Compositions and methods for obtaining organoids

Abstract

To provide a method for obtaining epithelial organoids, organoids obtained thereby, and uses thereof.SOLUTION: A method comprises the step of culturing one or more epithelial ducts, epithelial duct fragments and / or epithelial stem cells isolated therefrom in contact with an extracellular matrix in the presence of a basal medium, wherein the medium is free of FGF and / or nicotinamide.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims the benefit of U.S. Provisional Application No. 62 / 512,138, filed May 29, 2017, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to cell culture methods and cell culture medium formulations for use in said cell culture methods. More particularly, the present disclosure relates to cell culture methods and cell culture media for culturing primary tissues or cells thereof, and the maintenance, expansion, differentiation, and self-organization of primary tissues or cells thereof into epithelial three-dimensional (3D) structures. [Background technology]

[0003] background Since the discovery of adult stem and progenitor cells, the scientific community has focused on uncovering the molecular cues necessary to support the proliferation and differentiation of these cells ex vivo. Deriving adult stem cells avoids the ethical issues associated with the use of embryonic stem cells. Stem cell culture and differentiation are typically performed in 2D culture systems. Unfortunately, these 2D cultures of stem cells have many drawbacks when used as in vitro analogs of endogenous tissues. For example, 2D cultures are limited in their ability to induce cell polarization, acquire cellular functionality, and recapitulate human physiology in vitro. This reduces their accuracy as cellular models for pharmaceutical drug screening and therapeutic development. Therefore, improved culture systems for stem cells and their derived cell types that more accurately recapitulate in vivo tissues of interest are needed.

[0004] The design of artificial extracellular matrices based on natural adhesive proteins such as collagen, laminin, entactin, fibronectin, and vitronectin has driven research toward culturing systems in 3D environments. These natural and artificial extracellular matrices strive to mimic the in vivo cellular niche.

[0005] Organoids are 3D in vitro models of in vivo biological materials and processes, serving as a novel platform for addressing a wide variety of research questions related to embryonic development, tissue regeneration and functionality, drug toxicity and sensitivity, disease modeling, and adult stem cell biology. Epithelial organoids are derived by isolating and expanding stem and progenitor cells from the stem cell niche of an organ. They consist of epithelial monolayers or multilayers of stem / progenitor and differentiated cells that retain key physiological characteristics of the organ from which they originated. Epithelial organoids rely on an in vitro system that closely mimics the in vivo environment. Once established, epithelial organoids enable scientists to investigate specific research questions in a physiologically relevant context.

[0006] There is interest in developing cell culture methods to derive organoids representative of specific tissues. While some epithelial organs contain senescent and quiescent tissues, other epithelial tissues contain epithelia characterized by continuous turnover rates under homeostasis and even faster rates when exposed to damaging stimuli. Therefore, such tissues are particularly suitable for deriving organoids.

[0007] The intestinal epithelium was one of the first organoid systems to be established. However, there has been growing interest in developing cell culture methods to derive organoids from other tissues. Examples of these tissues include the liver and pancreas. Like the intestinal epithelium, the liver has a high regenerative capacity, especially under conditions of acute or chronic injury. The regeneration and turnover of liver tissue during homeostasis and injury has been thought to give rise to various cell types, including oval cells, hepatic progenitors, and mature hepatocytes. Facultative stem cells localized in the canals of Hering and bile ducts have also been implicated as a source of self-renewing cell types in terms of their function. Cultivating and expanding such cells ex vivo is more advantageous than primary hepatocytes, which are finite and limited in terms of proliferation and functionality once isolated from the liver.

[0008] Recently, single cells expressing adult stem cell markers Lgr5 or Epcam have been isolated from injured and uninjured mouse liver tissues, and when cultured in Expansion Medium, they have been shown to be capable of generating liver organoids in vitro that mimic endogenous in vivo liver epithelial structures (Huch et al., 2013, Nature, 494(7436):247-50 (Non-Patent Document 1); Huch et al. 2015, Cell, 160(1-2):299-312 (Non-Patent Document 2)). The method outlined in the related patent application (US20130189327 (Patent Document 1)) can be improved in many ways. First, toxin-mediated experimental liver injury, as described in US20130189327 (Patent Document 1), may be challenged by animal welfare standards. Second, even if damaged liver tissue were available, it may be desirable to avoid the tedious and technically challenging task of manually isolating hepatic ducts. Third, the expansion medium disclosed in US20130189327 (Patent Document 1) is complex and expensive, and therefore it may be desirable to quantify and / or remove numerous components, including various growth factors, small molecules, vitamins, and chemicals. Fourth, because US20130189327 (Patent Document 1) requires an undefined conditioned medium, which is technically difficult to prepare, the use of an unconditioned medium may offer certain advantages.

[0009] Therefore, there is a need for simple cell culture medium and method for producing epithelial organoids.In addition, there is a need for a cell culture medium that can support a faster epithelial organoid expansion rate.Most importantly, there is a need for a method for improving the yield of epithelial ducts and epithelial duct fragments from epithelial-lined organs, which allows organoid formation in cell culture medium that brings about the above-mentioned benefits.In addition, there is a need to establish a medium and protocol that can be used across various epithelial organs to establish organ-specific organoids. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] US20130189327 [Non-patent literature]

[0011] [Non-Patent Document 1] Huch et al., 2013, Nature, 494(7436):247-50 [Non-patent document 2] Huch et al. 2015, Cell, 160(1-2): 299-312 Summary of the Invention

[0012] overview The inventors have developed a method that allows the isolation of large numbers of cells derived from intact epithelial tissue that may be expanded and cultured long-term under minimal culture conditions and that may be suitable for downstream differentiation into mature cell types.

[0013] The present disclosure includes a method for obtaining organoids derived from one or more cells isolated from epithelial tissue, comprising culturing one or more cells in a culture medium of the present disclosure.

[0014] In one aspect, a method for obtaining a high yield of expandable cells includes enzymatically digesting epithelial tissue into epithelial ducts and / or epithelial duct fragments, optionally with frequent mechanical disruption of the digested tissue, and optionally thereafter filtering the digested tissue through a cell strainer (rather than manually picking the ducts under a microscope) to remove single cells. This high yield of epithelial ducts and / or epithelial duct fragments may be cultured in the media of the present disclosure.

[0015] In another embodiment, a method for obtaining a high yield of expandable cells comprises enzymatically digesting epithelial tissue into single epithelial stem and / or progenitor cells, optionally subjecting the digested tissue to frequent mechanical disruption, and optionally subsequently filtering the digested tissue through a cell strainer to separate large debris from the single epithelial stem and / or progenitor cells. This high yield of cells may be cultured in the media of the present disclosure.

[0016] Optionally, the epithelial ducts and / or epithelial duct fragments and / or epithelial stem and / or progenitor cells may also be supported by a mixture of culture medium and extracellular matrix of the present disclosure.

[0017] In one embodiment, the epithelial ducts and / or epithelial duct fragments and / or epithelial stem and / or progenitor cells may be seeded in a basal medium, which may be characterized by the absence of one, some, or all of EGF, R-spondin, fibroblast growth factor, and nicotinamide.

[0018] In another embodiment, the basal expansion medium comprises an activator of the Wnt-β-catenin pathway.

[0019] In another embodiment, the expansion medium comprises epidermal growth factor (EGF) and an activator of the Wnt-β-catenin pathway.

[0020] In another embodiment, the expansion medium comprises Noggin protein or a different bone morphogenetic protein (BMP) antagonist, an inhibitor of signaling downstream of BMP, eg, LDN193189, EGF, and an activator of the Wnt-β-catenin pathway.

[0021] Therefore, the present disclosure provides a method for obtaining epithelial organoids, comprising culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells isolated therefrom in the presence of a basal medium, wherein the medium does not contain FGF and / or nicotinamide.

[0022] In one embodiment, the medium comprises a Wnt agonist, optionally selected from one or more of CHIR99021 and / or Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor.

[0023] In one embodiment, the medium comprises EGF, hi another embodiment, the medium comprises B27 component and / or N2 component, and / or N-acetylcysteine.

[0024] In another embodiment, the epithelial stem or progenitor cell is a human cell, a mouse cell, a rat cell, a liver epithelial stem cell, a pancreatic epithelial stem cell, or an intestinal epithelial stem cell.

[0025] In another embodiment, the medium is effective for long-term culture of epithelial organoids, the long-term culture being greater than about 50 passages.

[0026] In another embodiment, the method further comprises culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem or progenitor cells in contact with an extracellular matrix.

[0027] In another embodiment, the method further comprises culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem or progenitor cells in contact with a reduced support derived from an extracellular matrix.

[0028] In one embodiment, the extracellular matrix concentration is 0.1 to 50% (v / v).

[0029] In another embodiment, the method further comprises culturing epithelial stem cells or epithelial progenitor cells derived from epithelial ducts and / or epithelial duct fragments in suspension. In one embodiment, the extracellular matrix concentration is about 0.1% (v / v) or less.

[0030] In another embodiment, the extracellular matrix comprises Matrigel™.

[0031] In another embodiment, the epithelial duct, epithelial duct fragment, and / or epithelial stem or progenitor cells isolated therefrom are obtained from intact tissue.

[0032] In another embodiment, the method further comprises subjecting the epithelial organoids to maturation and / or differentiation conditions.

[0033] The present disclosure also provides liver organoids obtained according to the methods disclosed herein.

[0034] The present disclosure also provides pancreatic organoids obtained according to the methods disclosed herein.

[0035] The present disclosure also provides intestinal organoids obtained according to the methods disclosed herein.

[0036] The present disclosure also provides methods for conducting drug discovery screening; assaying toxicity; investigating embryology, cell lineage, and differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in injury and repair; investigating inflammatory and infectious diseases; and studying the pathogenic mechanisms of cell transformation and the causes of cancer, comprising obtaining epithelial organoids according to the methods disclosed herein.

[0037] Furthermore, the present disclosure provides a method for treating liver damage, condition or disease in a subject or a method for regenerative medicine, comprising administering the liver organoid disclosed herein to a subject in need of the liver organoid disclosed herein.

[0038] Furthermore, the present disclosure provides a method for treating a pancreatic disorder, condition, or disease in a subject, or a method for regenerative medicine, comprising administering the pancreatic organoids disclosed herein to a subject in need thereof.

[0039] Furthermore, the present disclosure provides a method for treating an intestinal disorder, condition, or disease in a subject, or a method for regenerative medicine, comprising administering the intestinal organoids disclosed herein to a subject in need thereof.

[0040] The present disclosure also provides a method for obtaining epithelial organoids, comprising: isolating an organ or part thereof containing epithelial tissue; cutting and mechanically disrupting the isolated organ or part thereof to obtain one or more fragments of epithelial tissue; enzymatically digesting one or more fragments of epithelial tissue to obtain epithelial ducts and / or duct fragments; collecting the digested epithelial ducts and / or duct fragments with a cell strainer; plating the collected epithelial ducts and / or duct fragments; and Culturing the plated epithelial ducts and / or duct fragments in the presence of a basal medium, wherein said medium does not contain FGF and / or nicotinamide. Also provided is a method, including:

[0041] In one embodiment, the medium comprises a Wnt agonist, optionally CHIR99021, or the Wnt agonist is selected from one or more of Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor.

[0042] In one embodiment, the medium comprises EGF.

[0043] In another embodiment, the medium comprises B27 component and / or N2 component and / or N-acetylcysteine.

[0044] In another embodiment, the method further comprises the step of maintaining the intactness of ductal structures during digestion.

[0045] In another embodiment, the method further comprises treating the surface or device with a surfactant before contacting it with one or more fragments of epithelial tissue.

[0046] In another embodiment, the method further comprises plating the collected epithelial ducts and / or epithelial duct fragments in contact with an extracellular matrix.

[0047] In another embodiment, the method further comprises the step of plating the collected epithelial ducts and / or epithelial duct fragments in contact with a low concentration of support derived from an extracellular matrix.

[0048] In another embodiment, the extracellular matrix concentration is 0.1 to 50% (v / v).

[0049] In another embodiment, the method further comprises plating the collected epithelial ducts and / or epithelial duct fragments in suspension.

[0050] In another embodiment, the extracellular matrix concentration is about 0.1% (v / v) or less.

[0051] In another embodiment, the extracellular matrix comprises Matrigel™.

[0052] In another embodiment, the method further comprises subjecting the epithelial organoids to maturation and / or differentiation conditions.

[0053] The present disclosure also provides a medium for obtaining epithelial organoids, comprising a basal medium lacking FGF and / or nicotinamide.

[0054] In one embodiment, the medium further comprises a Wnt agonist, optionally CHIR99021.

[0055] In another embodiment, the Wnt agonist is selected from one or more of a Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor.

[0056] In another embodiment, the medium further comprises EGF.

[0057] In another embodiment, the medium further comprises B27 component and / or N2 component and / or N-acetylcysteine.

[0058] In another embodiment, the medium further comprises an extracellular matrix.

[0059] In one embodiment, the concentration of the extracellular matrix is ​​0.1 to 50% (v / v).

[0060] In another embodiment, the concentration of extracellular matrix is ​​about 0.1% (v / v) or less.

[0061] In another embodiment, the extracellular matrix comprises Matrigel™.

[0062] The present disclosure also provides a culture medium comprising a basal medium, HEPES, sodium bicarbonate, Rh insulin, progesterone, putrescine, sodium selenite, human apotransferrin, corticosterone, D-(+)-galactose, and BSA.

[0063] In one embodiment, said medium further comprises R-spondin-1 and / or CHIR99021.

[0064] In another embodiment, the medium further comprises EGF.

[0065] In another embodiment, the medium further comprises a BMP antagonist.

[0066] [The present invention 1001] A method for obtaining epithelial organoids, comprising the steps of: Culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells isolated therefrom in the presence of a basal medium, wherein the medium does not contain FGF and / or nicotinamide. [The present invention 1002] The method of claim 1001, wherein the culture medium comprises a Wnt agonist. [The present invention 1003] The method of claim 1002, wherein the Wnt agonist is CHIR99021. [The present invention 1004] 1003. The method of claim 1002, wherein the Wnt agonist is selected from one or more of Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor. [The present invention 1005] The method according to any one of claims 1001 to 1004, wherein the medium contains EGF. [The present invention 1006] The method according to any one of claims 1001 to 1005, wherein the medium comprises a B27 component and / or an N2 component and / or N-acetylcysteine. [The present invention 1007] The method of any one of claims 1001 to 1006, wherein said epithelial cells or progenitor stem cells are human cells. [The present invention 1008] The method of any one of claims 1001 to 1006, wherein the epithelial stem cells or epithelial progenitor cells are mouse cells. [The present invention 1009] The method of any one of claims 1001 to 1006, wherein the epithelial stem cells or epithelial progenitor cells are rat cells. [The present invention 1010] The method of any one of claims 1001 to 1009, wherein the epithelial stem cells or epithelial progenitor cells are liver epithelial stem cells. [The present invention 1011] The method of any of claims 1001 to 1009, wherein the epithelial stem cells or epithelial progenitor cells are pancreatic epithelial stem cells. [The present invention 1012] The method of any one of claims 1001 to 1009, wherein the epithelial stem cells or epithelial progenitor cells are intestinal epithelial stem cells. [The present invention 1013] The method of any of claims 1001 to 1012, wherein the medium is effective for long-term culture of epithelial organoids, and the long-term culture is about 50 or more passages. [The present invention 1014] The method of any of claims 1001 to 1013, further comprising the step of culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells in contact with an extracellular matrix. [The present invention 1015] Any of the methods of claims 1001 to 1014, further comprising the step of contacting and culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells with a low concentration support derived from an extracellular matrix. [The present invention 1016] The method of the present invention 1015, wherein the extracellular matrix concentration is 0.1 to 50% (v / v). [The present invention 1017] The method of any one of claims 1001 to 1013, further comprising the step of culturing epithelial stem cells or epithelial progenitor cells derived from epithelial ducts and / or epithelial duct fragments in suspension. [The present invention 1018] 1017. The method of claim 1017, wherein the extracellular matrix concentration is about 0.1% (v / v) or less. [The present invention 1019] The method of any of claims 1001 to 1018, wherein the extracellular matrix comprises Matrigel™. [The present invention 1020] 1020. The method of any of claims 1001 to 1019, wherein the epithelial duct, epithelial duct fragment, and / or epithelial stem cells or epithelial progenitor cells isolated therefrom are obtained from intact tissue. [The present invention 1021] The method of any of claims 1001 to 1020, further comprising the step of subjecting the epithelial organoid to maturation conditions and / or differentiation conditions. [The present invention 1022] A liver organoid obtained according to the method of the present invention 1010. [The present invention 1023] A pancreatic organoid obtained according to the method of the present invention. [The present invention 1024] Intestinal organoids obtained according to the method of the present invention. [The present invention 1025] A method for conducting drug discovery screening; assaying toxicity; investigating development, cell lineage, and differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in damage and repair; investigating inflammatory and infectious diseases; and studying the pathogenic mechanisms of cell transformation and the causes of cancer, the method comprising the step of obtaining an epithelial organoid of any of the present inventions 1001 to 1021. [The present invention 1026] A method for treating liver damage, condition or disease in a subject or a method for regenerative medicine, comprising administering the liver organoid of the present invention 1022 to a subject in need thereof. [The present invention 1027] A method for treating a pancreatic disorder, condition, or disease in a subject or a method for regenerative medicine, comprising administering the pancreatic organoid of the present invention 1023 to a subject in need thereof. [The present invention 1028] A method for treating an intestinal disorder, condition, or disease in a subject or a method for regenerative medicine, comprising administering the intestinal organoid of the present invention 1024 to a subject in need thereof. [The present invention 1029] 1. A method for obtaining epithelial organoids, comprising the steps of: isolating an organ or part thereof containing epithelial tissue; cutting and mechanically disrupting the isolated organ or part thereof to obtain one or more fragments of epithelial tissue; enzymatically digesting one or more fragments of epithelial tissue to obtain epithelial ducts and / or duct fragments; collecting the digested epithelial ducts and / or duct fragments with a cell strainer; plating the collected epithelial ducts and / or duct fragments; and Culturing the plated epithelial ducts and / or duct fragments in the presence of a basal medium, wherein the medium does not contain FGF and / or nicotinamide. [The present invention 1030] The method of claim 1029, wherein the culture medium comprises a Wnt agonist. [The present invention 1031] The method of claim 1030, wherein the Wnt agonist is CHIR99021. [The present invention 1032] 1030. The method of claim 1030, wherein the Wnt agonist is selected from one or more of a Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor. [The present invention 1033] The method of any one of claims 1029 to 1032, wherein the medium contains EGF. [The present invention 1034] The method of any one of claims 1029 to 1033, wherein the medium comprises a B27 component, an N2 component, and / or N-acetylcysteine. [This invention 1035] 1035. The method of any one of claims 1029 to 1034, further comprising the step of maintaining the intactness of ductal structures during digestion. [The present invention 1036] 1036. The method of any of claims 1029 to 1035, further comprising the step of treating the surface or device with a surfactant before contacting it with one or more fragments of epithelial tissue. [This invention 1037] The method of any of claims 1029 to 1036, further comprising the step of plating the collected epithelial ducts and / or epithelial duct fragments in contact with an extracellular matrix. [The present invention 1038] The method of any of claims 1029 to 1037, further comprising the step of plating the collected epithelial ducts and / or epithelial duct fragments in contact with a low concentration support derived from an extracellular matrix. [This invention 1039] The method of claim 1038, wherein the extracellular matrix concentration is 0.1 to 50% (v / v). [The present invention 1040] The method of any of claims 1029 to 1036, further comprising plating the collected epithelial ducts and / or epithelial duct fragments in suspension. [The present invention 1041] The method of claim 1040, wherein the extracellular matrix concentration is about 0.1% (v / v) or less. [The present invention 1042] 1042. The method of any one of claims 1029 to 1041, wherein the extracellular matrix comprises Matrigel™. [This invention 1043] The method of any of claims 1029 to 1042, further comprising subjecting the epithelial organoid to maturation conditions and / or differentiation conditions. [This invention 1044] A medium for obtaining epithelial organoids, comprising a basal medium without FGF and / or nicotinamide. [This invention 1045] The medium of the present invention 1044 further comprising a Wnt agonist. [The present invention 1046] The medium of the present invention 1045, wherein the Wnt agonist is CHIR99021. [This invention 1047] 1045. The culture medium of the present invention, wherein the Wnt agonist is selected from one or more of Wnt, Wnt3a, Norrin, R-spondin1, R-spondin2, R-spondin3, R-spondin4, and a GSK inhibitor. [This invention 1048] The medium of any one of 1044 to 1047 of the present invention, further comprising EGF. [This invention 1049] The medium of any of 1044 to 1048 of the present invention, further comprising component B27 and / or component N2 and / or N-acetylcysteine. [The present invention 1050] The medium of any one of 1044 to 1049 of the present inventions, further comprising an extracellular matrix. [This invention 1051] The medium of the present invention 1050, wherein the concentration of the extracellular matrix is ​​0.1 to 50% (v / v). [This invention 1052] 1050. The medium of the present invention, wherein the concentration of the extracellular matrix is ​​about 0.1% (v / v) or less. [This invention 1053] The medium of any one of 1050 to 1052, wherein the extracellular matrix comprises Matrigel (trademark). Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of example only, as various modifications and changes within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0067] The present disclosure will now be described with reference to the following drawings:

[0068] [Figure 1] (a) Expansion of epithelial tissue isolated from mouse liver into liver organoids, (b) expansion of epithelial tissue isolated from mouse pancreas into pancreatic organoids, and (c) expansion of epithelial tissue isolated from mouse intestine into intestinal organoids. [Figure 2] Shown is an epithelial organoid showing a single layer of epithelium, a hollow lumen, and characteristic intercellular junctions and morphology. [Figure 3]Processing of epithelial organs. (a) Generalized protocol for processing epithelium-containing organs to obtain epithelial ducts, epithelial duct fragments, and / or epithelial stem cells contained in epithelial ducts and / or epithelial duct fragments. EDC = enzymatic digestion cocktail. (b) Time course in minutes showing the progressive decrease in turbidity of the buffer containing digestive enzymes in successively digested samples. [Figure 4] Filtration of epithelial ducts and / or epithelial duct fragments is shown, showing an optional first pre-clearing step to filter out large debris. [Figure 5] Subconfluently plated epithelial ducts and / or epithelial duct fragments are shown. [Figure 6] Progressive epithelial organoid degeneration: (a) Healthy organoid at day 4, passage 15; (b) Organoid showing signs of increasingly darkened luminal spaces; (c) Organoid showing signs of dark and collapsed luminal spaces. [Figure 7] (a) Repeated digestion using a buffer containing digestive enzymes. Epithelial tissue can be digested in the presence of a buffer containing digestive enzymes. If digestion is incomplete, the supernatant can be collected and saved, and the pellet can be further digested one or more times. (b) Illustrative photographs of the appearance of epithelial ducts and / or epithelial duct fragments after digestion of epithelial tissue in a buffer containing digestive enzymes. [Figure 8] We demonstrate that liver organoids can be maintained and expanded regardless of whether the plated material is hepatic ducts and / or hepatic duct fragments, mechanically disrupted and / or enzymatically digested liver organoids, or single epithelial stem and / or progenitor cells contained within hepatic ducts and / or hepatic duct fragments. [Figure 9] We show that liver organoids can be passaged and expanded long-term. [Figure 10]Gene expression profiling of liver organoids, hepatic ducts and / or duct fragments, and primary mouse hepatocytes by RT-PCR: (a) Wnt target gene expression assessment, (b) liver progenitor gene expression assessment, (c) bile duct gene expression assessment, (d) liver gene expression assessment. [Figure 11] Protein synthesis in liver organoids using immunocytochemistry. [Figure 12] We demonstrate that pancreatic organoids can be maintained and expanded regardless of whether the plated material is pancreatic ducts and / or pancreatic duct fragments, mechanically disrupted and / or enzymatically digested pancreatic organoids, or single epithelial stem and / or progenitor cells contained within pancreatic ducts and / or pancreatic duct fragments. [Figure 13] We demonstrate that pancreatic organoids can be passaged and expanded long-term. [Figure 14] Gene expression profiling of pancreatic organoids, mouse fibroblasts, and liver organoids by RT-PCR: (a) Assessment of Wnt target gene expression, (b) assessment of pancreatic progenitor gene expression, (c) assessment of ductal gene expression, and (d) assessment of proliferation and non-ductal gene expression. [Figure 15] Showing protein synthesis in pancreatic organoids using immunocytochemistry. [Figure 16] We show that intestinal organoids can be maintained and expanded from intestinal crypts. [Figure 17] We demonstrate that intestinal organoids can be expanded from crypts or single cells. [Figure 18] Gene expression in intestinal and colonic organoids is shown. [Figure 19] Protein synthesis of intestinal markers in small intestinal organoids: (a) Lgr5 - stem cell marker, R - spondin receptor, (b) lysozyme - Paneth cells, (c) Villen - enterocytes, (d) chromogranin A - enteroendocrine cells. [Figure 20]Protein synthesis of colonic markers in colon organoids is shown: lysozyme - Paneth cells; villin - enterocytes; chromogranin A - enteroendocrine cells. DETAILED DESCRIPTION OF THE INVENTION

[0069] Detailed Description The present disclosure describes the medium and method for culturing epithelial organoid.The present disclosure also describes the medium and method for forming epithelial organoid.The epithelial organoid of the present disclosure can be derived from one or more epithelial organs or its part, epithelial tissue, epithelial duct, epithelial duct fragment and / or epithelial stem cell / progenitor cell according to the method disclosed herein, under the existence of the medium disclosed herein.

[0070] The disclosed medium and method can initiate and maintain epithelial organoids, including but not limited to organoids derived from liver tissue, pancreatic tissue, lung tissue and intestinal tissue.Epithelial organoids can be derived from human tissue or animal tissue.

[0071] Cell culture medium In one aspect, the present disclosure provides the culture medium for deriving and / or obtaining epithelial organoid.Epithelial organoid can be derived and / or obtained according to the method described herein below.

[0072] In one embodiment, the epithelial organoids are obtained by culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells isolated therefrom in the presence of the culture medium described below.

[0073] In one embodiment, the culture medium (also referred to herein as "medium" or "expansion medium") for forming and / or culturing epithelial organoid is a basal medium.The term "basal medium" used herein refers to a medium that contains the elements necessary for the growth of animal cells or human cells, namely, carbon source, water, salt, and amino acid source and nitrogen source (e.g., bovine or yeast extract).Exemplary basal media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), DMEM / nutrient mixture F-12 (DMEM / F-12), Advanced DMEM / F-12, RPMI1640, Iscove's Modified Dulbecco's Medium (IMDM), minimal essential medium (MEM), and / or basal medium Eagle (BME).In one embodiment, basal medium comprises a combination of various ratios of the above exemplary basal media.

[0074] The present inventors have shown that epithelial organoids can be obtained by culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells isolated therefrom in a medium that lacks FGF and / or nicotinamide.It has been shown that one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells cultured in the medium of the present disclosure can produce organoids that can undergo initial expansion (Fig. 1).Such organoids can show epithelial cell layer, hollow lumen, and typical intercellular junctions and morphology (Fig. 2).

[0075] Thus, in one embodiment, the medium does not contain or lacks FGF and / or its derivatives or agonists. As used herein, the term "FGF" or "fibroblast growth factor" refers to a member of the fibroblast growth factor family of signaling molecules. FGF may be a natural or synthetic FGF. In one embodiment, the FGF is a recombinant FGF. For example, 22 FGFs are known in humans. In another embodiment, the medium does not contain or lacks nicotinamide and / or its derivatives or agonists. In another embodiment, the medium does not contain FGF (and / or its derivatives or agonists) and / or nicotinamide (and / or its derivatives or agonists). In another embodiment, the medium does not contain or lack FGF and nicotinamide and / or its derivatives or agonists. In yet another embodiment, the medium has undetectable amounts of FGF and / or nicotinamide or does not have exogenously added FGF and / or nicotinamide.

[0076] In one embodiment, the medium is a plain medium, while in another embodiment, the medium is a medium supplemented with amino acids, vitamins, organic / inorganic salts, sugars, and / or antioxidants.

[0077] In another embodiment, the medium is supplemented with L-glutamine. L-glutamine is an essential amino acid that is unstable in liquid media at physiological pH. L-glutamine for adding to cell culture media is commercially available. In one embodiment, the medium is supplemented with 0.5 mM to 10 mM glutamine, or 1 mM to 5 mM glutamine, or 2 mM to 4 mM glutamine. In certain embodiments, the L-glutamine is a stabilized form of L-glutamine. One example of a stabilized form of L-glutamine is L-alanyl-L-glutamine.

[0078] The medium may also contain a buffering agent to maintain the appropriate pH of the medium. In one embodiment, the buffering agent is HEPES. Optionally, the medium contains up to 25 mM HEPES, about 20 mM HEPES, about 15 mM HEPES, about 10 mM HEPES, about 5 mM HEPES, about 1 mM HEPES, or less. In another embodiment, the buffering agent is sodium bicarbonate. As a guideline, sodium bicarbonate may be provided in the medium at about 1.0 g / L to 5.0 g / L per 4-10% CO2 cell culture conditions. In one embodiment, the medium is supplemented with about 1.5 g / L sodium bicarbonate for cells grown in 5% CO2, about 2 g / L for cells grown in 5% CO2, about 3 g / L for cells grown in 5% CO2, or about 4 g / L for cells grown in 5% CO2. In other embodiments, the basal medium is supplemented with multiple buffers, e.g., HEPES and sodium bicarbonate. In certain such embodiments, the basal medium comprises less than about 25 mM HEPES and less than about 5 g / L sodium bicarbonate.

[0079] In another embodiment, the medium includes at least one B-27 and / or N2 supplement. Such supplements and their components are well known in the field of mammalian cell culture. B-27 and N2 are commercially available from various suppliers. In another embodiment, the medium includes one or more subcomponents of B-27 and / or N2. In certain embodiments, the medium includes one or more of the following B-27 and / or N2 subcomponents: Rh insulin; progesterone; putrescine; sodium selenite; human apotransferrin; corticosterone; D-(+)-galactose; and BSA. Furthermore, when the medium includes several of the B-27 and / or N2 subcomponents, it may be desirable to deviate from the working concentrations of the complete B-27 and N2. For example, in certain embodiments, the medium includes lower concentrations of the B-27 and / or N2 subcomponents. In one embodiment of the medium, B-27 and / or N2 minor components may be used in the following final concentration ranges: 0.01-100 μg / mL Rh insulin; 2 nM-400 μM progesterone; 0.1-10 mM putrescine; 1 ng / mL-100 μg / mL sodium selenite; 0.2-200 μg / mL human apotransferrin; 1-500 ng / mL corticosterone; 30 μM-30 mM D-(+)-galactose; and 0.5 μg / mL-5 mg / mL BSA.

[0080] In one embodiment, the medium comprises DMEM / F-12 or Advanced DMEM / F-12, 10 to 20 mM HEPES, 0.5 to 1.5 g / L sodium bicarbonate, 55 to 75 μg / mL Rh insulin, 35 to 45 nM progesterone, 0.1 to 0.8 mM putrescine, 5 to 15 ng / mL sodium selenite, 60 to 100 μg / mL human apotransferrin, 10 to 30 ng / mL corticosterone, 10 to 20 μg / mL D-(+)-galactose, and 1 to 4 mg / mL BSA. In another embodiment, the medium comprises, consists of, or consists essentially of DMEM / F-12 or Advanced DMEM / F-12 and about 15 mM HEPES, 1.2 g / L sodium bicarbonate, 62 μg / mL Rh insulin, 38 nM progesterone, 0.4 mM putrescine, 10 ng / mL sodium selenite, 80 μg / mL human apotransferrin, 20 ng / mL corticosterone, 15 μg / mL D-(+)-galactose, and 2.6 mg / mL BSA. This medium is referred to herein as "Medium A."

[0081] In one embodiment, the epithelial organoid formed in the presence of medium A is cultured for about 5 or more passages.For example, the liver organoid formed according to the method described herein and in the presence of medium A can undergo initial expansion into organoid.

[0082] In other embodiments, the culture medium for obtaining epithelial organoid comprises the above-mentioned basic medium, such as medium A, and further comprises at least one, at least two, at least three, or at least four activators of Wnt-β-catenin pathway.As used herein, the term " activators of Wnt-β-catenin pathway " refers to any molecule, antibody, compound, or gene modifier (for example, siRNA) that leads to any β-catenin-induced and / or YAP / TAZ-induced transcriptional changes.For example, at least one or more activators of Wnt-β-catenin pathway can be any molecule or compound that positively regulates the signal transduction of Frizzled family receptor.Non-limiting examples of the molecule or compound that positively regulates the signal transduction of Frizzled family receptor include Wnt protein or agonist; and / or Norrin protein or agonist; and / or R-spondin protein or R-spondin agonist that binds to Lgr4 / 5 and RNF43 / ZNRF3.

[0083] In one embodiment, at least one, at least two, at least three, or at least four activators of the Wnt-β-catenin pathway are or include molecules or compounds that sequester negative regulators of the Wnt-β-catenin pathway.Non-limiting examples of molecules or compounds that sequester negative regulators of the Wnt-β-catenin pathway include, but are not limited to, SB-216763, CHIR99021, and CAS853220-52-7.

[0084] In another embodiment, the molecule or compound that sequesters a negative regulator of the Wnt-β-catenin pathway is a small molecule inhibitor. In a specific embodiment, the small molecule inhibitor is a GSK-3 inhibitor. For example, the GSK-3 inhibitor can be CHIR99021. In a specific embodiment, CHIR99021 is present at a concentration of 1 μM to 10 μM, or 3 μM to 8 μM, or 4 μM to 7 μM, or 5 μM to 6 μM.

[0085] In one embodiment, the Wnt protein is Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, or Wnt16. In another embodiment, the Wnt protein is Wnt3a. Wnt3a may be a recombinant protein or may be added as a conditioned medium. In another embodiment, Wnt3a may be added to the medium together with molecules and compounds that stabilize the protein, for example, its post-translational modification configuration, thereby enhancing its signaling ability.

[0086] In another embodiment, the R-spondin protein or agonist is R-spondin-1, R-spondin-2, R-spondin-3, or R-spondin-4. In a specific embodiment, the R-spondin protein or agonist is recombinant. The R-spondin protein or agonist in the culture medium may be present at a concentration of about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 μg / mL, or about 3 ng / mL to 150 ng / mL, or about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL. In a specific embodiment, the R-spondin is R-spondin-1.

[0087] In one embodiment, the medium comprises multiple activators of the Wnt-β-catenin pathway, hi another embodiment, the medium comprises R-spondin1 and CHIR99021.

[0088] In another embodiment, the Norrin protein or agonist is an ortholog of the invertebrate burs and pburs genes. In another embodiment, the Norrin protein or agonist acts as a cognate ligand for LGR4, Fzl4, LGR5, and LGR6. In another embodiment, the Norrin protein or agonist inhibits signaling via BMP. In certain embodiments, the Norrin protein or agonist is recombinant. In another embodiment, the Norrin protein or agonist in the culture medium is present at a concentration of about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 μg / mL, or about 3 ng / mL to 150 ng / mL, or about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0089] In one embodiment, the medium comprises Advanced DMEM / F-12 or DMEM / F-12, 10 to 20 mM HEPES, 0.5 to 1.5 g / L sodium bicarbonate, 0.005 to 0.025 μg / mL R-spondin-1 and / or 2.5 to 4.5 μM CHIR99021, 55 to 75 μg / mL Rh insulin, 35 to 45 nM progesterone, 0.1 to 0.8 mM putrescine, 5 to 15 ng / mL sodium selenite, 60 to 100 μg / mL human apotransferrin, 10 to 30 ng / mL corticosterone, 10 to 20 μg / mL D-(+)-galactose, and 1 to 4 mg / mL BSA. In another embodiment, the medium comprises, consists of, or consists essentially of Advanced DMEM / F-12 or DMEM / F-12, and about 15 mM HEPES, 1.2 g / L sodium bicarbonate, 0.015 μg / mL R-spondin-1 and / or 3.75 μM CHIR99021, 62 μg / mL Rh insulin, 38 nM progesterone, 0.4 mM putrescine, 10 ng / mL sodium selenite, 80 μg / mL human apotransferrin, 20 ng / mL corticosterone, 15 μg / mL D-(+)-galactose, and 2.6 mg / mL BSA. This medium is referred to herein as "medium B".

[0090] In one embodiment, the epithelial organoid formed in the presence of medium B is cultured for about 10 or more passages.For example, according to the method described herein, the liver organoid formed in the presence of medium B can undergo initial expansion, and its culture can be supported for at least 10 passages.In general, medium B can be used for the formation and culture of epithelial organoid.

[0091] In another embodiment, the culture medium for forming and / or culturing epithelial organoid comprises the above-mentioned basic medium (for example, medium A).In another embodiment, said medium further comprises at least one activator of the above-mentioned Wnt-β-catenin pathway (for example, medium B).In another embodiment, said medium further comprises one or more members of epidermal growth factor (EGF) family, for example, EGF, TGF-α, amphiregulin, betacellulin, epiregulin, heparin-binding EGF-like growth factor, epigen, and neuregulin-1, -2, -3 and -4.

[0092] In certain embodiments, the EGF family member is present at a concentration of about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 μg / mL, or about 3 ng / mL to 150 ng / mL, or about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0093] In one embodiment, the medium comprises Advanced DMEM / F-12 or DMEM / F-12, 10 to 20 mM HEPES, 0.5 to 1.5 g / L sodium bicarbonate, 0.005 to 0.025 μg / mL R-spondin-1 and / or 2.5 to 4.5 μM CHIR99021, 0.01 to 1.0 μg / mL EGF, 55 to 75 μg / mL Rh insulin, 35 to 45 nM progesterone, 0.1 to 0.8 mM putrescine, 5 to 15 ng / mL sodium selenite, 60 to 100 μg / mL human apotransferrin, 10 to 30 ng / mL corticosterone, 10 to 20 μg / mL D-(+)-galactose, and 1 to 4 mg / mL BSA. In another embodiment, the medium comprises, consists of, or consists essentially of Advanced DMEM / F-12 or DMEM / F-12, and about 15 mM HEPES, 1.2 g / L sodium bicarbonate, 0.015 μg / mL R-spondin-1 and / or 3.75 μM CHIR99021, 0.05 μg / mL EGF, 62 μg / mL Rh insulin, 38 nM progesterone, 0.4 mM putrescine, 10 ng / mL sodium selenite, 80 μg / mL human apotransferrin, 20 ng / mL corticosterone, 15 μg / mL D-(+)-galactose, and 2.6 mg / mL BSA. This medium is referred to herein as "medium C".

[0094] In one embodiment, the epithelial organoid formed in the presence of medium C is cultured for about 40 or more passages.For example, according to the method described herein, the liver organoid formed in the presence of medium C can undergo initial expansion, and the culture can be supported for about 40 or more passages.In general, medium C can be used for the formation and culture of epithelial organoid and the long-term culture of epithelial organoid.

[0095] In another embodiment, the culture medium for obtaining epithelial organoid comprises the above-mentioned basic medium (for example, medium A).The culture medium can also comprise at least one or more activators of the Wnt-β-catenin pathway (for example, medium B) and one or more members of the EGF family (for example, medium C).In another embodiment, the culture medium further comprises one or more inhibitors or antagonists of BMP or BMP signal transduction.

[0096] In one embodiment, the one or more inhibitors or antagonists of BMP are proteins, such as, but not limited to, noggin, chordin, follistatin, sclerostin, CTGF / CCN2, gremlin, cerberus, DAN, PRDC, decorin, alpha-2 macroglobulin protein, and derivatives thereof.

[0097] In certain embodiments, the concentration of protein for inhibiting a BMP, e.g., Noggin, is present at a concentration of about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 μg / mL, or about 3 ng / mL to 150 ng / mL, or about 5 ng / mL to 100 ng / mL, or about 10 ng / mL to 50 ng / mL.

[0098] In another embodiment, the one or more inhibitors or antagonists of BMP signaling are small molecule inhibitors capable of inhibiting the proteins identified above and / or signaling downstream of BMP, such as, but not limited to, LDN193189 or dorsomorphin.

[0099] In embodiments in which one or more inhibitors or antagonists of BMP or BMP signaling may be used to add to the culture medium, such inhibitors may be present at a concentration of 0.001 nM to 10 mM, optionally 0.0001 μM to 0.2 μM. In certain embodiments in which LDN193189 is added to the culture medium, LDN193189 may be present at a concentration of 0.001 nM to 10 mM, optionally 0.001 μM to 1 μM.

[0100] In one embodiment, the medium comprises Advanced DMEM / F-12 or DMEM / F-12, 10 to 20 mM HEPES, 0.5 to 1.5 g / L sodium bicarbonate, 0.005 to 0.025 μg / mL R-spondin-1 and / or 2.5 to 10 μM CHIR99021, 0.01 to 1.0 μg / mL EGF, 0.0.05 to 0.30 μg / mL noggin, 0.05 to 0.15 μM LDN, 55 to 75 μg / mL Rh insulin, 35 to 45 nM progesterone, 0.1 to 0.8 mM putrescine, 5 to 15 ng / mL sodium selenite, 60 to 100 μg / mL human apotransferrin, 10 to 30 ng / mL corticosterone, 10 to 20 μg / mL D-(+)-galactose, and 1-4 mg / mL BSA.

[0101] In another embodiment, the medium comprises, consists of, or consists essentially of Advanced DMEM / F-12 or DMEM / F-12, and about 15 mM HEPES, 1.2 g / L sodium bicarbonate, 0.015 μg / mL R-spondin-1 and / or 7.5 μM CHIR99021, 0.05 μg / mL EGF, 0.015 μg / mL noggin, 0.1 μM LDN, 62 μg / mL Rh insulin, 38 nM progesterone, 0.4 mM putrescine, 10 ng / mL sodium selenite, 80 μg / mL human apotransferrin, 20 ng / mL corticosterone, 15 μg / mL D-(+)-galactose, and 2.6 mg / mL BSA. This medium is referred to herein as "Medium D".

[0102] In one embodiment, the epithelial organoid formed in the presence of medium D is cultured for about 50 or more passages, and supports a split ratio of about 1:30.For example, the liver organoid formed according to the method described herein and in the presence of medium D can undergo initial expansion, and the culture can be supported for about 50 or more passages.In general, medium D can be used to form and culture epithelial organoid, and to further culture epithelial organoid for a long period of time.

[0103] In still other embodiments, the media described herein (including, but not limited to, Medium A, Medium B, Medium C, and / or Medium D) are further supplemented with other compounds or molecules that support the culture of human or animal cells. For example, in one embodiment, the media is supplemented with N-acetyl-L-cysteine. In certain embodiments, N-acetyl-L-cysteine ​​is present at a concentration of 750 μM to 1500 μM.

[0104] In further embodiments, the medium is supplemented with hepatocyte growth factor (HGF). In particular embodiments, HGF is present at a concentration of about 0.1 ng / mL to 1 mg / mL, or about 1 ng / mL to 1 μg / mL, or about 5 ng / mL to 150 ng / mL, or about 10 ng / mL to 100 ng / mL. In different embodiments, the medium lacks HGF.

[0105] In still further embodiments, the medium is supplemented with gastrin. In particular embodiments, gastrin is present at a concentration of 0.001 μM to 0.2 μM. In different embodiments, the medium lacks gastrin.

[0106] Prior to plating organoids, the media described herein (e.g., but not limited to, Medium A, Medium B, Medium C, and Medium D) may also be combined with 0.1-100% (v / v) Matrigel™, or other extracellular matrix or combinations of extracellular matrix components (i.e., laminin, collagen, proteoglycans, non-proteoglycan polysaccharides, fibronectin, vitronectin, elastin).

[0107] The media of the present disclosure, including but not limited to Medium A, Medium B, Medium C, and Medium D, may be supplemented with some, none, or all of the aforementioned additional supplements at concentrations suitable for a particular application. Additionally, any supplement described herein can be substituted with a small molecule analog of the corresponding protein at a concentration suitable for a particular application, even if a small molecule analog is not specifically described.

[0108] Any of the aforementioned supplements added to the media described herein may be prepared as 2- to 1000-fold concentrated stock solutions and stored at -20°C. Frozen stock solutions may be thawed and added to the disclosed culture media as appropriate. Complete media may be stored at 4°C for approximately 1-3 months.

[0109] method The present disclosure provides the method for obtaining epithelial organoid.Method comprises contacting one or more epithelial ducts, epithelial duct fragments and / or the epithelial stem cells separated therefrom with extracellular matrix and culturing in the presence of medium.In one embodiment, medium is as described above, the basal medium does not contain FGF and / or nicotinamide, or contains undetectable level of FGF and / or nicotinamide, or does not contain exogenously added FGF and / or nicotinamide.

[0110] Epithelial tissue lines the surfaces, cavities, and ducts of animal organs, glands, and blood vessels. Epithelia generally comprise tightly packed cell sheets polarized by opposing apical and basal membranes. One of the functions of epithelia is to protect the interface between two different environments. Epithelial tissues may contain stem cell niches that replenish differentiated somatic cell types during normal tissue maintenance and at a rapid rate during injury response.

[0111] In particular, epithelial ductal tissue structures generally contain stem / progenitor cells known to reside in the stem cell niche of organs and have colony-forming potential (Sato et al., 2009, Nature; Huch et al., 2013 Nature). Therefore, epithelial stem / progenitor cells (also referred to herein as "epithelial stem cells") are excellent feeder cells for generating epithelial organoids under appropriate culture conditions.

[0112] The term "organoid" as used herein refers to a three-dimensional in vitro model of an organ. Organoids can provide the realistic tissue structure of the organ from which they are derived. Therefore, the term "epithelial organoid" refers to organoids obtained from one or more epithelial stem cells / progenitor cells. Epithelial stem cells / progenitor cells can be derived from any organ, including but not limited to the liver, lung, pancreas, or intestine. The organoids obtained from one or more liver epithelial stem cells / progenitor cells are also referred to herein as "liver organoids", the organoids obtained from one or more pancreatic epithelial stem cells / progenitor cells are also referred to herein as "pancreatic organoids", and the organoids obtained from one or more intestinal epithelial stem cells / progenitor cells are also referred to herein as "intestinal organoids".

[0113] In one aspect, epithelial organoid is established from single epithelial stem cell / progenitor cell.Optionally, single epithelial stem cell / progenitor cell can be isolated by fluorescently labeled cell sorting of digested epithelial tissue.However, when epithelial stem cell / progenitor cell is plated as epithelial fragment, the efficiency of establishing organoid can be increased.As used herein, the term " epithelial fragment " refers to a fragment or part of epithelial tissue.Epithelial fragment can be obtained by partial enzymatic or mechanical destruction of epithelial tissue.Without being bound by theory, epithelial stem cell / progenitor cell integrated into epithelial fragment is still likely to be embedded in in vivo stem cell niche, and may benefit from paracrine signaling during the start of organoid culture.

[0114] In one embodiment, the epithelial fragment is an epithelial duct or a fragment thereof. As used herein, the term "epithelial duct" refers to a body passage or tube lined with epithelial cells that transports secretions or other substances. As noted above, epithelial ducts contain stem / progenitor cells (Sato et al., 2009, Nature; Huch et al., 2013 Nature).

[0115] Organs or parts thereof containing epithelium can be isolated from subjects using any method known to those skilled in the art of tissue dissection.For example, organs can be removed from freshly deceased subjects using conventional dissection.Alternatively, parts of organs can be obtained from subjects by biopsy.Regardless of the method used to isolate organs or parts thereof, subsequent processing of organs or parts thereof can be initiated in a timely manner.Subjects can be humans or animals, for example, rodents.

[0116] Isolated organs or their parts, and their derivatives, such as epithelial tissue, epithelial duct, epithelial duct fragment, or epithelial cell, or established organoid itself may be adhesive, and may tend to adhere to the surface or equipment used to process epithelial organs or their parts, such as plasticware and / or pipette or pipette tip.At any stage of the disclosed method, it may be desirable to rinse the surface or equipment that is in contact with isolated epithelium or its derivative or established organoid with detergent.The use of detergent, for example, AggreWell™ Rinsing Solution, helps to prevent the above from adhering to the surface or equipment.After rinsing the surface or equipment with detergent, it may be further desirable to wash the rinsed surface or equipment with basal medium, for example, Advanced DMEM / F-12 or DMEM / F-12.

[0117] The generalized protocol described below is illustrated in Figure 3. Further details of methods for culturing primary tissues or cells thereof, as well as the maintenance, expansion, differentiation, and self-organization of said primary tissues or cells thereof into epithelial 3D structures may be described in relevant sections herein or may be known to those skilled in the art.

[0118] To obtain fragments from epithelial tissue, fresh organs or portions thereof may be cut into small tissue fragments. Any suitable tool, such as a blade or edge, combined with or not combined with a pair of tweezers, may be used to cut fresh organs or portions thereof. The organ or portion of the tissue may be cut into pieces of a size suitable for downstream processing. For example, suitable-sized tissues may be about 1-5 mm. 3 or about 2 to 4 mm 3 or about 3 mm 3 It may correspond to the volume of

[0119] While the organ or portion thereof is being processed into epithelial tissue, both the organ or portion thereof and the epithelial tissue may be immersed in an ice-cold solution. The ice-cold solution may be a buffer solution, such as phosphate-buffered saline, or a standard tissue culture medium, such as Advanced DMEM / F-12 or DMEM / F-12. The ice-cold solution may be any solution that keeps the organ or portion thereof and the epithelial tissue cool and allows for downstream processing.

[0120] The ice-cold solution containing the epithelial tissue may be transferred to a container, where the epithelial fragments may sink to the bottom of the container. Once most or substantially all of the tissue has sunk to the bottom of the container, the ice-cold solution may be removed. The epithelial tissue pellet may be quickly immersed in an appropriate volume of a buffer containing a digestive enzyme. In one embodiment, the buffer containing the digestive enzyme may be an enzyme digestion cocktail. In certain embodiments, the enzyme digestion cocktail may include dispase and / or collagenase. When the enzyme digestion cocktail includes collagenase, the collagenase may be collagenase type XI, collagenase type IV, or any other collagenase capable of digesting epithelial tissue. In a further embodiment, the enzyme digestion cocktail may be supplemented with 1% fetal bovine serum. In another embodiment, the enzyme digestion cocktail may be supplemented with DNase I at a concentration of about 0.1 μg / mL to 100 μg / mL, or about 1 μg / mL to about 50 μg / mL, or about 5 μg / mL to 20 μg / mL. The concentration of the digestive enzyme may vary depending on the type of enzyme used, for example, the concentration may be 0.0001 to 10% (w / v).

[0121] The tube containing the epithelial tissue and the buffer containing the digestive enzyme may be placed under conditions for a sufficient period of time under which the digestive enzyme can function. For example, certain digestive enzymes function optimally at a temperature of approximately 37°C, e.g., 20°C to 40°C. Any environment that can maintain the temperature at which the digestive enzyme functions may be desirable. For example, the tube containing the epithelial tissue and the buffer containing the digestive enzyme may be placed in a heated water bath or a heated oven until the epithelial tissue is sufficiently digested. Digestion may be performed under static conditions or with movement, e.g., by rotating or shaking the tissue. The incubation time may depend on factors such as, but not limited to, the complexity and volume of the epithelial tissue and the volume of the digestive enzyme buffer. For example, approximately 3 mm 3 Epithelial tissue collections may require an initial incubation time of 5-60 minutes, 10-50 minutes, 20-40 minutes, or about 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes.

[0122] After incubating the epithelial tissue for a sufficient time to digest the epithelial tissue, the solution containing the digested epithelial tissue may be vigorously agitated. Any method of vigorously agitating may be used as long as it does not irreparably damage the digested epithelial tissue and render it ineffective for downstream applications. For example, the solution containing the digested epithelial tissue may be pipetted up and down a sufficient number of times. As another example, the solution containing the digested epithelial tissue may be vigorously mixed, for example, by vortexing.

[0123] After sufficient vigorous agitation of the digested epithelial tissue contained in the buffer containing digestive enzymes, the agitated undigested epithelial tissue may sink to the bottom of the container, and the buffer containing digestive enzymes may be removed from the agitated partially digested epithelial tissue, as further described below.

[0124] The removed solution containing the digestive enzyme may further contain suspended, digested epithelial ducts, epithelial duct fragments, and single cells, which may or may not be visible in the digestive enzyme buffer. The aforementioned digestion cycle may need to be repeated multiple times until all or substantially all of the epithelial tissue is digested and destroyed to produce epithelial ducts. Therefore, the removed solution containing suspended particulate matter may be pooled and stored on ice. Alternatively, the remaining epithelial tissue pellet may be subjected to additional digestion cycles by intermittent mechanical disruption of the digested tissue and supernatant collection. While continuing to collect and pool the digestive enzyme supernatant, the digestion cycle may be repeated until no traces of undigested tissue remain.

[0125] Repeated digestion cycles may be necessary depending on factors such as, but not limited to, the complexity and volume of the epithelial tissue, the volume of the digestive enzyme-containing buffer, the concentration and composition of the digestive enzyme, the duration of incubation in the digestive enzyme-containing buffer, or the incubation temperature in the digestive enzyme-containing buffer. In certain embodiments, up to five or more digestion cycles of approximately 10, 20, 30, 40, or 50 minutes may be required to sufficiently digest the epithelial tissue into epithelial ducts and / or epithelial duct fragments. In other embodiments, a single long digestion may be selected instead of several digestion cycles. Figure 3b illustrates that the appearance of the digestive enzyme-containing buffer and the supernatant of the digested material gradually becomes lighter after successive digestion cycles.

[0126] It may be desirable to obtain a solution containing only epithelial ducts and / or epithelial duct fragments in the supernatant, or a supernatant containing substantially only epithelial ducts and / or epithelial duct fragments. The appearance of epithelial ducts and / or epithelial duct fragments is known to those skilled in the art of processing epithelial tissue. For example, some characteristics of epithelial ducts and / or epithelial duct fragments may include a whitish color. This whitish color may not readily fall off due to gravity in aqueous solution and therefore may not be captured and pooled in the spent enzyme supernatant during the digestion cycle described above. Smaller epithelial duct fragments are often invisible to the naked eye.

[0127] To isolate and concentrate epithelial ducts and / or epithelial duct fragments from the digestive enzyme-containing buffer, the solution may be passed through a filter with a sufficient mesh size (Figure 4). The size of intact epithelial ducts may typically be approximately 70 μm or larger. The size of epithelial duct fragments may typically be approximately 37-70 μm. Thus, in certain embodiments, providing a filter with a mesh size of approximately 30 μm-80 μm may be sufficient to remove only single cells and capture all epithelial ducts and / or epithelial duct fragments. In other embodiments, it may be desirable to pass the epithelial ducts and / or epithelial duct fragments in the digestive enzyme-containing buffer through a 40 μm filter. In further embodiments, it may be desirable to preclear larger debris from the digestive enzyme-containing buffer by passing the solution through a filter with a larger mesh size. For example, a filter with a mesh size of approximately 70-80 μm may be used for this purpose. Nevertheless, the mesh size of the filter used in the pre-clearing step must be of an appropriate size to allow all epithelial ducts and / or epithelial duct fragments to pass through while retaining larger debris such as undigested epithelial tissue. In this particular embodiment, the flow-through, free of large debris, may then be passed through a second, even smaller mesh size filter (i.e., about 40 μm) to capture the duct fragments in the filtrate as described above.

[0128] The filtrate or pre-cleared filtrate may be collected from the filter surface. Any method of removing the filtrate or pre-cleared filtrate from the filter surface is contemplated by the present disclosure. In an exemplary method of removing the filtrate or pre-cleared filtrate from the filter surface, the filter surface may be inverted or upside down, and the contents of the filter surface may be collected in a suitable container by placing the inverted or upside down filter surface over the container and rinsing the other side of the filter surface with a suitable solution. The suitable solution may include a buffer solution, such as phosphate-buffered saline, or another solution, such as a basal medium or cell culture medium. The collected filtrate or pre-cleared filtrate should sink to the bottom of the container, and the solution should be removed from the pellet containing the filtrate or pre-cleared filtrate. It may be necessary to subject the container and its contents to centrifugal forces sufficient to pellet the filtrate or pre-cleared filtrate but not otherwise damaging the filtrate or pre-cleared filtrate, rendering it useless for downstream applications. In one embodiment, the filtrate and pre-cleared filtrate may be centrifuged at about 300 x g for about 5 minutes.

[0129] Once the pellet of epithelial tubes and / or epithelial duct fragments has settled to the bottom of the container and been separated from the supernatant, the pellet of epithelial tubes and / or epithelial duct fragments, or portions thereof, may be plated in a culture vessel. The culture vessel may be any culture vessel suitable for culturing cells. For example, the culture vessel may be a Petri dish, a culture flask, or a culture plate in 6-, 12-, 24-, 48-, or 96-well format. The number / density of epithelial tubes and / or epithelial duct fragments plated in the culture vessel may depend on the particular type of culture vessel used. In embodiments where epithelial tubes and / or epithelial duct fragments are plated in a 24-well plate, it may be desirable to divide the epithelial tubes and duct fragments of a given mouse epithelial organ into, for example, 3-10 wells. While the number of epithelial tubes and / or epithelial duct fragments collected per organ may vary, those skilled in the art will understand that in certain embodiments, the epithelial tubes and / or epithelial duct fragments may be plated at a density that prevents the epithelial tubes and / or epithelial fragments from coalescing during subsequent culture. FIG. 5 illustrates exemplary subconfluent densities of plated fragments or single cells.

[0130] It may be desirable to suspend the epithelial ducts and / or epithelial duct fragments in an extracellular matrix prior to plating. As used herein, the term "extracellular matrix" refers to a collection of extracellular molecules that provide structural and biochemical support for surrounding cells. Both natural and synthetic extracellular matrices are contemplated within the present disclosure. In one embodiment, the matrix comprises an extracellular matrix protein. Examples of extracellular matrix proteins include, but are not limited to, laminin, collagen, fibronectin, vitronectin, and entactin. Various matrices containing extracellular matrix proteins, such as Matrigel™, are commercially available.

[0131] In one embodiment, a pellet of epithelial ducts and / or epithelial duct fragments from a mouse liver may be combined with approximately 100 μL of extracellular matrix (e.g., Matrigel™) and 5-100 μL, 10-90 μL, 20-80 μL, 30-70 μL, or 40-60 μL of suspension and plated in the center of a well of a 24-well culture plate. In another embodiment, multiple extracellular matrix domes containing epithelial ducts and epithelial duct fragments (prepared as described above) may be plated in a single well. Those skilled in the art will appreciate that any number of extracellular matrix domes, each consisting of any volume of extracellular matrix, may be plated in a culture vessel, provided that each dome is distinct or substantially distinct.

[0132] In another embodiment, epithelial ducts and / or epithelial fragments may be contacted with a mixture of culture medium and extracellular matrix as disclosed herein in a ratio of about 1:2. However, the final concentration of extracellular matrix (e.g., Matrigel™) may be 10-99.9% (v / v). The resulting suspension may then be plated as described above.

[0133] In yet another embodiment, epithelial tubes and / or epithelial tube fragments may be added to a culture medium disclosed herein containing 0.1-50% (v / v) extracellular matrix, optionally 1-20% (v / v) or 5-10% (v / v) extracellular matrix, such as Matrigel™. In particular, plating a mixture of epithelial tubes and / or epithelial tube fragments and culture medium containing 0.1-50% (v / v) extracellular matrix, optionally 1-20% (v / v) or 5-10% (v / v) extracellular matrix, into an appropriate culture vessel may allow for the culture of organoids using a low concentration of extracellular matrix support (i.e., in a suspension or suspension-like state). The low concentration of extracellular matrix support provides a semi-solid environment for organoids in which the culture medium is combined with the extracellular matrix in the appropriate ratio. In such embodiments, the culture vessel may be a low-attachment culture vessel, such as a low-attachment 6-well plate, 12-well plate, or 24-well plate, or may not be. In one embodiment, culture medium may first be added cold to each cold well, followed by the addition and mixing of thawed extracellular matrix. As a next step, stem cell-containing material isolated from an organ (e.g., epithelial ducts and / or epithelial duct fragments) may be added to the wells. The 6-well plate containing the plated mixture of epithelial ducts and / or epithelial duct fragments and culture medium containing 0.1-50% (v / v) extracellular matrix may be placed on a 1.9 cm diameter orbital shaker at, for example, about 70 rpm and cultured at 37°C. The 12-well plate containing the plated mixture of epithelial ducts and / or epithelial duct fragments and culture medium containing 0.1-50% (v / v) extracellular matrix may be placed on a 1.9 cm diameter orbital shaker at, for example, about 80 rpm. In general, organoids cultured with low concentrations of extracellular matrix support support faster expansion rates and organoid yields in shorter times than organoids cultured in static extracellular matrix domes.

[0134] In yet another embodiment, epithelial duct and / or epithelial duct fragment can be added to the culture medium disclosed herein, which comprises about 0.1% (v / v) or less extracellular matrix, for example, Matrigel (trademark).In particular, when the mixture of epithelial duct and / or epithelial duct fragment and the culture medium which comprises about 0.1% (v / v) or less extracellular matrix is ​​plated in suitable culture vessel, organoid may be able to be suspension cultured.

[0135] In embodiments using ≥10% (v / v) extracellular matrix, once one or more extracellular matrix domes containing epithelial tubes, epithelial tube fragments, and / or epithelial stem / progenitor cells isolated therefrom have polymerized, a sufficient amount of culture medium may be added to the well. The culture medium for culturing the domes containing epithelial tubes, epithelial tube fragments, or epithelial stem / progenitor cells isolated therefrom, or nascent therein, may be any medium described herein, e.g., Medium A, Medium B, Medium C, or Medium D. A sufficient volume of culture medium may be added to the culture vessel to cover the domes and ensure that the epithelial tubes, epithelial tube fragments, and / or epithelial stem / progenitor cells are adequately nourished.

[0136] Seeding efficiency of organoid fragments can typically approach 50%. While low-density organoid fragments can be efficiently expanded in the culture medium of the present disclosure, seeding 200 fragments typically results in 100 fragments being incorporated into a Matrigel™ dome. This fragment density may be suitable for expanding fragments over 5-7 days, for example, within the volume provided by a 30 μL Matrigel™ dome. However, Matrigel™ domes with different volumes may also be suitable for specific applications. As a non-limiting example, Matrigel™ domes with volumes ranging from 10 μL to 1 mL may be used as desired.

[0137] In embodiments where it is desired to form and expand epithelial organoids from single epithelial stem cells and / or progenitor cells, the filtered fraction containing epithelial ducts and / or epithelial duct fragments can be exposed to single cell dissociation enzymes, such as Accutase™, trypsin, Gentle Cell Dissociation Reagent, or TrypLE™. The enzyme can be dissolved in basal medium, and the concentration and duration can be adjusted to the amount of material exposed to the enzyme. DNase I can also be added to the single cell dissociation enzyme. After digestion is complete, enzyme activity can be removed by adding basal medium containing FBS and / or by diluting the enzyme. The single cell digestion suspension can also be passed through a single cell strainer to remove all cells that are not completely dissociated into single units. In one embodiment, epithelial fragments may be collected from a mouse organ, captured as filtrate on a 40 μm filter, pelleted, and digested with about 1-10 mL of TrypLE™ and about 0.1 μg / mL-100 μg / mL, or about 1 μg / mL-50 μg / mL, or about 5 μg / mL-20 μg / mL of DNase I at 37°C for about 3-45 minutes. The enzymatic digestion reaction may be stopped by adding DMEM / F-12 supplemented with 10% FBS. The cell suspension may then be filtered through a 40 μm filter, pelleted, and plated as a cell-Matrigel™ mixture.

[0138] Single epithelial stem / progenitor cells prepared in this manner may be plated in domes containing >10% (v / v) extracellular matrix, suspended in low concentrations of 0.1-50% (v / v) extracellular matrix, or suspended in approximately 0.1% (v / v) or less extracellular matrix, as previously described. For example, 5,000-50,000 cells may be seeded in domes composed of 100% (v / v) extracellular matrix or in suspension wells containing 10% (v / v) extracellular matrix.

[0139] Organoid expansion from epithelial ducts and / or epithelial duct fragments or single epithelial stem / progenitor cells may be monitored daily to track their growth. Generally, organoids are subcultured within a week to prevent them from forming dense, black cell clusters and before the luminal lumen darkens and collapses (Figure 6). During early passages, the organoid luminal lumen may darken and collapse around days 4–6. During later passages, the organoid luminal lumen may darken and collapse around days 6–7. Established organoids may be mechanically subcultured into organoid fragments or enzymatically subcultured into organoid fragments or single cells. Higher organoid yields may be achieved if established organoids are mechanically subcultured and seeded as organoid fragments.

[0140] To mechanically divide the dome cultures, the integrity of the extracellular matrix domes may be verified using an optical microscope. If the extracellular matrix domes containing epithelial organoids are substantially intact, the medium may be removed from the culture vessel, for example, by aspiration. Using a p1000 pipette, approximately 1000 μL of cold culture medium, such as basal culture medium (i.e., Advanced DMEM / F-12), may be forcibly directed toward the approximate center of each extracellular matrix dome. To disrupt and fragment the extracellular matrix domes and organoids but avoid completely destroying the organoids to single cells, a volume of cold culture medium may be vigorously pipetted up and down in the culture vessel approximately 5, 10, 15, 20, or 30 times. A portion of the resulting suspension may be transferred to an empty culture vessel for counting.

[0141] In an exemplary embodiment, three 10 μL volumes of cell suspension may be plated into empty wells of a 6-well plate as individual volume domes. The number of organoid aggregates in each 10 μL volume dome may be counted using an optical microscope, and the number of organoid aggregates in the cell suspension may be determined. Once the density of the organoid aggregates in the cell suspension has been determined, a volume containing approximately 200 aggregates may be added to a tube containing 1 mL of the culture medium disclosed herein, e.g., basal medium, and centrifuged at 300×g for 5 minutes. The supernatant may be carefully aspirated without disrupting the pellet, and the pellet may be resuspended in at least 10 μL to approximately 100 μL of extracellular matrix. The suspension of extracellular matrix containing the organoid aggregates may be plated into one well of a pre-warmed 24-well plate and allowed to polymerize. When the extracellular matrix dome containing organoid aggregates polymerizes, about 750 μ L of pre-warmed culture medium as disclosed herein can be added to each dome (i.e., well).In order to avoid destroying the extracellular matrix dome, it may be desirable to add pre-warmed culture medium along the side of well.For well-established organoid culture, organoid can be subcultured at an appropriate and constant split ratio, for example, 1:30, so that the determination of the organoid fragment density in well can be omitted.

[0142] For enzymatic dissection, the integrity of the extracellular matrix domes may be verified using an optical microscope. If the extracellular matrix domes containing epithelial organoids are substantially intact, the medium may be removed from the culture vessel, for example, by aspiration. Using a p1000 pipette, approximately 500 μL of cold culture medium, such as basal culture medium (i.e., Advanced DMEM / F-12), may be forcibly directed toward the approximate center of each extracellular matrix dome and left for approximately 1 minute. Using a p1000 pipette tip, for example, by pipetting up and down and / or compressing, the extracellular matrix domes may be drawn into the solution, and the volume may be transferred to a fresh tube. Wells with disrupted extracellular matrix domes may be rinsed with an additional volume of culture medium and pooled with the contents of the fresh tube.

[0143] The contents of the fresh tube may be vigorously pipetted up and down approximately 5, 10, 15, or 20 times. The vigorously pipetted solution may be centrifuged at approximately 300 x g for 5 minutes, and the supernatant may be carefully removed and discarded. The pellet may then be contacted with an appropriate enzyme solution to dissociate the organoids or portions thereof into substantially single cells, and the digestive enzyme may be allowed to function for a sufficient period of time. For example, certain digestive enzymes work optimally at temperatures of approximately 37°C, e.g., 20°C to 40°C. Any environment capable of maintaining the temperature at which the digestive enzyme functions may be desirable. Digestion may also be performed under static conditions, or with movement, e.g., by rotating or shaking the tissue. For example, the tube may be placed in a heated water bath or heated oven until the organoids or portions thereof are sufficiently dissociated. In one embodiment, incubating the organoids or portions thereof in Single Cell Dissociation Buffer in a 37°C water bath for about 10 or 20 minutes may be sufficient to dissociate the organoids or portions thereof into single cells.

[0144] Once the organoid or a portion thereof has been sufficiently exposed to the enzyme solution, this solution may be combined with about 3 mL of culture medium, for example, the basal medium described herein, and the number of cells therein may be determined using a hemocytometer. The desired number of viable cells may be transferred to a tube containing culture medium, for example, the basal medium described herein, and centrifuged at about 300 x g for 5 minutes. The pellet containing single cells may be resuspended in any appropriate volume of extracellular matrix, and plated as a single or multiple domes into one well of a pre-warmed 24-well plate and allowed to polymerize. Once the extracellular matrix domes containing dissociated single cells have polymerized, about 750 μL of pre-warmed culture medium as disclosed herein may be added to each dome (i.e., well). In order to avoid destroying the Matrigel™ domes, it may be desirable to add the pre-warmed culture medium along the side of the well.

[0145] To mechanically split the suspension culture, a cell scraper may be used to release the organoids and Matrigel™ and transfer them to a new container. In one embodiment, the container may be a 15 mL Falcon tube. Using a p1000 pipette, approximately 1000 μL of cold culture medium, such as basal culture medium (i.e., Advanced DMEM / F-12), may be forced to the bottom of the tube containing the submerged organoids. To break down the extracellular matrix domes and organoids into fragments but avoid completely breaking down the organoids to single cells, a volume of cold culture medium may be vigorously pipetted up and down in the culture vessel approximately 5, 10, 15, 20, or 30 times. A portion of the resulting suspension may be transferred to an empty culture vessel for counting. The desired number of organoid fragments may then be transferred to a new culture plate already containing cold culture medium and extracellular matrix. The culture medium may then be returned to an orbital shaker at 37 °C.

[0146] In order to avoid organoid lumen collapse, subcultured organoids plated in the culture medium of the present disclosure according to the method described herein may be replaced about every 2-7 days, or as needed.However, if organoids are embedded in extracellular matrix, it may be possible to restore the lumen of collapsed organoids to a bulging state.In particular, even collapsed organoids that have been cultured for more than one month without subculture or medium replacement may be restored using the culture medium disclosed.

[0147] In certain embodiments, it may be desirable to use different media at different stages of culture. For example, it may be desirable to initiate culture in one type of medium, e.g., one of medium A, medium B, medium C, or medium D. After a certain number of passages, or after passage for a certain period of time, it may be desirable to use a different type of medium, e.g., medium A, medium B, medium C, or medium D. In other embodiments, it may be desirable to alternate between one type of medium, e.g., medium A, medium B, medium C, or medium D, and a different type of medium, e.g., medium A, medium B, medium C, or medium D, during different stages of culture.

[0148] Particularly with regard to liver organoids, the initiation of liver organoids may depend on the expression of Lgr5+ cells and / or other positive markers corresponding to stem / progenitor cells.However, epithelial stem cell marker Lgr5 may not generally be active under homeostatic conditions.More precisely, when tissue is subjected to obstruction injury, mechanical injury, and / or toxic injury, epithelial stem cell marker Lgr5 may typically be activated.Therefore, epithelial organoids are often obtained from damaged liver tissue.In one embodiment of the present disclosure, epithelial ducts, epithelial duct fragments, and / or epithelial stem cells isolated therefrom are obtained from intact tissue.As used herein, the term "intact tissue" refers to tissue that has never been subjected to obstruction injury, mechanical injury, and / or toxic injury.

[0149] In one embodiment of the present disclosure, the epithelial organoids are derived from one or more freshly isolated epithelial ducts, epithelial duct fragments, and / or epithelial stem / progenitor cells or epithelial fragments, including epithelial stem / progenitor cells derived from mouse tissue obtained from previously uninjured male or female mice that are about 1 year old or older.

[0150] In one embodiment, the epithelial organoid obtained by the method described herein is further subjected to maturation condition and / or differentiation condition.As used herein, the term " maturation condition and / or differentiation condition " refers to the culture condition that promotes the maturation and differentiation of epithelial organoid into mature cell type.A variety of maturation condition and differentiation condition are known in the art, and can be specifically selected according to the mature cell type of interest.In one embodiment, the liver and / or pancreas organoid obtained by the method described herein is further subjected to maturation condition and / or differentiation condition.

[0151] Organoids and Uses Thereof The present disclosure provides the epithelial organoid obtained by the method described herein.In one aspect, organoid is liver organoid, pancreatic organoid or intestinal organoid.

[0152] The organoid obtained by the method described herein is optionally human organoid.Non-human animal organoid is also contemplated herein.

[0153] The epithelial organoid formed by using the method described herein can express one or more gene or protein markers that characterize the epithelial tissue from which organoid originates.For example, liver organoid can express one or more of the following genes: Lgr5, Axin2, Hnf4a, Epcam, ZO1, Krt19 and Sox9.As another example, pancreatic organoid can express one or more of the following genes: Lgr5, Sox9, Pdx1, Krt7, Muc1 and Car2.As another example, intestinal organoid can express one or more of the following genes: Lgr5, Lyz, Vil1, ChgA and Muc2.

[0154] The self-renewal of liver organoids formed using the methods and media described herein may be driven primarily by Axin2 rather than Lgr5, thus providing a more physiologically relevant culture system for liver homeostasis (Wang et al., 2015 Nature).

[0155] The present disclosure also provides pharmaceutical compositions comprising the organoids described herein and a pharmaceutically acceptable carrier.

[0156] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, isotonic agents, absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard textbook in this field, which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.

[0157] A pharmaceutical composition is formulated to be compatible with its intended route of administration, which includes parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.

[0158] Various uses of epithelial organoids are known in the art.

[0159] In particular, the organoids and / or pharmaceutical compositions of the present disclosure are useful for treating a disorder, condition, or disease in a subject or in regenerative medicine.

[0160] In one aspect, the organoids of the present disclosure are used in a method for treating a disorder, condition, or disease or a method for regenerative medicine, comprising administering said organoids to a subject in need thereof.

[0161] In another aspect, provided is the method for treating the liver damage, condition or disease of object or the method for regenerative medicine, comprising administering to object the liver organoid obtained by the method described herein.In one aspect, the liver damage, condition or disease is Alagille syndrome, Wilson's disease, type 1 glycogen storage disease, alpha 1 antitrypsin deficiency, fibrosis, cirrhosis, neonatal / viral (A, B, C) / autoimmune / toxic hepatitis, fatty liver disease, tyrosinemia, sarcoidosis, lysosomal acid lipase deficiency, liver tumor, cystic liver disease, biliary atresia, galactosemia, primary biliary cholangitis, porphyria, Reye's syndrome, hemochromatosis, Gilbert's syndrome, fatty liver and gallstone.

[0162] In a further aspect, there is provided a method for treating a pancreatic disorder, condition, or disease in a subject, or a method for regenerative medicine, comprising administering to the subject a pancreatic organoid obtained by the method described herein. In one aspect, the pancreatic disorder, condition, or disease is acute / chronic / hereditary pancreatitis, pancreatic tumor, diabetes, cystic fibrosis, exocrine pancreatic insufficiency, congenital malformation (pancreatic ductal dysplasia, annular pancreas), Zollinger-Ellison syndrome, and pancreatic cyst / pseudocyst.

[0163] In another aspect, provided is the method for treating the intestinal disorder, condition or disease of object or the method for regenerative medicine, comprising administering to object the intestinal organoid obtained by the method described herein.In one aspect, the intestinal disorder, condition or disease is cystic fibrosis, ulcerative colitis, Crohn's disease, colorectal cancer, colon polyp, irritable bowel syndrome, celiac disease, fecal incontinence, lactose intolerance, diverticulosis / diverticulitis, acid reflux, diarrhea, gastrointestinal ulcer, short bowel syndrome, curling ulcer, blind loop syndrome, Milroy's disease, Whipple's disease, carcinoid syndrome, Hirschsprung's disease and anal cancer.

[0164] In another aspect, the effective amount of organoid disclosed herein is used in preparing the pharmaceutical preparation for treating disorder, condition or disease or for regenerative medicine.The effective amount of organoid disclosed herein generally relates to the amount required to achieve therapeutic purpose.

[0165] As used herein, the term "subject" includes all members of the animal kingdom. In one aspect, the subject is a mammal. In a further aspect, the subject is a human.

[0166] As used herein, "treating a disorder, condition, or disease" includes, but is not limited to, reversing, alleviating, or inhibiting the progression of the disorder, condition, or disease, or the symptoms or conditions associated with the disorder, condition, or disease.

[0167] The organoids described herein may also be useful in methods for conducting drug discovery screens; assaying toxicity; investigating embryology, cell lineage, and differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in injury and repair; investigating inflammatory and infectious diseases; and studying the mechanisms of cell transformation and the causes of cancer. [Example]

[0168] The following non-limiting examples illustrate the present disclosure.

[0169] Example 1 - Digestion of organs or parts thereof Whole organs containing epithelium from male and female mice (i.e., C57BL / 6) were dissected into approximately 1–3 mm pieces. 3 The tissue was cut into pieces of approximately 1-3 mm 3 The epithelial tissue fragments were immersed in approximately 25 mL of ice-cold DMEM / F-12. The same volume of ice-cold DMEM / F-12 containing the epithelial fragments was transferred to a 50 mL Falcon tube using a 25 mL serological pipette. The epithelial tissue fragments were allowed to settle by gravity for approximately 2 minutes, and the supernatant was discarded. The pellet of epithelial tissue fragments was then contacted with 5 mL of Enzyme Digestion Cocktail containing dispase (0.0125% (w / v)) and collagenase (0.0125% (w / v)). The epithelial tissue fragments were incubated in the Enzyme Digestion Cocktail in a 37°C water bath for 20 minutes. After the 20-minute incubation, the solution was vigorously pipetted and vortexed approximately seven times using a 10 mL serological pipette, and the digested epithelial tissue fragments were allowed to settle by gravity for approximately 1 minute. The initial supernatant was discarded.

[0170] To obtain epithelial ducts and / or epithelial duct fragments from the epithelial tissue fragments, the digestion cycle was repeated as described above (Figure 7a). Early after the second digestion cycle, epithelial ducts and / or epithelial duct fragments may be present in the supernatant (Figure 7b). Once epithelial ducts and / or epithelial duct fragments were present in the supernatant, the supernatant was removed and placed in an appropriate tube. The supernatants from the subsequent digestion cycles of the remaining epithelial tissue fragments were pooled.

[0171] After five to seven digestion cycles of 20 minutes each, substantially all of the epithelial tissue fragments were digested into epithelial ducts and / or epithelial duct fragments.

[0172] Example 2 - Products of digestion of organs or parts thereof To isolate epithelial ducts and / or epithelial duct fragments from single cells, e.g., highly differentiated cells and debris, the pooled supernatants were filtered (as shown in Figure 4). The pooled supernatants were passed through a 40 μm filter, and the flow-through was discarded. The filtrate on the filter surface was collected by carefully inverting the filter surface and placing it on top of a 50 mL Falcon tube. Approximately 10 mL of ice-cold Advanced DMEM / F-12 was passed over the underside of the filter surface, thereby washing the filtrate off the filter surface and collecting in the tube. Washing was repeated until all or substantially all of the filtrate had been washed off the filter. Scraping the filter may aid collection. The tubes were centrifuged at 300 x g for 5 minutes to pellet the epithelial ducts and / or epithelial duct fragments, and the supernatant was discarded. Optionally, 10 mL of the filtrate suspension containing the epithelial ducts and / or epithelial duct fragments was aliquoted into 3–10 tubes and then centrifuged.

[0173] Example 3 - Preclearing of products after digestion of organs or parts thereof To isolate epithelial ducts and / or epithelial duct fragments from single cells, e.g., highly differentiated cells and debris, the pooled supernatants that were set aside (as shown in Figure 4) were subjected to filtration. First, the pooled supernatants were passed through a 70 μm filter, and the flow-through was retained. The filtrate, containing debris, e.g., undigested tissue and ducts, was discarded (or, alternatively, it could be subjected to further digestion and / or extracellular matrix embedding, as outlined in Examples 4 or 5). A portion of the pooled supernatants that had passed through the 70 μm filter was then passed through a 40 μm filter as described in Example 2. The filtrate on the filter surface was collected by carefully inverting the filter surface and placing it on top of a 50 mL Falcon tube. Approximately 10 mL of ice-cold Advanced DMEM / F-12 was passed over the underside of the filter surface, thereby washing the filtrate off the filter surface and collecting in the tube. Washing was repeated until all, or substantially all, of the filtrate had been washed from the filter. Scraping the epithelial ducts from the filter may aid collection. The tubes were centrifuged at 300 x g for 5 minutes to pellet the epithelial ducts and / or epithelial duct fragments, and the supernatant was discarded. Optionally, 10 mL of the filtrate suspension containing the epithelial ducts and / or epithelial duct fragments was equally divided into 3-10 tubes and then centrifuged.

[0174] Example 4 - Plating epithelial tubes and / or epithelial tube fragments into Matrigel™ domes The pelleted epithelial tubes and / or epithelial tube fragments were resuspended in an appropriate volume of thawed Matrigel™. If the epithelial tubes and / or epithelial tube fragments from a mouse organ were not equally divided into 3-10 tubes, they were resuspended in approximately 30 μL to 300 μL of Matrigel™. Otherwise, the epithelial tubes and / or epithelial duct fragments aliquoted into each of the 3-10 tubes were resuspended in approximately 10 μL to 30 μL of Matrigel™.

[0175] It was also possible to dilute the mixture of Matrigel™ and epithelial material to contain up to 90% (v / v) cell culture medium without compromising the stability of the subsequently plated Matrigel™ domes.

[0176] A suspension of epithelial ducts and / or duct fragments was deposited as 25-60 μL droplets on the bottom of a pre-warmed 24-well plate and allowed to polymerize into a dome at 37°C for 10 minutes. The eight central wells of the 24-well plate were the least tilted and therefore most suitable for depositing the Matrigel™ droplets.

[0177] While dispensing the suspension of epithelial tubes and / or epithelial tube fragments, the pipette tip was moved slightly upward to prevent the epithelial tubes and / or epithelial tube fragments from adhering to the outer surface of the pipette tip and to distribute the epithelial tubes and / or epithelial tube fragments evenly within the dome.

[0178] Example 5 -Plating epithelial tubes and / or epithelial tube fragments suspended in a low concentration of extracellular matrix support Epithelial tubes and / or epithelial tube fragments were grown in suspension with a low concentration of support from extracellular matrix. Epithelial tubes and / or epithelial tube fragments obtained as outlined in Example 2 or Example 3 were mixed with culture medium. This mixture was cooled to 4°C when combined with a low concentration of extracellular matrix (e.g., 0.1% to 50% (v / v) Matrigel™). Using low-adhesion plates (e.g., 12-well plates), epithelial material suspended in cell culture medium (and low Matrigel™ concentration) was added to each well and maintained at 37°C with rotation at 70-80 rpm.

[0179] Example 6 -Cultivation of epithelial ducts and / or epithelial fragments in culture medium Without disrupting the polymerized Matrigel™ domes, approximately 750 μL of culture medium was added along the sidewall of the well containing one or more Matrigel™ domes. Wells without Matrigel™ domes received 750 μL of sterile liquid, such as PBS, culture medium, etc.

[0180] Images of each Matrigel™ dome were taken on day 0 and periodically thereafter (Figure 1). The medium was changed every 2-7 days for up to 1 week by carefully removing the medium from each well. A 750 μL volume of pre-warmed (i.e., 20-37°C) culture medium was added to each well.

[0181] Example 7 -Culture of epithelial ducts or epithelial fragments in medium A The method described in Example 6 was used to form and expand liver organoids using DMEM / F-12 and medium containing approximately 15 mM HEPES, 1.2 g / L sodium bicarbonate, 0.5 mM L-alanyl-L-glutamine, 61.5 μg / mL Rh insulin, 0.012 μg / mL progesterone, 64 μg / mL putrescine, 0.0104 μg / mL sodium selenite, 80 μg / mL human apotransferrin, 0.02 μg / mL corticosterone, 15 μg / mL D-(+)-galactose, and 2600 μg / mL BSA.

[0182] Such media supported the formation and expansion of liver organoids for approximately five passages.

[0183] Example 8 -Culture of epithelial ducts or epithelial fragments in medium B The method described in Example 6 was used to form and expand epithelial organoids using culture medium A of Example 7 supplemented with 0.016 μg / mL R-spondin-1 and / or 3.75 μM CHIR99021.

[0184] In the presence of R-spondin-1 and CHIR99021, the formation and expansion of epithelial organoids was supported for approximately 20 or more passages. In the presence of either R-spondin-1 or CHIR99021 alone, the formation and expansion of epithelial organoids was supported for approximately 10 or more passages.

[0185] Example 9 -Culture of epithelial ducts or epithelial fragments in medium C The method described in Example 6 was used to form and expand epithelial organoids using the culture medium of Example 8 supplemented with 0.05 μg / mL EGF.

[0186] Such media supported the formation and expansion of epithelial organoids for approximately 40 or more passages.

[0187] Example 10 -Culture of epithelial ducts or epithelial fragments in medium D The method described in Example 6 was used to form and expand epithelial organoids using culture medium C of Example 9 supplemented with 0.016 μg / mL Noggin and 0.1 μM LDN193189.

[0188] Such media supported the formation and expansion of epithelial organoids for approximately 50 or more passages.

[0189] Example 11 -Hepatic organoid formation and expression of its markers Liver organoid formation and expansion occurred when livers were treated according to Examples 1 and 2, optionally according to Example 3, plated according to Examples 4 or 5, and cultured in the presence of media described in Examples 7 to 10. In one example, livers were treated according to Examples 1, 4, and 7.

[0190] Regardless of plating hepatic ducts, hepatic duct fragments, single epithelial stem and / or progenitor cells, or mechanically disrupted or enzymatically digested liver organoids, liver organoid formation and expansion occurred (Figure 8). Once formed, liver organoids were maintained and expanded long-term over multiple passages (Figure 9).

[0191] Formed and expanded liver organoids show hepatobiliary gene expression, as shown in Figure 10.Furthermore, according to the method described above, liver organoids are formed and expanded.The medium disclosed herein synthesizes liver and duct proteins (Figure 11).

[0192] Example 12 -Pancreatic organoid formation and marker expression Pancreatic organoid formation and expansion occurred when pancreases were treated according to Examples 1 and 2, optionally according to Example 3, plated according to Examples 4 or 5, and cultured in the presence of media described in Examples 7-10. In one example, pancreases were treated according to Examples 1, 4, and 7.

[0193] Regardless of plating pancreatic ducts, pancreatic duct fragments, single epithelial stem and / or progenitor cells, or mechanically disrupted or enzymatically digested pancreatic organoids, pancreatic organoid formation and expansion occurred (Figure 12). Once formed, pancreatic organoids were maintained and expanded long-term over multiple passages (Figure 13).

[0194] The formed and expanded pancreatic organoids show the expression of pancreatic duct genes, as shown in Figure 14. Furthermore, the formed and expanded pancreatic organoids according to the methods and media disclosed herein synthesize pancreatic duct proteins (Figure 15).

[0195] Example 13 -Formation of small intestinal and colonic organoids and their marker expression When small intestine or colon is treated according to Example 1 and 2, optionally according to Example 3, plated according to Example 4 or 5, and cultured in the presence of the medium described in Example 7 or 8, small intestinal organoid and colonic organoid form and expand (Figure 16).In one example, intestine is treated according to Example 1, 4 and 7.

[0196] Intestinal organoid formation and expansion occurred regardless of plating intestinal crypts, intestinal crypt fragments, single epithelial stem and / or progenitor cells, or mechanically disrupted or enzymatically digested intestinal organoids (Figure 17).

[0197] The formed and expanded intestinal and colonic organoids express intestinal and colonic markers as shown in Figures 18-20.

[0198] Example 14 -Subculture of organoids using mechanical disruption The expansion of organoids plated according to Examples 4 or 5 was monitored daily using a light microscope to follow their growth, in particular to ensure that the lumen of the organoids was not darkened or collapsed.

[0199] When cells were cultured within Matrigel™ domes, the culture medium covering the Matrigel™ domes was aspirated. Approximately 1000 μL of Advanced DMEM / F-12 was forced into the center of each Matrigel™ dome. The volume of Advanced DMEM / F-12 was vigorously pipetted up and down 15 times.

[0200] When cultured in suspension (i.e., without extracellular matrix or with a low concentration of extracellular matrix support), the organoid suspension was vigorously pipetted up and down 15 times.

[0201] For counting, triplicate 10 μL volumes of the cell suspension were plated into empty wells of a 6-well plate as individual volume domes. A volume of the cell suspension containing approximately 200 clumps was transferred to a tube containing 1 mL of Advanced DMEM / F-12 and centrifuged at 300 x g for 5 minutes. The supernatant was carefully aspirated without disrupting the pellet. The pellet was resuspended and plated according to Examples 4 or 5. Culture medium was added to each well containing a Matrigel™ dome as described in Example 6, specifically using culture medium formulated as described in any one of Examples 7-10.

[0202] Example 15 -Subculture of organoids using enzymatic digestion The expansion of organoids plated according to Examples 4 or 5 was monitored daily using a light microscope to follow their growth, in particular to ensure that the lumen of the organoids was not darkened or collapsed.

[0203] When cells were cultured within Matrigel™ domes, the culture medium covering the Matrigel™ domes was aspirated. Approximately 500 μL of cold Advanced DMEM / F-12 was forcefully added to the center of each Matrigel™ dome and allowed to sit for approximately 1 minute. The Matrigel™ domes were drawn into the solution by pipetting up and down with a p1000 pipette tip and compressing, and the volume was transferred to a fresh tube. The wells were rinsed with 500 μL of Advanced DMEM / F-12, and this volume was pooled with the contents of the fresh tube.

[0204] If cultured in suspension (i.e., without extracellular matrix or with a low concentration of extracellular matrix), the organoid suspension was transferred to a fresh tube, the well was rinsed with 500 μL of Advanced DMEM / F-12, and this volume was pooled with the contents of the fresh tube.

[0205] The contents of the fresh tube were vigorously pipetted 15 times, centrifuged at 300 x g for 5 minutes, and the supernatant was carefully removed and discarded. The pellet was contacted with 3 mL of single-cell dissociation buffer and incubated in a 37°C water bath for 10-20 minutes. After the incubation period, 3 mL of Advanced DMEM / F-12 was added to the tube, and the number of cells in the suspension was determined using a hemocytometer. A volume containing approximately 1,000-8,000 viable cells was transferred to a 15 mL Falcon tube and centrifuged at 300 x g for 5 minutes. The pellet was resuspended and plated according to Examples 4 or 5. Culture medium was added to each well containing a Matrigel™ dome as described in Example 6, specifically using culture medium with a formulation as described in any one of Examples 7-10.

[0206] Example 16 -Cryopreservation and recovery of epithelial organoids Epithelial organoids were cryopreserved in liquid nitrogen for at least one year. Organoids were collected as outlined in Examples 14 or 15 and resuspended in cryopreservation medium containing up to 10% DMSO. The non-DMSO fraction of the cryopreservation medium included one of the media described in Examples 7-10, commercially available basal medium, or PBS. Vials of cryopreservation medium containing epithelial organoids or organoid fragments were first frozen to -80°C using a controlled freezing method and then transferred to liquid nitrogen for long-term storage. To thaw, the vials were removed from liquid nitrogen and placed in a 37°C water bath. The contents were added dropwise to the medium described in Examples 7-10 and centrifuged at 300 x g for 5 minutes. Adding BSA to the collection medium increased the number of organoids or organoid fragments recovered. The supernatant was removed, and the pelleted epithelial material was resuspended and plated as described in Examples 4 or 5.

[0207] Table 1. Selected components of FGF- and / or nicotinamide-free culture medium for forming and expanding epithelial organoids. TIFF0007752651000001.tif74145 "x" indicates that the corresponding factor exists.

Claims

1. A method for obtaining epithelial organoids, comprising the steps of: Culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells isolated therefrom in the presence of a medium comprising: (a) a basal medium comprising a carbon source, water, salts, an amino acid source, and a nitrogen source, and a plurality of buffers; and (b) at least one of B27, N2, and / or insulin, progesterone, putrescine, sodium selenite, apotransferrin, corticosterone, galactose, and BSA; the medium does not contain FGF and nicotinamide; and The medium is (i) Supporting organoids for more than five passages without CHIR99021, R-spondin1, EGF, Noggin, and LDN193189; (ii) without EGF, noggin, and LDN193189, and with the addition of CHIR99021 and / or R-spondin1, supporting organoids for 10 or more passages; or (iii) noggin and LDN193189 are not included, and (i) EGF and (ii) CHIR99021 and / or R-spondin1 are added, supporting organoids for more than 30 passages; Process.

2. The method of claim 1 , wherein the culture medium contains N-acetylcysteine.

3. The method of any one of claims 1 to 2, wherein the epithelial stem cells or epithelial progenitor cells are human cells, mouse cells, or rat cells.

4. The method of any one of claims 1 to 3, wherein the epithelial stem cells or epithelial progenitor cells are liver epithelial stem cells, pancreatic epithelial cells, or intestinal epithelial cells.

5. The method of any one of claims 1 to 4, wherein the culture medium is effective for long-term culture of the epithelial organoid, and the long-term culture is about 50 or more passages.

6. The method of any one of claims 1 to 5, further comprising culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem or progenitor cells in contact with an extracellular matrix.

7. The method of any one of claims 1 to 6, further comprising culturing one or more epithelial ducts, epithelial duct fragments, and / or epithelial stem cells or epithelial progenitor cells in contact with a low concentration of support derived from an extracellular matrix.

8. 8. The method of claim 7, wherein the extracellular matrix concentration is 0.1 to 50% (v / v).

9. The method of any one of claims 1 to 5, further comprising culturing epithelial stem cells or epithelial progenitor cells derived from epithelial ducts and / or epithelial duct fragments in suspension.

10. 10. The method of claim 9, wherein the suspension comprises an extracellular matrix having a concentration of about 0.1% (v / v) or less.

11. The method of any one of claims 6 to 8 and 10, wherein the extracellular matrix comprises Matrigel™.

12. 12. The method of any one of claims 1 to 11, wherein the epithelial duct, epithelial duct fragment, and / or epithelial stem or progenitor cells isolated therefrom are obtained from intact tissue.

13. The method of any one of claims 1 to 12, further comprising subjecting said epithelial organoid to maturation and / or differentiation conditions.

14. The organoid obtained according to the method of claim 4, wherein said organoid is liver organoid, pancreatic organoid or intestinal organoid.

15. A method for conducting drug discovery screening; assaying toxicity; investigating embryology, cell lineage, and differentiation pathways; studying gene expression, including recombinant gene expression; investigating mechanisms involved in damage and repair; investigating inflammatory and infectious diseases; and studying the pathogenic mechanisms of cell transformation and the causes of cancer, comprising obtaining an epithelial organoid described in any one of claims 1 to 13.

16. The liver organoid of claim 14, for use in treating liver disorders, conditions or diseases in a subject in need thereof or in regenerative medicine.

17. The pancreatic organoid of claim 14, for use in treating pancreatic disorders, conditions or diseases in a subject in need thereof or in regenerative medicine.

18. 15. The intestinal organoid of claim 14 for use in treating an intestinal disorder, condition, or disease in a subject in need thereof or in regenerative medicine.

19. A medium for obtaining epithelial organoids, comprising: The medium is (a) a basal medium containing a carbon source, water, salts, an amino acid source, and a nitrogen source, as well as a plurality of buffers; (b) B27, N2, and at least one of their minor components, including insulin, progesterone, putrescine, sodium selenite, apotransferrin, corticosterone, galactose, and BSA; Including, the medium does not contain FGF and nicotinamide; and The medium is (i) Supporting organoids for more than five passages without CHIR99021, R-spondin1, EGF, Noggin, and LDN193189; (ii) without EGF, noggin, and LDN193189, and with the addition of CHIR99021 and / or R-spondin1, supporting organoids for 10 or more passages; or (iii) noggin and LDN193189 are not included, and (i) EGF and (ii) CHIR99021 and / or R-spondin1 are added, supporting organoids for more than 30 passages; The medium.

20. 20. The medium of claim 19, further comprising N-acetylcysteine.

21. The medium of any one of claims 19 to 20, further comprising an extracellular matrix.

22. 22. The medium of claim 21, wherein the concentration of the extracellular matrix is ​​0.1 to 50% (v / v).

23. 22. The medium of claim 21, wherein the concentration of extracellular matrix is ​​about 0.1% (v / v) or less.

24. 24. The medium of any one of claims 21 to 23, wherein the extracellular matrix comprises Matrigel™.

25. (i) the concentration of insulin is in the range of 0.01 to 100 μg / mL; (ii) the concentration of progesterone ranges from 2 nM to 400 μM; (iii) the concentration of putrescine is in the range of 0.1 to 10 mM; (iv) the concentration of sodium selenite is in the range of 1 ng / mL to 100 μg / mL; (v) the concentration of apotransferrin is in the range of 0.2 to 200 μg / mL; (vi) the concentration of corticosterone is in the range of 1 to 500 ng / mL; (vii) the concentration of galactose is in the range of 30 μM to 30 mM; and (viii) the concentration of BSA is in the range of 0.5 μg / mL to 5 mg / mL; 14. The method of any one of claims 1 to 13.

26. (i) the concentration of insulin is in the range of 0.01 to 100 μg / mL; (ii) the concentration of progesterone ranges from 2 nM to 400 μM; (iii) the concentration of putrescine is in the range of 0.1 to 10 mM; (iv) the concentration of sodium selenite is in the range of 1 ng / mL to 100 μg / mL; (v) the concentration of apotransferrin is in the range of 0.2 to 200 μg / mL; (vi) the concentration of corticosterone is in the range of 1 to 500 ng / mL; (vii) the concentration of galactose is in the range of 30 μM to 30 mM; and (viii) the concentration of BSA is in the range of 0.5 μg / mL to 5 mg / mL; The medium according to any one of claims 19 to 24.

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