximately 3 years.

[0292] I1-1. The method described in I1, in which the drug is brought into contact with a cell monolayer for approximately 15 minutes to approximately 3 years.

[0293] I2. The method according to I, I1, or I1-1, wherein the drug is a therapeutic agent.

[0294] I3. The method according to I2, wherein the therapeutic agent is a polypeptide-based therapeutic agent, a small molecule therapeutic agent, a cell therapeutic agent, a gene editing system, a nucleic acid-based therapeutic agent, or a combination thereof.

[0295] J. A method for performing genome screening, a) Prepare a cell monolayer produced by one of the methods described in H to H3. b) to induce mutations in the genome of one or more cells in a cell monolayer, and c) The method, which includes analyzing changes in the cellular monolayer related to the mutation.

[0296] J1. The method described in J, which involves inducing a mutation using a gene regulatory system.

[0297] J2. The method described in J1, wherein the gene regulatory system is a gene editing system.

[0298] J3. The method described in J2, where the gene editing system is the CRISPR system.

[0299] J4. The method according to any one of J-J3, wherein the change is a change in a property selected from the group consisting of cell viability, cell metabolism, reduction potential, cell proliferation, cell morphology, organoid morphology, organoid size, protein expression level, nucleic acid expression level, nucleic acid modification, post-translational modification, activation of cell signaling pathways, suppression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

[0300] J1. The composition according to any one of A-C21 and F-J4 or any one of E-E9, wherein the tissue fragment is a fragment from tissue selected from the group consisting of lacrimal gland, tonsil, salivary gland, gastrointestinal tissue, thyroid gland, lung, mammary gland, liver, bile duct, stomach, kidney, pancreas, endometrium, fallopian tube, cervix, prostate, bladder, taste bud, ovary, placenta, and combinations thereof.

[0301] J5. A composition according to any one of A to C21 and F to J4 or any one of E to E9, wherein tissue-derived epithelial stem cells are obtained from fragments of organoids selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblast organoids, and combinations thereof.

[0302] K. A system for culturing tissue-derived epithelial organoids, comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium.

[0303] K1. The system described in K, in which tissue-derived epithelial organoids are intestinal organoids.

[0304] K2. The system according to K or K1, wherein the hydrogel has a geometric shape having a length, width and / or diameter greater than approximately 0.1 mm.

[0305] K3. The system according to any one of K to K2, wherein the hydrogel has a geometric shape having a length, width, and / or diameter of approximately 0.1 mm to approximately 1,000 mm.

[0306] K4. A system described in any one of K to K3, wherein the hydrogel is a liquid droplet.

[0307] K5. The system described in any one of K to K3, wherein the hydrogel has a filamentous structure.

[0308] K6. The system according to K5, wherein the filamentous structure has a linear, serpentine, or helical shape.

[0309] K7. The system according to any one of K to K6, wherein a stem cell marker and / or proliferation marker is expressed at a higher level in a population of tissue-derived epithelial organoids compared to a population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

[0310] K8. The system according to K7, wherein the stem cell marker 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 combinations thereof.

[0311] The system according to any one of K to K6, wherein the differentiation marker is expressed at a lower level in the tissue-derived epithelial organoid population compared to the reference tissue-derived epithelial organoid population, and the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate.

[0312] K10. The system described in K9, 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

[0313] K11. The system according to any one of K to K10, wherein the tissue-derived epithelial organoid is selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, trophoblast organoids, and combinations thereof.

[0314] K12. The system described in any one of K to K11, further comprising robots and / or automated components.

[0315] K13. The system according to K12, wherein the robot and / or automated components include a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0316] L. The method according to any one of A-C21 and F-J5, wherein one or more steps of the method are performed by a robot and / or automated components.

[0317] L1. The method according to L, wherein the robot and / or automated components include a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0318] L2. The method according to L or L1, wherein the robot and / or automated component is a liquid handling robot.

[0319] A method for producing tissue-derived epithelial organoids described in MD-D1 or compositions described in E-E9, wherein one or more steps of the method are carried out by a robot and / or automated components.

[0320] M1. The method according to M, wherein the robot and / or automated components include a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0321] The method according to any one of B to B18, wherein the preparation of a mixture containing N. hydrogel and tissue-derived epithelial stem cells by introducing it into a culture medium to produce a suspension mixture is performed by a robot and / or automated component.

[0322] N1. The method according to any one of B to B18, wherein the suspension mixture is cultured in a culture medium to produce turbid tissue-derived epithelial organoids, which is performed by robotic and / or automated components.

[0323] N2. The method according to N or N1, wherein the robot and / or automated components include a liquid handling robot, a 3D printer, a syringe pump, or a combination thereof.

[0324] N3. The method according to N2, wherein the robot and / or automated components include one or more liquid handling robots. [Examples]

[0325] Examples The subject matter disclosed herein will be better understood by referring to the following examples provided not as an limitation, but as illustrations of the subject matter disclosed herein.

[0326] Example 1: Formation of intestinal organoids This example provides a method for culturing intestinal organoids in suspended basement membrane extract (BME) hydrogels of various shapes. This method streamlines the protocol, increases scalability, enables dynamic sampling, and improves culture uniformity without requiring specialized equipment or additional expertise. The method is adaptable to multiple culture formats, and organoids produced by this method can be used for downstream applications such as implementation in medium-throughput drug screening and the production of Transwell monolayers for barrier evaluation, as shown in Examples 2 and 3. The suspended BME hydrogel culture method enables the more widespread and high-throughput use of intestinal organoids than previously possible.

[0327] method Induction of human gut organoids Organoids were induced with several modifications as previously reported (Pleguezuelos-Manzano et al. (2020)). Anonymized human colon and ileal tissue samples from deceased donors were procured by Donor Network West. The tissue was washed in Advanced DMEM / F12 medium (ThermoFisher), cut into 5 cm × 5 cm segments, then the epithelium was scraped from the submucosa into the medium and chopped with a razor blade. The solution was pelletized at 450 × g for 5 minutes, and then Mg 2+ or Ca 2+ The crypts were incubated in 2.5 μM EDTA in PBS without ions at 37°C for 9 minutes (ileum) or 12 minutes (colon), vortexed every 3-4 minutes until the crypts were released. The crypts were pelletized at 450 × g for 5 minutes, washed in PBS, filtered through sterile gauze and then a 100 μm cell strainer to remove debris, and pelletized again at 450 × g for 5 minutes. The crypts were resuspended on ice in CULTREX® Reduced Growth Factor Basement Membrane Matrix, Type II (BME, R&D Systems catalog no. 3533-010-02), seeded into 50 μL domes in 24-well plates, cured at 37°C for 15–30 minutes, and then overlaid with 500 μL of colonic transit medium (Intesticult Organoid Growth Medium (OGM, StemCell Technologies catalog no. 06010) + 10 μM Y27632) or ileal medium (OGM + 10 μM Y27632 + 2.5 μM CHIR99021). After the first 2–3 days of culture, the medium was replaced every 2–3 days, or when the medium turned yellow, using plain OGM for colonic culture or ileal medium for ileal culture.

[0328] Maintenance of organoids Organoid cultures were subcultured every 1-2 weeks by digestion at 37°C for 10 minutes using TrypLE Express (ThermoFisher), followed by trituration with a P1000 pipette. Incubation was repeated up to two times as needed to obtain single-cell suspensions. TrypLE Express was inactivated by dilution with PBS, and cells were pelleted at 450 × g for 3 minutes. Cells were then placed on ice in 6 × 10⁶ units. 5 Cells were resuspended in BME at a concentration of 1 / mL and seeded into 24-well plates in 50 μL domes, then cured at 37°C for 15–30 minutes. The domes were overlaid with colonic transit medium or ileal medium for the first 2–3 days, with the medium replaced every 2–3 days with plain OGM for colonic or ileal medium. For colonic organoid differentiation, the cultures were washed with Advanced DMEM / F12 medium, then overlaid with Intesticult Organoid Differentiation Media (ODM, StemCell Technologies catalog no. 100-0214) + 5 μM DAPT for 5 days, with the medium replaced every 2–3 days.

[0329] For the suspension hydrogel BOBA culture, single organoid cells in BME were prepared on ice as described above. Preheated colonic transit medium or ileal medium 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 maintained on a 37°C warm bead bath. Similar results were obtained with ultra-low adhesion (ULA) or standard tissue culture plates. Figure 10 shows an exemplary schematic diagram illustrating BOBA formation. To generate suspension BME droplets or BOBA, the organoid cell-BME solution was directly dispensed in 10 μL volumes into the warm medium at a slow to moderate speed using an electronic repeater pipette (Integra VIAFLO 300) with a wide-mouth or cut pipette tip (approximately 2 mm opening) to avoid the formation of filaments or thin strands instead of droplets. During dispensing, the tip was immersed just below the liquid surface, and then lifted after each dispensing to ensure droplet separation. For larger format cultures, BOBA was transferred to the flask using a serum pipette or decant. The culture medium was changed every 2-3 days using plain OGM for colonic or ileal medium. For 6-well plate cultures, a sterile 70 μm cell strainer was placed in each well, the plate was tilted, and 4 mL of medium was gently aspirated through the strainer. For flask cultures, the flask was tilted at an angle to allow the BOBA to settle in the corner, and then approximately 2 / 3 volume of used medium was replaced using a serum pipette.

[0330] Suspension hydrogel SOBA and SOBA fragment culture Single organoid cells in BME were prepared on ice as described above. Preheated colonic transit medium was added to 6-well plates (5 mL / well) and 100 cm Petri dishes (15-30 mL / dish) and maintained on a warm bead bath. Figure 11 shows an exemplary schematic diagram illustrating SOBA filament generation. To generate suspended SOBA filaments, the cell-BME solution was gently drawn into a syringe equipped with a 15-gauge blunt-tipped needle, and then the immersed needle was directly extruded into the warm medium while moving it in a linear, serpentine, or spiral motion in the XY plane. The extrusion speed and motion in the XY plane may affect the length and / or width of the filament. To generate SOBA fragments, the SOBA filament culture was gently triturated twice using a 10 mL serum pipette or a wide-mouth P1000 pipette tip. Further medium was added to achieve a final BME-to-medium ratio of 1:10. The SOBA culture was changed as described above.

[0331] Bright-field microscopy and image analysis Cultures were imaged by bright-field microscopy using a THUNDER DMi8 inverted optical microscope (Leica) equipped with 2.5x, 4x, or 10x objective lenses and a DFC9000 GTC camera (Leica). Images were analyzed using Imaris Image Analysis software and Imaris Batch software packages (Oxford Instruments). For automated organoid diameter measurement, the Imaris Surface Detection module was used in conjunction with the inverted bright-field images. Background and out-of-focus organoids were excluded using a background subtraction process (rolling ball with a diameter of 19.5 μm). The detected surfaces were then filtered based on four criteria assigned to the software: quality metric (greater than 3000 A.U.), minor axis length (greater than 35 μm, excluding fragments), oval roundness (greater than 0.2, excluding shadows), and major axis length (between 35 μm and 600 μm, excluding false detections of overlapping organoids). The diameters of the remaining surfaces (at least 40 per analyzed image) are reported as the longest side of the smallest object-oriented bounding box. This process was then performed in batches across all analyzed images. The average per image was calculated, and the average of all three experiments was plotted for n=3 iterations.

[0332] Spatial organoid uniformity and organoid diameter analysis were performed using FIJI (ImageJ). A horizontal rectangular (1.5 mm × 9 mm) ROI was set across the center of each image and divided into 1 mm sections along the X-axis. For each section, the diameter was manually measured between the widest ends of each organoid.

[0333] Preparation of immunofluorescence samples and confocal microscopy The dome cultures in 24 wells were fixed with 2% paraformaldehyde (PFA) in PBS. The domes were removed from the plate using a spatula and transferred to microcentrifuge tubes using a cut P1000 pipette tip. For BOBA culture, 500 μL of culture was transferred to a microcentrifuge tube using a cut P1000 pipette tip, the medium was removed, and 2% PFA in PBS was added. The samples were incubated in fixative at room temperature for 15–30 minutes, then washed three times in PBS. The samples were stained at room temperature for at least 4 hours in a microcentrifuge tube containing primary antibody diluted in blocking / permeabilization buffer (3% BSA, 0.1% Triton X-100, 0.02% sodium azide in PBS), then washed three times in PBS. The primary antibodies used were as follows: α-Ki67 (Invitrogen catalog number MA5-14520), α-MUC2 (Millipore catalog number MABF1989), α-FABP1 (Novus catalog number NBP-87695), and α-CHGA (Novus catalog number NB120-15160). The samples were then incubated at room temperature for at least 2 hours with secondary antibodies (donkey α-rabbit Alexa Fluor 488 (ThermoFisher catalog number A-21206) or goat α-mouse Alexa Fluor 594 (ThermoFisher catalog number A-11032)), DAPI, and AlexaFluor 660 Phalloidin diluted in blocking / permeabilization buffer. Images were acquired using a Stellaris 8 confocal microscope (Leica) with a 40x objective lens, and 3D reconstruction was performed using Imaris Image Analysis software (Oxford Instruments).

[0334] Transcriptome analysis For RNA isolation, the RNeasy Micro Plus kit (Qiagen) was used. RLT+ lysis buffer was added to either a BME dome or pelletized BOBA and stored at -80°C. RNA isolation was performed using QiaCube Connect (Qiagen), and RNA was quantified using Nanodrop 8000 (ThermoFisher). Bulk mRNA-seq (NovaSeq PE150) and analysis were performed using 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 was calculated using a negative binomial distribution model with Benjamini-Hochberg FDR correction.

[0335] Variability of BME organoids suspended in a 96-well plate 225cm 2 Colon organoid SOBA fragments were collected from flasks after 9 days of culture and gently triturated twice using a serum pipette to homogenize the samples without destroying intact organoids. The organoid mixture was transferred to a reagent reservoir and then seeded at 100 μL / well into a 96-well plate using a P200 multichannel pipette with a wide-mouth pipette tip. Dome cultures were prepared as described above and seeded into 5 μL domes in the center of each well of the 96-well plate using a repeater pipette. After growing the cultures for 7 days, viability measurements were performed. All viability measurements were performed using a Cell Titer Glo 3D Assay Kit (Promega), and luminescence was measured with an Ensight plate reader (Perkin Elmer).

[0336] statistical analysis Unless otherwise specified, all statistical analyses were performed using Prism 9 software (Graphpad). Statistical tests, n, and p-values ​​are shown in the legend of the figures. result:

[0337] Suspension BME hydrogel method for human intestinal organoid culture Conventional intestinal organoid culture methods require depositing a solution of organoid cells in cold ECM onto a plastic surface, curing it in an incubator to form a hydrogel dome, and then overlaying it with growth medium (Figures 1A-1B) (Mahe et al. (2013), Pleguezuelos-Manzano et al. (2020), Sato et al. (2009), Sato et al. (2011)). To address the scaling limitations of this technique, a method has been developed to immediately cure the cold cell-ECM solution in the form of a suspended hydrogel suspended in warm medium. A method using suspension droplets of BME, an Engelbreth-Holm-Swarm (EHS) cell-derived ECM equivalent to MATRIGEL®, has been demonstrated and is called BOBA (BME-embedded organoid bead assembly) culture.

[0338] Intestinal organoid proliferation was first evaluated using the suspension BOBA culture method, which was then compared to the conventional surface-attached BME dome method, also known as dome culture. For dome culture, single cells from digested organoids were suspended in cold BME, seeded as a 50 μL dome in a 24-well plate, cured in a 37°C incubator for 15–30 minutes, and then overlaid with growth medium (Intesticult Organoid Growth Media). The medium was changed individually for each well every 2–3 days. For BOBA culture, the single-cell BME solution was deposited directly onto preheated medium in the form of 10 μL droplets using an electronic repeater pipette with a wide-mouth tip, and immediately cured onto BOBA suspended in hydrogel. BOBA could be generated in petri dishes, plates, or conical tubes and easily transferred to larger containers such as cell culture flasks via serum pipettes or decant (Figures 1A–1B). The BOBA was allowed to settle, and then the entire flask was replaced by replacing the top 75% of the volume of the used medium with fresh medium.

[0339] Human intestinal organoid cells-BME solution were plated in parallel with dome or BOBA cultures and grown in growth medium for 9 days. Organoid growth and size were similar in both methods, as observed by bright-field microscopy (Figure 1C) and quantified by organoid diameter measurement (Figure 1D). Organoid cell growth was also comparable, as measured by quantifying the abundance of cells expressing the growth marker Ki-67 (Figures 1E-1F). The BOBA method was effective for a surface area of ​​1 cm². 2 While it appeared to allow for greater organoid cell proliferation per well, statistical significance was observed only for small intestinal (ileal) organoids (Figures 1G-1H). In the case of colon organoids, dome culture yielded an average of 2.9 × 10⁶ cells per well in a 24-well plate. 5 individual living cells or 1.5 × 10⁶ 5 cells / cm 2 On the other hand, BOBA culture yields 75 cm 2 In the flask, the average was 2.2 × 10⁻⁶ 7 A single living cell, or 2.9 × 10⁶ 5 cells / cm 2 This resulted in (Figure 1G). In the case of ileal organoids, dome culture yielded an average of 4.8 × 10⁴ 5 individual living cells or 2.6 × 10⁶ 5 cells / cm 2 On the other hand, BOBA culture yields 75 cm 2 In the flask, the average was 2.9 × 10⁻⁶ 7 Individual living cells or 3.9 × 10⁶ 5 cells / cm 2 This resulted in (Figure 1H). The number of viable cells per 1 μL of BME hydrogel was similar, indicating that the amount of proliferation was comparable for both methods when a fixed seeding density was given (Figure 1G).

[0340] Organoid differentiation in BOBA culture A key advantage of the intestinal organoid model is its ability to differentiate into various intestinal epithelial cell types by altering the culture medium composition, for example, by extracting stem cell-promoting factors (Clevers (2016), Schutgens and Clevers (2019), Zachos et al. (2016)). We compared the differentiation of organoids in dome and BOBA cultures. Organoids were grown in growth medium for 7 days, then washed and transferred to differentiation medium (Intesticut Organoid Differentiation Medium containing 5 μM DAPT) for 5 days. Bright-field microscopy showed that in both culture formats, proliferative organoids in growth medium exhibited a cystic morphology with large lumens (Figure 2A), while differentiated organoids exhibited a dense spheroid morphology with elongated columnar cells and small lumens (Figure 2A).

[0341] Bulk RNA-seq analysis was performed on proliferative and differentiated organoids in dome and BOBA cultures. In both culture methods, differentiated organoids downregulated the expression of stem cells and proliferation markers (MKI67, LGR5, SOX9, and CD44) and upregulated the expression of differentiation markers for goblet cells (MUC2, MUC5B, TFF3) and intestinal cells (KRT20, FABP1, ALPI, and CEACAM7) compared to proliferative organoids (Figure 2B). Differentiated cell types were also observed by immunofluorescence (IF) confocal microscopy in both culture formats (Figure 2C).

[0342] Characterization and optimization of BOBA culture conditions Next, we evaluated how various culture parameters affect organoid proliferation in the BOBA method. Cultures were placed in 6-well plates (Figures 3A-3B) or 25 cm². 2 Place various volumes of BME into one of the flasks (Figure 3C), 6 × 10 5Cells were seeded at a constant BME density of cells / mL. All BOBA was generated in 10 μL droplets containing 5 mL of growth medium, and organoid growth was evaluated by quantifying organoid diameter and the number of viable cells after 9 days of culture (Figures 3B-3C). In 6-well plates, increasing the total BME volume per well from 0.5 mL to 2 mL resulted in a decrease in organoid diameter and a decrease in the ratio of viable cells to BME volume (although not statistically significant). The total number of viable cells and 1 cm³ were also evaluated. 2 The ratio of viable cells per surface area was similar under all conditions, and the higher BME volume conditions showed lower proliferation per seeded cell despite a larger number of seeded cells. In summary, these data suggest that organoid proliferation is impaired when the BME volume exceeds a threshold for a certain amount of culture medium in a 6-well plate (Figure 3B).

[0343] Interestingly, 25cm 2 For BOBA cultures in flasks, organoids grew equally well under all test conditions (Figure 3C). As the total BME volume per flask increased from 0.5 mL to 2 mL, organoid diameter and the ratio of viable cells to BME volume remained similar. 2 The ratio of viable cells per surface area appears to increase as the volume of BME in the culture increases (though not statistically significant due to inter-experimental variability), and the threshold for BME volume relative to the medium is 25 cm³ for 6-well plates and 25 cm³. 2 This suggests that the results may differ in flasks. Both the BME-to-culture medium ratio and the container type should be optimized for specific applications.

[0344] Homogeneity of organoids in BOBA culture Conventional dome cultures are known to cause organoid heterogeneity (Pleguezuelos-Manzano et al. (2020), Ringel et al. (2020)). Natural ECM hydrogels restrict 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)). Organoids were observed to grow more uniformly in BOBA culture than in dome culture (Figure 4). Colon organoid cultures were imaged by bright-field microscopy at the deepest point of each format (the z-plane at the bottom of the dome or the z-plane at the center of the BOBA), and the average organoid diameter across the rectangular ROI at the center of each hydrogel was quantified (Figure 4C). Consistent with previous reports (Park et al. (2022), Shin et al. (2020)), dome-cultured organoids grew larger at the edges of the hydrogel and smaller in the core (Figure 4B-4E). However, organoids in BOBA grew to a comparable size across the entire hydrogel (Figure 4B-E).

[0345] Organoids in the core of dome cultures were also observed to often exhibit a morphology lacking a dense spheroid lumen, indicating differentiation (Figure 4B). Supporting the hypothesis that a subpopulation of differentiated organoids may exist within the dome cultures, bulk RNA-seq analysis showed that dome-cultured organoids had lower expression of stem cell and proliferation markers and higher expression of intestinal cell markers compared to their BOBA-cultured counterparts (Figure 5).

[0346] Alternative geometries of suspended BME hydrogels While the BOBA method offers significant upscaling advantages over the conventional dome method, large-scale culture and growth of suspended BOBA hydrogel droplets without automated liquid handlers can still be a labor-intensive undertaking. To reduce the time and effort required for suspended BME hydrogel culture, we devised an alternative hydrogel shape, specifically hydrogel filaments. Extruded filaments are used in the bioprinting field to spatially control cell proliferation or to construct layered assemblies of 3D hydrogel structures, but typically depend on surface adhesion (Kolesky et al. (2014) Adv. Mater. 26, 2966-2966, Kolesky et al. (2016) Proc National Acad Sci 113, 3179-3184). To generate organoid cell-containing hydrogel filaments called SOBA (Syringe Extrusion Organoid BME Assembly), a cold cell-BME solution was filled into a syringe with a 15-gauge (1.37 mm inner diameter) blunt-tipped needle, and then injected directly into a warm medium while moving the tip across the XY plane (e.g., in a linear, serpentine, or spiral pattern). By gently triturating the SOBA culture using a wide-mouth P1000 tip or a 10 mL serum pipette, filamentary fragments more similar in shape to BOBA, called SOBA fragments, were also generated.

[0347] Organoids grown in BOBA, SOBA, or SOBA fragment cultures over a 9-day period showed similar growth, as confirmed by bright-field microscopy (Figure 6A), organoid diameter measurement (Figures 6B-6C), and viable cell count (Figure 6D). Compared 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 uniformly dispersed in the medium, and therefore single cultures can be more easily divided, sampled, or dispensed.

[0348] Consideration: This example describes the development of a suspension BME hydrogel culture method for human intestinal epithelial organoids that overcomes several challenges associated with conventional surface-attached dome hydrogel culture methods. Compared to the dome method, the BOBA, SOBA, and SOBA filament methods simplify and accelerate the protocol, enable compatibility with scalable culture vessels, allow dynamic culture sampling, and improve the uniformity of organoid culture.

[0349] Several suspension culture protocols have been proposed for the scaling of intestinal organoids and tumor organoids, but these methods cultured the organoids in liquid medium containing low concentrations of solubilized MATRIGEL® (similar to BME) that did not form intact hydrogels (Hirokawa et al. (2021) Commun Biology 4, 1067, Price et al. (2022) Sci Rep-Uk 12, 5571). This is in contrast to the method of this disclosure, which relies on the formation of fully cured insoluble hydrogels. Furthermore, previous studies have shown that organoids and tumor organoids can be grown in a 5% solution of soluble MATRIGEL®, but concentrations above 5% resulted in decreased growth. Furthermore, intestinal organoids grown in 5% MATRIGEL® showed inverted epithelial polarity (Hirokawa et al. (2021)), which is consistent with previous reports of organoid polarity inversion under low ECM conditions (Co et al. (2019), Cell Reports, 26, 2509-2520.e4).

[0350] The BOBA method offers several advantages over the conventional dome method. First, it simplifies the protocol. Since the hydrogel hardens directly in the culture medium, the need for a separate hardening incubation step is eliminated, resulting in time and labor savings. Second, it reduces the amount of container surface area and technical precision required to plate the dome. By reducing reliance on available surface area, the BOBA method utilizes all three 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 is available in larger sizes, easier to handle, and compatible with flasks that allow for rapid medium changes. For example, BOBA culture can be performed in a single 225 cm³ vessel. 2 A 10 mL BME can be cultured in a flask, and the medium can be changed simply by replacing the supernatant with a serum pipette. However, using the dome method, an equivalent culture requires nine 24-well plates (200 wells with 50 μL domes), and the medium must be changed individually for each well. This also reduces the amount of plastic consumed by more than 70% (225 cm³). 2 The flask contains 152.7 g of plastic, replacing 562.5 g of plastic in nine 24-well plates (data not shown). Further scale-up can be achieved using multi-wall flasks or “cell factories” capable of accommodating several liters of culture volume. Table 1 shows general guidelines for the suspension BME hydrogel culture setup of this disclosure for several container types. The type of culture vessel has been reported to affect parameters such as gas transfer (Allen et al. (2001) Am J Physiol-Lung C 281, L1021-L1027), and factors such as culture medium formulation, hydrogel composition, and organoid strain-specific growth rate can all affect organoid growth. [Table 1] Seeding conditions for suspended BME hydrogel cultures

[0351] The BOBA method also overcomes the culture heterogeneity arising from the hydrogel diffusion limit in dome culture (Park et al. (2022), Shin et al. (2020)). Several protocols suggest plating smaller 10–15 μL hydrogel domes (Pleguezuelos-Manzano et al. (2020), Stewart et al. (2020), Methods Mol Biology, 2121, 185–198) to thereby reduce the diffusion pathway for molecular transport. However, plating smaller domes reduces the total hydrogel volume per well, even when multiple domes are plated per well. Another approach to overcome the limitations of hydrogel diffusion is to invert the plate during the hydrogel curing process so that gravity causes cells to settle near the top surface of the dome, leaving few or no cells in the core of the dome (Pleguezuelos-Manzano et al. (2020)). This results in the suboptimal use of expensive hydrogel volume, ultimately leading to fewer organoid cells that can be seeded per μL of hydrogel. While complex bioengineering techniques have been developed to increase hydrogel surface area to improve molecular transport (Park et al. (2022)), these are difficult to scale and still rely on immobilizing the hydrogel on a 2D surface. The BOBA method produces a more homogeneous culture without sacrificing the amount of hydrogel per well or the number of cells per μL of hydrogel. Although not limited to a specific theory, the observed improvement in the homogeneity of organoid cultures can be explained by the fact that BOBA hydrogels have (1) smaller diameters and thus shorter diffusion pathways, and (2) all outer surfaces exposed to the medium that allow for uniform molecular diffusion into the hydrogel.

[0352] Another reported challenge regarding the morphological heterogeneity of organoids in hydrogel domes is that organoids located near the bottom of the plate may adhere to the plate surface, spread out, flatten, and lose their 3D structure (Pleguezuelos-Manzano et al. (2020), Price et al. (2022)). Since suspended hydrogel droplets do not come into direct contact with the plate surface, organoid spreading and flattening do not occur.

[0353] In addition to BOBA hydrogel droplets, organoids can be grown as SOBA filaments and SOBA filament fragments. Organoids proliferate similarly in all three configurations, demonstrating the robustness of the suspension BME culture method. The SOBA method facilitates and accelerates culture preparation, as large volumes of cell-BME solution can be filled into a single syringe to generate SOBA hydrogel filaments. Approximately 10 mL of SOBA can be generated in under 1 minute, compared to an equivalent dome culture which requires over 15 minutes to plate and another 15–30 minutes to harden. SOBA is much longer than BOBA, but presumably its smaller diameter (typically less than 2 mm) allows for efficient nutrient transport from the medium, so heterogeneity in organoid growth (similar to that observed in dome cultures) was not observed. SOBA fragments are more similar in size and geometric shape to BOBA, but are generated much faster. Similar to BOBA, SOBA fragments are uniformly dispersed throughout the culture, which may be useful for dynamic sampling or splitting the culture for multiple applications or readouts.

[0354] Overall, suspension hydrogel culture methods enable large-scale organoid scaling, improve the uniformity of organoid cultures, and save labor, time, and resources. These culture improvements make intestinal organoids more suitable for high-throughput applications such as compound or genome screening. This method can be extended to culture diseased and tumor organoids, intestinal organoids from other species, and organoids from different tissue types. Therefore, BOBA, SOBA, and SOBA filaments have the potential to accelerate and expand the adoption of organoid technology.

[0355] Example 2: Use of organoids in a screening method This example discloses the use of intestinal organoids produced by the method of Example 1 in a cytotoxicity assay.

[0356] method: 96-well plate suspension BME organoid cytotoxicity assay Two 96-well plates were used to extract 225 cm³ of homogenized colon organoid SOBA fragments. 2 Cells were seeded in flasks at 90 μl / well. Compound stock was diluted 10-fold to the final concentration in Intesticult OGM, and 10 μL was added to each well. An eight-dose dilution series, starting at 100 μM and involving five-fold dilutions, was evaluated using a technical quadruple series. Three days after treatment, viability was measured using the Cell Titer Glo 3D Assay Kit (Promega), and luminescence was measured using an Ensight plate reader (Perkin Elmer). 4PL fitted curves were created using Prism (GraphPad).

[0357] result: Application of suspended BME hydrogel organoids in drug toxicity screening assays Intestinal organoids generated in BOBA, SOBA, or SOBA fragment format can be used directly in downstream assays without additional organoid digestion or subculturing steps. As proof of concept, the implementation of these organoids was demonstrated in drug toxicity screening. SOBA fragment cultured organoids were cultured at 225 cm². 2 The cells were grown in flasks (Figures 7A-7B), homogenized by trituration using a serum pipette, and then transferred to 96-well plates (Figures 7A-7C). Inter-well variability, as measured by a Cell Titer Glo 3D ATP-based viability assay, was comparable for SOBA filament growth organoids and dome cultures plated in 96-well plates (Figure 7D).

[0358] For drug toxicity screening, SOBA filament growth organoids in 96-well plates were treated for 3 days with compounds known to cause enterotoxicity (diacerine, sorafenib, SN-38, or docetaxel) or a DMSO vehicle control. Survival rates were determined using ATP as an indicator, and dose-response curves were constructed (Figure 7E). This is an example of a method for directly using suspended BME hydrogel cultures for downstream applications.

[0359] The usefulness of suspended BME hydrogel-grown cultures is demonstrated here in two applications. First, SOBA filament cultures were homogenized to produce 96-well plate cultures with low inter-well organoid variability. As a proof of concept, drug toxicity screening was performed, but this method can be used for other medium- to high-throughput screenings.

[0360] Example 3: Generation of organoid-derived models This embodiment discloses the use of intestinal organoids produced by the method of Example 1 in providing alternative organoid-derived models.

[0361] method: Transwell single layer 225cm2 The colon organoid SOBA fragments from the flask were collected in a 50 mL conical tube and pelletized at 800 × g for 3 minutes. The supernatant was removed, and the organoids were digested into single cells by incubation in TrypLE Express in a 37°C water bath for 10 minutes, followed by trituration with a P1000 pipette. Incubation and trituration were repeated up to two times as needed. The cells were pelleted, washed with PBS, and resuspended in either monolayer growth medium (Intesticult OGM + 10 μM Y-27632) or monolayer differentiation medium (Intesticult ODM + 10 μM Y-27632). For each well of a 96-well PET Transwell plate (pore size 0.4 μm, Corning catalog number 3450), 2.0 × 10¹⁶ cells were added in 100 μL of medium. 5 Cells were seeded in the apical chamber, and 200 μL of culture medium was added to the basal chamber. The culture medium in both chambers was changed every 2-3 days. Bright-field imaging was performed using a THUnder microscope (Leica) with a 10x objective lens and a DFC9000 GTC camera (Leica). Transepithelial electrical resistance (TEER) was measured by volt / ohmmeter (EVOM3, WPI), and then the resistance was multiplied by the Transwell surface area (0.143 cm²). 2 This was obtained by multiplying by ).

[0362] result: Suspended BME hydrogel culture facilitates alternative organoid-derived models. In addition to directly using organoids in suspended BME hydrogel form, these organoids can facilitate the generation of other organoid-derived models, particularly those requiring large cellular inputs such as monolayers and microphysiological systems (MPS) devices. Colonic epithelial Transwell monolayers were generated using suspended BME hydrogel organoids, and epithelial barrier function was evaluated under two conditions. (225 cm²) 2SOBA filament organoids cultured in flasks were digested into single cells, seeded confluence onto 96-well Transwell inserts, and cultured for 7 days in monolayer growth medium or monolayer differentiation medium (Figure 8A). The two conditions resulted in Transwell epithelial cultures with different morphologies. In growth medium, the resulting epithelium formed 3D structures protruding from the monolayer surface, while in differentiation medium, these structures were absent, and a "cobblestone" cell morphology typical of differentiated epithelial cells with mature tight junctions was observed (Figure 8B). Consistent with this morphology, epithelial barrier function, quantified by transepithelial electrical resistance (TEER), was higher in the Transwell monolayer in differentiation medium than in growth medium until day 3 (Figure 8C). This experiment is an example of how organoids grown using BOBA, SOBA, or the SOBA filament method may enable the production of alternative intestinal organoid-derived in vitro models.

[0363] SOBA filament organoids can be used to produce organoid-derived Transwell monolayers, which offers the advantage of simultaneous access to both the apical and basal sides, but scale-up is difficult due to the need to seed large cell numbers. By enabling substantial organoid proliferation, the suspension BME hydrogel culture method can facilitate the development and implementation of complex intestinal organoid-derived models with higher tissue fidelity and experimental advantages.

[0364] Example 4: Generation of lung organoids This example discloses the generation of lung organoids using the BME suspension method described herein.

[0365] method: Isolation of human lung ATII cells and induction of organoids Anonymized human lung tissue from deceased donors was procured by Donor Network West. Lung dissection into single cells and establishment of organoids had been previously reported (Konishi et al., 2022). Briefly, the tissue was washed with HBSS buffer and swollen with digestion buffer (collagenase type I: 450 units / mL, dispase: 5 units / mL, DNase I: 10 units / mL). After removing the pleura and small airways, the remaining tissue was finely chopped with a single-edged blade and transferred to a conical tube containing warm digestion buffer. It was incubated at 37°C for a total of 1 hour, stirring vigorously every 15 minutes. This solution was passed through a 100 μm cell strainer and pelletized to 450 g over 10 minutes at 4°C. The cell pellet was resuspended in 5 mL of ACK buffer for hemolysis and incubated at room temperature for 3–5 minutes. The reaction was stopped by adding DMEM / F12 + 10% FBS. Next, the cell suspension was filtered through a 40 μm cell strainer and pelleted at 450 g for 5 minutes at 4°C. The cells were then labeled with CD45 magnetic microbeads (Miltenyi Biotech, catalog number 130-045-801) and loaded onto a MACS® column placed in the magnetic field of a MACS separator (according to the manufacturer's protocol). The magnetically labeled CD45+ cells were re...

Claims

1. A method for generating tissue-derived epithelial organoids, (a) To produce a mixture of tissue-derived epithelial stem cells and hydrogel by contacting the hydrogel with the tissue-derived epithelial stem cells. (b) Suspending the mixture of the hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a mixture of the suspended hydrogel and tissue-derived epithelial stem cells, and (c) To produce tissue-derived epithelial organoids by culturing the mixture of the suspended hydrogel and tissue-derived epithelial stem cells in the culture medium. Methods that include...

2. The method according to claim 1, wherein a mixture of the hydrogel and tissue-derived epithelial stem cells is produced by contacting a plurality of tissue-derived epithelial stem cells with the hydrogel.

3. The aforementioned multiple tissue-derived epithelial stem cells are approximately 1 × 10 ４ Individual tissue-derived epithelial stem cells / hydrogel 1 ml to approximately 1 x 10 ７ The method according to claim 2, comprising 1 ml of tissue-derived epithelial stem cells / hydrogel.

4. The method according to claim 2 or 3, wherein the plurality of tissue-derived epithelial stem cells are included in a tissue fragment, an organoid fragment, or a combination thereof.

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

6. The method according to any one of claims 1 to 5, wherein the hydrogel solidifies upon contact with the culture medium.

7. The method according to any one of claims 1 to 6, wherein suspending the mixture of the hydrogel and tissue-derived epithelial stem cells in the culture medium includes immersing a dispensing device containing the mixture of the hydrogel and tissue-derived epithelial stem cells in the culture medium and dispensing the mixture of the hydrogel and tissue-derived epithelial stem cells into the culture medium.

8. The method according to any one of claims 1 to 7, wherein the temperature of the culture medium is approximately 25°C to approximately 50°C.

9. The method according to any one of claims 1 to 8, wherein the temperature of the culture medium is approximately 30°C to approximately 50°C.

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

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

12. The method according to any one of claims 1 to 11, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

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

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

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

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

17. The method according to any one of claims 1 to 16, wherein a mixture of the hydrogel and tissue-derived epithelial stem cells is suspended in the culture medium in droplet form.

18. The method according to any one of claims 1 to 16, wherein the mixture of the suspended hydrogel and tissue-derived epithelial stem cells has a filamentous structure.

19. The method according to claim 18, wherein the filamentous structure has a linear, snake-like, or spiral shape.

20. The method according to any one of claims 1 to 19, further comprising fragmenting the mixture of the suspended hydrogel and tissue-derived epithelial stem cells to produce a fragmented structure containing the tissue-derived epithelial organoid.

21. A method for producing a suspension culture of tissue-derived epithelial organoids, (a) Introducing a mixture containing a hydrogel and tissue-derived epithelial stem cells into a culture medium to produce a suspension mixture, and (b) Culturing the suspension mixture in a culture medium to produce the tissue-derived epithelial organoids in a suspended state. Methods that include...

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

23. The aforementioned multiple tissue-derived epithelial stem cells are approximately 1 × 10 ４ Individual tissue-derived epithelial stem cells / hydrogel 1 ml to approximately 1 x 10 ７ The method according to claim 22, comprising 1 ml of tissue-derived epithelial stem cells / hydrogel.

24. The method according to claim 22 or 23, wherein the plurality of tissue-derived epithelial stem cells are included in a tissue fragment, an organoid fragment, or a combination thereof.

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

26. The method according to any one of claims 21 to 25, wherein the hydrogel solidifies upon contact with the culture medium.

27. The method according to any one of claims 21 to 26, wherein introducing the mixture into the culture medium includes immersing a dispensing device containing the mixture into the culture medium and dispensing the mixture into the culture medium.

28. The method according to any one of claims 21 to 27, wherein the temperature of the culture medium is approximately 25°C to approximately 50°C.

29. The method according to any one of claims 21 to 28, wherein the temperature of the culture medium is approximately 30°C to approximately 50°C.

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

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

32. The method according to any one of claims 21 to 31, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

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

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

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

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

37. The method according to any one of claims 21 to 36, wherein the mixture is introduced into the culture medium in droplet form.

38. The method according to any one of claims 21 to 36, wherein the mixture is introduced into the culture medium in a filamentous structure.

39. The method according to claim 38, wherein the filamentous structure has a linear, snake-like, or spiral shape.

40. The method according to any one of claims 21 to 39, further comprising fragmenting the suspension mixture to produce a fragmented structure containing the tissue-derived epithelial organoid.

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

42. A method for producing a suspension culture of tissue-derived epithelial organoids, (a) To produce a mixture of tissue-derived epithelial stem cells and hydrogel by contacting the hydrogel with the tissue-derived epithelial stem cells. (b) Depositing the mixture of the hydrogel and tissue-derived epithelial stem cells onto a substrate, (c) Solidifying the mixture of the hydrogel and tissue-derived epithelial stem cells to produce a mixture of the solidified hydrogel and tissue-derived epithelial stem cells. (d) Suspending the mixture of the solidified hydrogel and tissue-derived epithelial stem cells in a culture medium to produce a mixture of the suspended hydrogel and tissue-derived epithelial stem cells, and (e) To produce tissue-derived epithelial organoids by culturing the mixture of the suspended hydrogel and tissue-derived epithelial stem cells in the culture medium. Methods that include...

43. The method according to claim 42, wherein a mixture of the hydrogel and tissue-derived epithelial stem cells is produced by contacting a plurality of tissue-derived epithelial stem cells with the hydrogel.

44. The aforementioned multiple tissue-derived epithelial stem cells are approximately 1 × 10 ４ Individual tissue-derived epithelial stem cells / hydrogel 1 ml to approximately 1 x 10 ７ The method according to claim 43, comprising 1 ml of tissue-derived epithelial stem cells / hydrogel.

45. The method according to claim 43 or 44, wherein the plurality of tissue-derived epithelial stem cells are included in a tissue fragment, an organoid fragment, or a combination thereof.

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

47. The method according to any one of claims 32 to 46, further comprising removing the mixture of the solidified hydrogel and tissue-derived epithelial stem cells from the substrate before suspending the mixture in the culture medium.

48. The method according to any one of claims 42 to 47, wherein the hydrogel is selected from the group consisting of synthetic hydrogels, natural hydrogels, and combinations thereof.

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

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

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

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

53. The method according to any one of claims 42 to 52, wherein a mixture of the hydrogel and tissue-derived epithelial stem cells is deposited on the substrate as droplets.

54. The method according to any one of claims 42 to 52, wherein the mixture of the hydrogel and tissue-derived epithelial stem cells is deposited on the substrate so as to have a filamentous structure.

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

56. The method according to any one of claims 42 to 55, further comprising fragmenting a mixture of the hydrogel and tissue-derived epithelial stem cells in the culture medium to produce a fragmented structure containing the tissue-derived epithelial organoid.

57. The method according to any one of claims 1 to 56, wherein the tissue-derived epithelial organoid has a more uniform morphology compared to a reference tissue-derived epithelial organoid, and the reference tissue-derived epithelial organoid is a tissue-derived epithelial organoid embedded in a hydrogel attached to a substrate.

58. The method according to claim 57, wherein the tissue-derived epithelial organoid has a uniform size.

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

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

61. The method according to 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 combinations thereof.

62. The method according to any one of claims 1 to 61, wherein the differentiation marker is expressed at a lower level in the population of tissue-derived epithelial organoids compared to the population of reference tissue-derived epithelial organoids, and the reference tissue-derived epithelial organoids are tissue-derived epithelial organoids embedded in a hydrogel attached to a substrate.

63. The method according to 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

64. A tissue-derived epithelial organoid produced by the method described in any one of claims 1 to 63.

65. A composition comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium.

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

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

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

69. The composition according to any one of claims 65 to 67, wherein the hydrogel has a filamentous structure.

70. The composition according to claim 69, wherein the filamentous structure has a linear, snake-like, or spiral shape.

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

72. The composition according to claim 71, 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 combinations thereof.

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

74. The composition according to 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

75. A method for screening drugs, (a) Contacting a tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids described in claim 64 or a composition described in any one of claims 65 to 74 with a drug, and (b) Analyze the changes in the tissue-derived epithelial organoids or the aggregate of the tissue-derived epithelial organoids that indicate the efficacy, pharmacokinetics, and / or toxicity of the drug. Methods that include...

76. The method according to claim 75, wherein the drug is brought into contact with the tissue-derived epithelial organoid or an aggregate of the tissue-derived epithelial organoid for about 15 minutes to about 3 years.

77. The method according to claim 75 or 76, wherein the drug is a therapeutic agent.

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

79. A method for performing genome screening, (a) Prepare the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids described in claim 64 or the composition described in any one of claims 65 to 74. (b) to induce mutations in the genome of one or more cells of the tissue-derived epithelial organoid, and (c) Analyze the changes in the tissue-derived epithelial organoid or the population of tissue-derived epithelial organoids that are related to the mutation. This method includes the following.

80. The method according to claim 79, wherein the mutation is induced using a gene regulatory system.

81. The method according to claim 80, wherein the gene regulatory system is a gene editing system.

82. The method according to claim 81, wherein the gene editing system is a CRISPR system.

83. The method according to any one of claims 75 to 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 modification, post-translational modification, activation of cell signaling pathways, suppression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

84. A method for generating an epithelial cell model, (a) Prepare the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids described in claim 64. (b) Digesting the tissue-derived epithelial organoid or a group of tissue-derived epithelial organoids into single cells, and (c) Culturing the single cell in a culture medium to produce a cell monolayer. Methods that include...

85. The method according to claim 84, wherein the single cell is cultured on a permeable cell culture insert.

86. The method according to claim 84 or 85, wherein the culture medium is a differentiation medium.

87. The method according to claim 84 or 85, wherein the culture medium is a cell proliferation medium or a stem cell promoting medium.

88. A method for screening drugs, (a) Contacting a cell monolayer produced by the method of any one of claims 84 to 87 with a drug, and (b) Analyzing changes in the cell monolayer that indicate the efficacy, pharmacokinetics, and / or toxicity of the drug. Methods that include...

89. The method according to claim 88, wherein the drug is brought into contact with the cell monolayer for about 15 minutes to about 3 years.

90. The method according to claim 88 or 89, wherein the drug is a therapeutic agent.

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

92. A method for performing genome screening, (a) Prepare a cell monolayer produced by the method described in any one of claims 84 to 87, (b) to induce a mutation in the genome of one or more cells in the cell monolayer, and (c) Analyze the changes in the cell monolayer that are related to the mutation. Methods that include...

93. The method according to claim 92, wherein the mutation is induced using a gene regulatory system.

94. The method according to claim 93, wherein the gene regulatory system is a gene editing system.

95. The method according to claim 94, wherein the gene editing system is a CRISPR system.

96. The method according to any one of claims 84 to 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 modification, post-translational modification, activation of cell signaling pathways, suppression of cell signaling pathways, enzyme activity, barrier integrity, and combinations thereof.

97. The method according to any one of claims 1 to 63 and 75 to 96, or the composition according to any one of claims 65 to 74, wherein the tissue fragment is a fragment from tissue selected from the group consisting of lacrimal glands, tonsils, salivary glands, gastrointestinal tissue, thyroid gland, lungs, mammary glands, liver, bile ducts, stomach, kidneys, pancreas, endometrium, fallopian tubes, cervix, prostate gland, bladder, taste buds, ovaries, placenta, and combinations thereof.

98. The method according to any one of claims 1 to 63 and 75 to 96 or the composition according to any one of claims 65 to 74, wherein the tissue-derived epithelial stem cells are obtained from fragments of organoids selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cell trophoblast organoids and combinations thereof.

99. A system for culturing tissue-derived epithelial organoids, comprising tissue-derived epithelial organoids and a culture medium, wherein the tissue-derived epithelial organoids are embedded in a hydrogel suspended in the culture medium.

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

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

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

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

104. The system according to any one of claims 99 to 102, wherein the hydrogel has a filamentous structure.

105. The system according to claim 104, wherein the filament-like structure has a linear, snake-like, or spiral shape.

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

107. The system according to claim 106, wherein the stem cells and / or proliferation markers are selected from the group consisting of MKI67, EpCAM, CD49f, ASCL2, CD133, LGR5, SOX9, ALDH1A1, NEUROG3, NKX6.1, SMOC2, PDX1, CD44, and combinations thereof.

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

109. The system according to 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 (K18), cytokeratin 5 (K5), cytokeratin 14 (K14), smooth muscle actin (SMA), and combinations thereof.

110. The system according to any one of claims 99 to 109, wherein the tissue-derived epithelial organoid is selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cell trophoblast organoids, and combinations thereof.

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

112. The system according to claim 111, wherein the one or more robots and / or automated components include a liquid handling robot.

113. A system for carrying out the method according to any one of claims 1 to 63 and 75 to 98, comprising one or more robots and / or automated components.

114. The system according to claim 113, wherein the one or more robots and / or automated components include a liquid handling robot.

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

116. The method according to claim 115, wherein the one or more robots and / or automated components include a liquid handling robot.

117. The tissue-derived epithelial organoid according to claim 64, wherein the tissue-derived epithelial organoid is selected from the group consisting of 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, cervical organoids, prostate organoids, bladder organoids, ovarian organoids, taste bud organoids, cell trophoblast organoids, and combinations thereof.

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