Intestinal epithelial-like cell monolayer and uses thereof

Abstract

The disclosure provides methods for producing epithelial-like monolayers and uses of the monolayers produced according to the provided methods that include, for example, their use as a model system for evaluating the bioavailability and pharmacokinetics of orally administered drugs in humans. The disclosure also provides the epithelial-like monolayers produced by the provided methods as well as kits for preparing the monolayers.

Description

INTESTINAL EPITHELIAL-LIKE CELL MONOLAYER AND USES THEREOF

[0001] The disclosure relates to methods for producing epithelial-like monolayers and uses of the monolayers produced according to the claimed methods that include, for example, there use as a model system for evaluating the bioavailability and pharmacokinetics of orally administered drugs in humans.

[0002] The prediction of bioavailability in humans is essential in the development of drugs that can be orally administered. The small intestine, the site of drug absorption, is an important organ that can limit oral bioavailability. The small intestine possesses a selective barrier function that prevents undesired substances and microbes from entering the human body. The intestinal epithelium not only forms a physical barrier by tight cell-cell adhesion and a mucus layer comprising cells such as goblet cells that produce the MUC2 protein, but also actively excretes xenobiotics through drug efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP) and degrades them through drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which accounts for 30% of drug metabolism in humans (NPL1). Nuclear receptors regulate the expression of these drug efflux transporters and metabolizing enzymes. Some commercial drugs are substrates for nuclear receptors, causing drug-drug interactions. Thus, the effects of the drugs are attenuated because drug absorption is reduced, and the toxicities are enhanced due to the increased exposure to the reactive metabolites of drugs. Drug absorption in humans is frequently predicted from non-clinical species (NPL2). Pharmacokinetics studies are often performed on rodents and dogs; however, sometimes, these models are not suitable for preclinical studies because of species differences such as differences in terms of drug efflux transporters, metabolizing enzymes, and nuclear receptors (NPL3).

[0003] Multiple attempts have been made to construct in vitro systems to understand the complex drug absorption processes. Monolayer cultures derived from Caco-2, a human colon adenocarcinoma cell line, are the most commonly utilized model for the prediction of drug absorption in humans (NPLs 4 and 5). This model is useful with respect to the expression of functional drug efflux transporters and the formation of a stable and reproducible barrier; however, it lacks drug metabolic enzymes and nuclear receptor function (NPL6). A straightforward approach to overcoming these challenges is the use of primary cells, but it has come inherent limitations in terms of short culture period, lot-to-lot variation, and limited resources of human intestinal tissues (NPL7). Recently, protocols have been modified to induce iPSC-derived enterocyte-like cells to emulate absorption process in humans (NPLs 8-10). Although remarkable progress has been achieved, room for improvement exists in terms of the maturation status of drug transporters and expression of metabolizing enzymes compared with those in vivo. Furthermore, these in vitro systems cannot simulate the epigenetic status of the original patient. The other approach is to utilize adult stem cell-derived organoids, which include organ specific cell types and can recapitulate several aspects of the in vivo organ function (NPL11). The establishment of intestinal organoids was initially reported in a mouse study (NPL12) and then in humans a few years later (NPLs 13 and 14). These organoids derived from human biopsy or surgical specimens encompass intestinal stem cells, which can be differentiated into mature intestinal cells such as absorptive enterocytes, goblet cells, enteroendocrine cells, and Paneth cells that retain the characteristics of the original intestinal tissue region (NPLs 13 and 15-20). Although microinjection and polarity controlling technologies have been reported, they have limited application for drug absorption studies (NPLs 21 and 22). This is mainly because it is difficult to assess drug absorption using intestinal organoids because they form closed lumen, from which collecting sufficient liquid to detect the drug become difficult.

[0004] Accordingly, there is a need to develop reproducible and robust methodologies for assessing oral drug absorption that evaluates key factors affecting intestinal drug absorption such as drug transporters and metabolizing enzymes, and their applicability to the intestinal drug absorption in humans. The provided methods and the monolayers produced by the methods address this need.

[0005] [NPL1] Zanger et al., Pharmacol. Ther. 138:103-141 (2013). [NPL2] Choi et al., Pharmaceutics. 11 (2019). [NPL3] Musther et al., Eur. J. Pharm. Sci. 57:280-291 (2014). [NPL4] Hubatsch et al., Nat. Protoc. 2:2111-2119 (2007). [NPL5] Kus et al., Pharmaceutics. 15:(2023). [NPL6] Sun et al., Expert Opin. Drug Metab. Toxicol. 4:395-411 (2008). [NPL7] Li, et al., Drug Metab. Dispos. 46:1562-1571 (2018). [NPL8] Kwon et al., Sci. Adv. 7 (2021). [NPL9] Leo et al., Stem Cells. 41:775-791 (2023). [NPL10] Inui et al., Stem Cell Res. Ther. 15 (2024). [NPL11] Clevers et al., Cell. 165:1586-1597 (2016). [NPL12] Sato et al., Nature. 459:262-265 (2009). [NPL13] Sato et al., Gastroenterology. 141:1762-1772 (2011). [NPL14] Jung et al., Nat. Med. 17:1225-1227 (2011). [NPL15] Yin et al., Nat. Methods. 11:106-112 (2014). [NPL16] Beumer et al., Nat. Cell Biol. 20:909-916 (2018). [NPL17] Fujii et al., Cell Stem Cell. 23:787-793.e6 e786 (2018). [NPL18] Kraiczy et al., Gut. 68:49-61 (2019). [NPL19] He et al., Cell Stem Cell (2022). [NPL20] Zeve et al., Nat. Commun. 13:261 (2022). [NPL21] Williamson et al., Cell. Mol. Gastroenterol. Hepatol. 6:301-319 (2018). [NPL22] Co et al., Nat. Protoc. 16:5171-5192 (2021).

[0006] [PTL 1] Japanese Patent Appl. No. JP 2021-122208

[0007] The disclosure relates to methods for producing epithelial-like monolayers and uses of the monolayers produced according to the claimed methods that include, for example, there use as a model system for evaluating the bioavailability and pharmacokinetics of orally administered drugs in humans.

[0008] In some embodiments, the disclosure provides: [A01] A method for preparing an intestinal organoid-derived monolayer, comprising, (a) culturing a cell derived from an intestinal organoid on an extracellular matrix in a cell culture expansion medium that comprises lysophosphatidic acid (LPA), sphingolipid, albumin, a BMP signaling inhibitor, an ALK5 inhibitor, a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, gastrin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF / FGF2), and IGF-I (insulin-like growth factor), to form a monolayer of expanded cells; (b) culturing the monolayer of expanded cells in a differentiation medium comprising LPA, sphingolipid, albumin, gastrin, a BMP signaling inhibitor, an ALK5 inhibitor, and EGF to form a monolayer of intestinal epithelial-like cells; optionally, (c) culturing the monolayer of intestinal epithelial-like cells in a medium containing the differentiation medium and vitamin D3 or an analog thereof (e.g., 1alpha, 25-dihydroxy-vitamin D3 (VD3)) and / or rifampicin, to form a treated monolayer of intestinal epithelial-like cells. [A02] The method of [A01], wherein (b) further comprises culturing the monolayer up to 1 or 2 days in the differentiation medium without EGF. [A03] The method of [A01] or [A02], wherein the extracellular matrix comprises one or more C-terminal fragments of laminin E8 isoforms (e.g., iMatrix-511) and fibronectin. [A04] The method of [A01] to [A03], wherein the culturing of (a), (b), and / or (c) is conducted in a Transwell (registered trademark). [A05] The method of any one of [A01] to [A04], wherein the expansion medium and / or differentiation medium comprises LPA at a total concentration of 2 micro M or higher. [A06] The method of any one of [A01]- [A05], wherein the expansion medium and / or differentiation medium comprises a member selected from palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof. [A07] The method of any one of [A01] or [A06], wherein the expansion medium and / or differentiation medium comprises sphingolipid at a concentration of 1 micro M or higher, or from about 1-5 micro M. [A08] The method of any one of [A01] to [A07], wherein the expansion medium and / or differentiation medium comprises sphingosine-1-phosphate (S1P), optionally at a concentration of 1 micro M or higher. [A09] The method of any one of [A01] to [A08], wherein the albumin in the expansion medium and / or differentiation medium is bovine serum albumin or human serum albumin isolated from biological serum, or recombinant bovine serum albumin or human serum albumin. [A10] The method of any one of [A01] to [A09], wherein the expansion medium and / or differentiation medium comprises albumin at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher, or from about 1 mg / mL to about 10 mg / mL. [A11] The method of any one of [A01] to [A10], wherein the expansion medium and / or differentiation medium comprises albumin at a concentration of 4 mg / mL or higher, lysophosphatidic acid at a concentration of 2 micro M or higher, and sphingosine-1-phosphate at a concentration of 10 nM or higher. [A12] The method of any one of [A01] to [A11], wherein the expansion medium and / or differentiation medium further comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof. [A13] The method of any one of [A01] to [A12], wherein the expansion medium and / or differentiation medium comprises LPC, LPE, LS, and / or LPG, at a concentration of 2 micro M or higher. [A14] The method of any one of [A01] to [A13], wherein the expansion medium and / or differentiation medium comprises a BMP signaling inhibitor selected from noggin, DAN, and a DAN-like protein such as, cerberus and gremlin, or a combination thereof. [A15] The method of any one of [A01] to [A14], wherein the expansion medium and / or differentiation medium comprises a BMP signaling inhibitor at a concentration of about 10 to about 200 ng / ml. [A16] The method of any one of [A01] to [A15], wherein the expansion medium and / or differentiation medium comprises noggin, optionally, at a concentration of about 10-200 ng / ml (e.g., 100 ng / ml). [A17] The method of any one of [A01] to [A16], wherein the ALK5 inhibitor is A83-01. [A18] The method of any one of [A01] to [A17], wherein the expansion medium and / or differentiation medium comprises an ALK5 inhibitor at a concentration of about 0.2-2 micro M. [A19] The method of any one of [A01] to [A18], wherein the expansion medium and / or differentiation medium comprises recombinant human, bovine, or murine EGF. [A20] The method of any one of [A01] to [A19], wherein the expansion medium and / or differentiation medium comprises EGF at a concentration of about 10-100 ng / ml (e.g., about 40-60 ng / ml or 50 ng / ml). [A21] The method of any one of [A01] to [A20], wherein the expansion medium and / or differentiation medium comprises gastrin (e.g., [Leu15]-Gastrin I human) at a concentration of about 5-15 nM. [A22] The method of any one of [A01] to [A21], wherein the expansion medium comprises the ROCK inhibitor Y-27632. [A23] The method of any one of [A01] to [A22], wherein the expansion medium comprises a ROCK inhibitor at a concentration of about 5-100 micro M. [A24] The method of any one of [A01] to [A23], wherein the expansion medium comprises a Wnt pathway signaling agonist at a concentration of about 25 ng / ml to about 150 ng / ml. [A25] The method of any one of [A01] to [A24], wherein the expansion medium comprises a Wnt protein (e.g., Wnt3a). [A26] The method of any one of [A01] to [A25], wherein the expansion medium comprises R-spondin (e.g., R-spondin-1, R-spondin-2, R-spondin-3 or R-spondin-4), optionally at a concentration of 0.5-1.5 micro g / ml. [A27] The method of any one of [A01] to [A26], wherein Wnt pathway signaling agonist is Afamin / Wnt3a CM and / or R-spondin-1. [A28] The method of any one of [A01] to [A27], wherein the expansion medium comprises recombinant human, bovine or murine bFGF (FGF2). [A29] The method of any one of [A01] to [A28], wherein the expansion medium comprises bFGF at a concentration of about 25-500 ng / ml (e.g., 50 ng / ml). [A30] The method of any one of [A01] to [A29], wherein the expansion medium comprises recombinant human, bovine, or murine IGF-1. [A31] The method of any one of [A01] to [A30], wherein the expansion medium comprises IGF-1 at a concentration of about 50-500 ng / ml (e.g., 100 ng / ml). [A32] The method of any one of [A01] to [A31], wherein the differentiation medium does not contain Wnt protein (e.g., Wnt3a), R-spondin (e.g., R-spondin1), nicotinamide, SB202190; IGF-1 and / or FGF2. [A33] The method of any one of [A01] to [A32], wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; and (ii) separating the intestinal organoid into single cells. [A34] The method of [A33], wherein the intestinal organoid is a duodenal organoid. [A35] The method of any one of [A01] to [A34], wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is derived from a human. [A36] The method of any one of [A01] to [A35], wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is derived from a small intestine or a large intestine. [A37] The method of [A36], wherein the small intestine is a duodenum, a jejunum, or an ileum. [A38] The method of [A33] or [A37], wherein the intestine-derived cell is a duodenum-derived crypt cell. [A39] The method of any one of [A01] to [A38], wherein the culturing of (a) takes place over about 3-5 days (e.g., about 4 days) at 37 degrees C and 5% CO2and the culturing of (b) takes place over about 5-7 days (e.g., about 6 days) at 37 degrees C and 5% CO2. [A40] The method of any one of [A01] to [A39], wherein up to about 1 day or about 2 days of the culturing of (b) is performed in differentiation medium that does not contain EGF. [A41] The method of [A01] or [A40], wherein VD3 and / or rifampicin is added to the differentiation medium during the last about 1.5 -2.5 days (e.g., about 2 days) of the culturing of (b). [A42] The method of any one of [A01] to [A41], which further comprises (d) evaluating a barrier function of the cell. [A43] The method of [A42], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [A44] The method of any one of [A01] to [A43], wherein the monolayer of intestinal epithelial-like cells formed in (b) has an increased barrier function compared to the monolayer of expanded cells formed in (a). [A45] The method of any one of [A01] to [A44], wherein cells of the monolayer of intestinal epithelial-like cells formed in (b) have an increased expression of at least one gene selected from the group consisting of ABCG2, CES2, CYP2D6, UGT1A1, and UGT1A10 compared to cells of the monolayer of expanded cells formed in (a). [A46] The method of any one of [A01] to [A45], wherein the monolayer of intestinal epithelial-like cells formed in (b) has a 10-fold or more, 9-fold or more, 8-fold or more, 7-fold or more, 6-fold or more, 5-fold or more, 4-fold or more, 3-fold or more, 2-fold or more, 1.9-fold or more, 1.8-fold or more, 1.7-fold or more, 1.6-fold or more, or 1.5-fold or more increased transepithelial electrical resistance (TEER) value compared to that the monolayer of expanded cells formed in (a). [A47] The method of any one of [A01] to [A46], wherein the monolayer of intestinal epithelial-like cells formed in (b) has a transepithelial electrical resistance (TEER) value of 500 omega x cm2or higher, 600 omega x cm2or higher, 700 omega x cm2or higher, 800 omega x cm2or higher, 900 omega x cm2or higher, 1000 omega x cm2or higher, 1100 omega x cm2or higher, 1200 omega x cm2or higher, 1300 omega x cm2or higher, 1400 omega x cm2or higher, 1500 omega x cm2or higher, 1600 omega x cm2or higher, 1700 omega x cm2or higher, 1800 omega x cm2or higher, 1900 omega x cm2or higher, or 2000 omega x cm2or higher. [A48] The method of any one of [A01] to [A47], wherein the monolayer of intestinal epithelial-like cells formed in (b) is a monolayer of duodenal epithelial-like cells. [A49] A method for preparing an intestinal organoid-derived monolayer, comprising, (a) culturing a cell derived from an intestinal organoid on an extracellular matrix in a cell culture expansion medium that comprises (i) a member selected from lysophosphatidic acid (LPA), sphingolipid, albumin, a fatty acid, a PPAR-delta (Peroxisome proliferator-activated receptor delta) agonist, and a GPR40 / FFAR1 (G protein-coupled receptor 40 / free fatty acid receptor 1) agonist, or a combination thereof and (ii) a BMP signaling inhibitor, an ALK5 inhibitor, a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, gastrin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF / FGF2), and IGF-I (insulin-like growth factor), to form a monolayer of expanded cells; (b) culturing the monolayer of expanded cells in a differentiation medium comprising (i) a member selected from LPA, sphingolipid, albumin, a fatty acid, a PPAR-delta agonist, and a GPR40 / FFAR1 agonist, or a combination thereof and (ii) gastrin, a BMP signaling inhibitor, an ALK5 inhibitor, and EGF to form a monolayer of intestinal epithelial-like cells; optionally, (c) culturing the monolayer of intestinal epithelial-like cells in a medium containing the differentiation medium and vitamin D3 or an analog thereof (e.g., 1alpha, 25-dihydroxy-vitamin D3 (VD3)) and / or rifampicin, to form a treated monolayer of intestinal epithelial-like cells.

[0009] In some embodiments, the disclosure provides: [B01] The monolayer of intestinal epithelial-like cells formed according to the method of any one of [A01] to [A49]. [B02] The treated monolayer of intestinal epithelial-like cells formed according to the method of any one of [A01] to [A49].

[0010] In some embodiments, the disclosure provides: [C01] A method of screening an agent for the ability to modify a barrier function of an intestinal epithelial cell, comprising contacting the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells obtained by the method of any one of [A01] to [A49], the monolayer of [B01], or the treated monolayer of [B02] with a test agent. [C02] The method of [C01], which further comprises evaluating a barrier function of the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells after the monolayer is contacted with the test agent. [C03] The method of [C01] or [C02], which further comprises selecting a test agent that increases or decreases a barrier function of the monolayer of intestinal epithelial-like cells compared to a control. [C04] The method of any one of [C01] to [C03], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [C05] The method of any one of [C01] to [C04], wherein the monolayer of intestinal epithelial-like cells is a monolayer of duodenal epithelial-like cells. [C06] The method of any one of [C01] to [C05], wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

[0011] In some embodiments, the disclosure provides: [D01] A method for evaluating pharmacokinetics and / or toxicity of an agent in an intestinal epithelial cell, comprising contacting the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells obtained by the method of any one of [A01] to [A49], the monolayer of [B01], or the treated monolayer of [B02] with a test agent. [D02] The method of [D01], which further comprises detecting a change in expression of a protein in cells of the monolayer of intestinal epithelial-like cell or the treated monolayer of intestinal epithelial-like cells after the cells are contacted with the test agent. [D03] The method of [D02], wherein a change is detected in the expression of a protein selected from the group consisting of ALPI, ABCB1, ABCC2, ABCG2, SLC15A1, SLCO2B1, CES2, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, UGT1A1, UGT1A10, UGT2B7, and UGT2B17. [D04] The method of any one of [D01] to [D03], wherein the monolayer of intestinal epithelial-like cells is a monolayer of duodenal epithelial-like cells. [D05] The method of any one of [D01] to [D04], wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

[0012] In some embodiments, the disclosure provides: [E01] A kit for producing an intestinal organoid-derived monolayer comprising, lysophosphatidic acid (LPA), a sphingolipid, and albumin. [E02] The kit of [E01], which further comprises an extracellular matrix. [E03] The kit of [E02], wherein the extracellular matrix comprises C-terminal fragments of laminin E8 isoforms (e.g., iMatrix-511) and fibronectin. [E04] The kit of any one of [E01] to [E03], which further comprises gastrin, EGF, a BMP signaling inhibitor, an ALK5 inhibitor, or a combination thereof. [E05] The kit of any one of [E01] to [E04], which further comprises a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, or a combination thereof. [E06] The kit of any one of [E01] to [E05], which further comprises bFGF and / or IGF-I. [E07] The kit of any one of [E01]- [E06], wherein the kit comprises a member selected from palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof. [E08] The kit of any one of [E01] to [E07], which comprises a solution containing LPA at a total concentration of 2 micro M or higher. [E09] The kit of any one of [E01] to [E08], which comprises sphingosine-1-phosphate (S1P). [E10] The kit of any one of [E01] or [E09], which comprises a solution containing sphingolipid at a concentration of 1 micro M or higher. [E11] The kit of any one of [E01] to [E10], which comprises bovine serum albumin or human serum albumin isolated from biological serum, or recombinant bovine serum albumin or human serum albumin. [E12] The kit of any one of [E01] to [E11], which comprises a solution containing albumin at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. [E13] The kit of any one of [E01] to [E12], which comprises a solution containing lysophosphatidic acid at a concentration of 2 micro M or higher, sphingolipid at a concentration of 1 micro M or higher and albumin at a concentration of 4 mg / mL or higher. [E14] The kit of any one of [E01] to [E13], which comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof. [E15] The kit of any one of [E01] to [E14], which contains a solution comprising LPC, LPE, LS, and / or LPG, at a concentration of 2 micro M or higher. [E16] The kit of any one of [E01] to [E15], which comprises a BMP signaling inhibitor selected from noggin, DAN, and a DAN-like protein such as, cerberus and gremlin, or a combination thereof. [E17] The kit of any one of [E01] to [E16], which contains a solution comprising a BMP signaling inhibitor at a concentration of about 10 to about 200 ng / ml. [E18] The kit of any one of [E01] to [E17], which comprises noggin. [E19] The kit of any one of [E01] to [E18], which comprises the ALK5 inhibitor A83-01. [E20] The kit of any one of [E01] to [E19], which comprises ALK5 inhibitor at a concentration of about 0.2-2 micro M. [E21] The kit of any one of [E01] to [E20], which comprises recombinant human, bovine, or murine EGF. [E22] The kit of any one of [E01] to [E21], which contains a solution comprising EGF at a concentration of about 10-100 ng / ml. [E23] The kit of any one of [E01] to [E22], which comprises the ROCK inhibitor Y-27632. [E24] The kit of any one of [E01] to [E23], which contains a solution comprising a ROCK inhibitor at a concentration of at a concentration of about 5-100 micro M. [E25] The kit of any one of [E01] to [E24], which contains a solution comprising a Wnt pathway signaling agonist that is a Wnt protein selected from Wnt3a, Wnt5, and Wnt6a. [E26] The kit of any one of [E01] to [E25], which comprises Wnt pathway signaling agonist R-spondin (e.g., R-spondin-1, R-spondin-2, R-spondin-3, R-spondin-4, or a combination thereof). [E27] The kit of any one of [E01] to [E26], which comprises the Wnt pathway signaling agonist Afamin / Wnt3a CM and / or R-spondin-1. [E28] The kit of any one of [E01] to [E27], which comprises recombinant human, bovine, or murine bFGF (FGF2). [E29] The kit of any one of [E01] to [E28], which contains a solution comprising bFGF at a concentration of about 25-250 ng / ml. [E30] The kit of any one of [E01] to [E29], which comprises recombinant human, bovine, or murine IGF-1. [E31] The kit of any one of [E01] to [E30], which contains a solution comprising IGF-1 at a concentration of at a concentration of about 50-250 ng / ml. [E32] The kit of any one of [E01] to [E31], which further comprises vitamin D3 or an analog thereof (e.g., VD3) and / or rifampicin. [E33] The kit of any one of [E01] to [E32], which further comprises a cell derived from an intestine or an intestine-derived organoid. [E34] The kit of [E33], wherein the cell derived from an intestine or intestine-derived organoid is derived from a human. [E35] The kit of [E33] or [E34], wherein the cell derived from an intestine or intestine-derived organoid is derived from a small intestine or a large intestine. [E36] The kit of [E35], wherein the small intestine is a duodenum, a jejunum, or an ileum. [E37] The kit of any one of [E33] to [E36], wherein the cell derived from an intestine is a duodenum-derived crypt cell. [E38] The kit of any one of [E33] to [E37], wherein the intestine-derived organoid is a duodenal organoid. [E39] A kit for producing an intestinal organoid-derived monolayer comprising a member selected from lysophosphatidic acid (LPA), a sphingolipid, albumin, a fatty acid, a PPAR-delta agonist, and a GPR40 / FFAR1 agonist, or a combination thereof.

[0013] The disclosure relates to a method for producing intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function. The disclosure also relates to intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function obtained by the provided method. The intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function provided in the disclosure have significantly increased expression of ABCG2 (which is a drug transporter) and CES2, CYP2D6, UGT1A1, and UGT1A10 (which are drug metabolizing enzymes). The barrier function of intestinal epithelial-like cells (intestinal epithelial-like monolayers) obtained in the disclosure is similar to that of natural intestinal epithelial cells, especially duodenal epithelial cells. The intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function obtained in the disclosure have significantly increased barrier function compared to intestinal epithelial-like cells obtained by conventional methods. By using the intestinal epithelial-like cells (intestinal epithelial-like monolayers) provided in the disclosure, it is possible to efficiently evaluate the kinetics and / or toxicity of drugs in the intestinal tract.

[0014] Fig. 1 shows changes in proliferation properties of intestinal organoid-derived cells on Transwell (registered trademark) by addition of AlbuMAX (registered trademark).Fig. 2 shows changes in EdU-positive cells on Transwell (registered trademark) by addition of AlbuMAX (registered trademark).Fig. 3-1 shows changes in the barrier function of the monolayer model by addition of AlbuMAX (registered trademark).Fig. 3-2 is a continuation of Fig. 3-1.Fig. 4 shows fasted state simulated intestinal fluid (FaSSIF) resistance of the monolayer model in the presence of AlbuMAX (registered trademark).Fig. 5-1 shows changes in expression of intestine-related genes by addition of AlbuMAX (registered trademark).Fig. 5-2 is a continuation of Fig. 5-1.Fig. 6 shows dose-response of barrier function improvement by addition of AlbuMAX (registered trademark).Fig. 7 shows effect of albumin on the barrier function of the monolayer model.Fig. 8 shows effect of fatty acids (all 50 mM) on the barrier function of the monolayer model.Fig. 9 shows effect of PPAR-delta agonist on the barrier function of the monolayer model.Fig. 10 shows effect of GPR40 agonist on the barrier function of the monolayer model.Fig. 11 shows effect of LPA and S1P on the barrier function of the monolayer model.

[0015] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the provided compositions, suitable methods and materials are described below. Each publication, patent application, patent, and other reference mentioned herein is herein incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0016] Other features and advantages of the disclosed methods and compositions will be apparent from the following disclosure, drawings, and claims.

[0017] It is understood that wherever embodiments, are described herein with the language “comprising” otherwise analogous embodiments, described in terms of “containing” “consisting of” and / or “consisting essentially of” are also provided. However, when used in the claims as transitional phrases, each should be interpreted separately and in the appropriate legal and factual context (e.g., in claims, the transitional phrase “comprising” is considered more of an open-ended phrase while “consisting of” is more exclusive and “consisting essentially of” achieves a middle ground).

[0018] As used herein, the singular form “a”, “an”, and “the”, include plural forms unless it is expressly stated or is unambiguously clear from the context that such is not intended.

[0019] As used herein, the terms "approximately" and "about," as applied to one or more values of interest, refer to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0020] The term “and / or” as used in a phrase such as “A and / or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0021] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0022] Where embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the disclosed composition or method encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The disclosed methods and compositions also envisage the explicit exclusion of one or more of any of the group members in the disclosed compositions or methods.

[0023] As used herein, an organoid is defined as a 3D culture of stem cells and their differentiated progeny, initiated from a single stem cell or a multicellular aggregate of cells with at least one stem cell.

[0024] The term “expressed” is used to describe the presence of a marker within or on a cell or cells (e.g., a cell monolayer). In order to be considered as being expressed, a marker must be present at a detectable level. By “detectable level” is meant that the marker can be detected using one of the standard laboratory methodologies such as PCR, blotting or FACS analysis. A gene is considered to be expressed by a cell of the population of the invention if expression can be reasonably detected after 30 PCR cycles, which corresponds to an expression level in / on the cell of at least about 100 copies per cell. The terms “express” and “expression” have corresponding meanings. At an expression level below this threshold, a marker is considered not to be expressed. The comparison between the expression level of a marker in a cell, and the expression level of the same marker in another cell, for example a cell that has not been contacted with a test agent can routinely be conducted using a reverse transcriptase polymerase chain reaction (RT-PCR) or other techniques known in the art.

[0025] The explanation of invention provided in the disclosure The disclosure provides methods for producing epithelial-like monolayers and uses of the monolayers produced according to the provided methods that include, for example, their use as a model system for evaluating the bioavailability and pharmacokinetics of orally administered drugs in humans. The disclosure also provides, inter alia, the epithelial-like monolayers produced by the provided methods and kits for preparing the monolayers.

[0026] In some embodiments, the disclosure provides a method for preparing an intestinal organoid-derived monolayer, that comprises (a) culturing a cell derived from an intestinal organoid on an extracellular matrix in a cell culture expansion medium that comprises lysophosphatidic acid (LPA), sphingolipid, albumin, a BMP signaling inhibitor, an ALK5 inhibitor, a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, gastrin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and IGF-I (insulin-like growth factor-1), to form a monolayer of expanded cells; and (b) culturing the monolayer of expanded cells in a differentiation medium comprising LPA, sphingolipid, albumin, gastrin, a BMP signaling inhibitor, an ALK5 inhibitor, and EGF to form a monolayer of intestinal epithelial-like cells; optionally, (c) further culturing the monolayer of intestinal epithelial-like cells in the differentiation medium and vitamin D3 or an analog thereof and / or rifampicin, to form a treated monolayer of intestinal epithelial-like cells. In some embodiments, culturing step (b) further comprises culturing the monolayer up to 1 or 2 days in modified differentiation medium wherein the differentiation medium is modified to not contain EGF.

[0027] Cell expansion preparation In some embodiments, the cell derived from the intestinal organoid is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; and (ii) separating the intestinal organoid into single cells.

[0028] In the disclosure, organoids generally refer to three-dimensional structures constructed by self-assembly of cells. The intestinal organoids refer to those having hollow sections and the tissue structures found in the intestinal tract. In some embodiments, intestinal organoids can be prepared by culturing intestinal-derived cells by methods known to those skilled in the art. As an example, intestine-derived cells isolated from a subject can be embedded in Matrigel and cultured in a culture medium known to those skilled in the art, as described in the examples in the specification. In some embodiments, the established intestinal organoids may be subjected to further culturing step as needed for maintenance of the intestinal organoids.

[0029] In the disclosure, single cells can be separated from the intestinal organoids and thus obtained. The separation of single cells from intestinal organoids can also be performed by methods known to those skilled in the art.

[0030] In some embodiments, the cell is derived from an organoid derived from a small intestine or a large intestine. In further embodiments, the small intestine is a duodenum, a jejunum, or an ileum. In some embodiments, the cell is derived from an intestinal organoid that is a duodenal organoid. In some embodiments, the cultured cell is derived from an organoid that is derived from a duodenum-derived crypt cell.

[0031] In some embodiments, the cultured cell is derived from an organoid derived from a human. In some embodiments, the cell is derived from an organoid derived from a human organoid derived from a human small intestine or a large intestine. In further embodiments, the cell is derived from an organoid derived from a human duodenum, jejunum, or ileum. In some embodiments, the cell is derived from an intestinal organoid that is a human duodenal organoid. In some embodiments, the cultured cell is derived from an organoid that is derived from a human duodenum-derived crypt cell.

[0032] In some embodiments, the cell is derived from the intestinal organoid in a step that includes treating fragments and / or cells of the organoid in a solution that comprises trypsin, trypsin / EDTA or TrypE. In some embodiments, the cell is derived from the intestinal organoid in a step that includes treating fragments and / or cells of the organoid in a solution that comprises TrypE, but does not comprise trypsin.

[0033] In some embodiments, the cell derived from the intestinal organoid is cultured on an extracellular matrix that comprises one or more C-terminal fragments of laminin E8 isoforms and fibronectin. In some embodiments, the extracellular matrix comprises the E8 fragment of laminin-511. In some embodiments, the extracellular matrix comprises iMatrix-511 and fibronectin. In further embodiments, the extracellular matrix comprises iMatrix-511 as its major component and fibronectin.

[0034] In some embodiments, one or more of culturing steps (a)-(c) is performed on Transwell (registered trademark). In some embodiments, the Transwell (registered trademark) contains an extracellular matrix coating that comprises one or more C-terminal fragments of laminin E8 isoforms and fibronectin. In some embodiments, the extracellular matrix coating comprises the E8 fragment of laminin-511. In some embodiments, the extracellular matrix coating comprises iMatrix-511 and fibronectin. In further embodiments, the extracellular matrix coating comprises iMatrix-511 as its major component and fibronectin.Cell expansion

[0035] In some embodiments, the cell derived from the intestinal organoid is cultured in an expansion medium that comprises LPA, sphingolipid, albumin, a BMP signaling inhibitor, an ALK5 inhibitor, a ROCK inhibitor, one or more Wnt pathway signaling agonists, gastrin or a gastrin analog, EGF, bFGF (FGF2), and IGF-I.

[0036] In some embodiments, the expansion medium comprises LPA at a concentration of 2 micro M or higher. In some embodiments, the expansion medium comprises LPA at a concentration of about 2-50, 4-40, 5-30, 5-20, or 7-15 micro M. In some embodiments, the expansion medium comprises LPA at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0037] In some embodiments, the expansion medium comprises a saturated fatty acid selected from palmitic acid and stearic acid, or a combination thereof. In some embodiments, the expansion medium comprises palmitic acid and stearic acid at a concentration of 2 micro M or more. In some embodiments, the expansion medium comprises a saturated fatty acid selected from palmitic acid and stearic acid, or a combination thereof at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the expansion medium comprises one or more saturated fatty acids at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0038] In some embodiments, the expansion medium comprises an unsaturated fatty acid selected from palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof. In some embodiments, the expansion medium comprises one or more unsaturated fatty acids at a concentration of 2 micro M or more. In some embodiments, the expansion medium comprises an unsaturated fatty acid selected from palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the expansion medium comprises one or more saturated fatty acids at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0039] In some embodiments, the expansion medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a total concentration of 2 micro M or more. In some embodiments, the expansion medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the expansion medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a total concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0040] In some embodiments, the expansion medium comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof. In some embodiments, the expansion medium comprises LPC, LPE, LS, or LPG, at a concentration of 2 micro M or higher. In some embodiments, the expansion medium comprises LPC, LPE, LS, and LPG, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the expansion medium comprises LPC and / or LS, at a concentration of 2 micro M or higher. In some embodiments, the expansion medium comprises LPC and / or LS, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M.

[0041] In some embodiments, the expansion medium comprises sphingolipid at a concentration of 1 micro M or higher. In some embodiments, the concentration of sphingolipid in the expansion medium is from about 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 micro M. In further embodiments, the expansion medium comprises sphingosine-1-phosphate (S1P). In some embodiments, the expansion medium comprises S1P at a concentration of 1 micro M or higher. In further embodiments, the expansion medium comprises S1P at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0042] In some embodiments, the expansion medium comprises albumin at a concentration of 1 mg / mL or higher. The albumin contained in the expansion medium may be purified from a natural source (e.g., human serum or bovine serum) or recombinantly produced. In some embodiments, the expansion medium comprises bovine serum albumin (BSA). In some embodiments, the expansion medium comprises human serum albumin (HSA). In some embodiments, the albumin is recombinantly produced, for example, in plants (e.g., rice), bacteria (e.g., E. coli), or yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae). In some embodiments, the expansion medium comprises recombinant bovine serum albumin (rBSA). In some embodiments, the expansion medium comprises recombinant human serum albumin (rHSA). In some embodiments, the albumin is purified from animal serum (e.g., bovine or human). In some embodiments, the albumin is purified from bovine serum. Albumin is the main fatty acid binding protein in extracellular fluids. In some embodiments, the albumin is purified from bovine serum using a procedure (e.g., chromatography) that does not disrupt or eliminate the albumin-fatty acid complexes present in the serum (i.e., the purified albumin contains fatty acids). In further embodiments, the expansion medium comprises albumin at a concentration 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. In some embodiments, the concentration of albumin in the expansion medium is from about 1-10, or 2-5 mg / ml. In some embodiments, the expansion medium comprises bovine serum albumin at a concentration of from about 1-10, or 2-5 mg / ml. In some embodiments, the expansion medium comprises human serum albumin at a concentration of from about 1-10, or 2-5 mg / ml. In some embodiments, the expansion medium comprises albumin and fatty acids isolated together from bovine serum or human serum at a total concentration of about 1-10, or 2-5 mg / ml.

[0043] In some embodiments, the expansion medium comprises fatty acids at a total concentration of 30, 40, 50, 60, 70 micro M or higher. In some embodiments, the expansion medium comprises fatty acids at a total concentration of 50 micro M or higher.

[0044] In some embodiments, the expansion medium comprises a PPAR-delta agonist. In some embodiments, the expansion medium comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. In further embodiments, the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid) at a concentration of 1 micro M or higher, or 10 micro M or higher.

[0045] In some embodiments, the expansion medium comprises a GPR40 / FFAR1 agonist. In some embodiments, the expansion medium comprises a GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. In further embodiments, the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl] methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid) at a concentration of 100 nM or higher, or 1 micro M or higher.

[0046] In some embodiments, the expansion medium comprises a BMP signaling inhibitor at a concentration of 1 ng / mL or higher. In some embodiments, the BMP signaling inhibitor is selected from noggin, gremlin, DAN, a DAN-like protein containing a DAN cysteine knot domain, cerberus, chordin, a chordin-like protein containing a chordin domain, follistatin, a follistatin-related protein containing a follistatin domain, alpha-2 macroglobulin, and sclerostin / SOST, or a combination thereof. In some embodiments, the BMP signaling inhibitor is a recombinant protein (e.g., a recombinant human, bovine or murine BMP inhibitor polypeptide). In some embodiments, the concentration of the BMP signaling inhibitor in the expansion medium is about 10-200 ng / ml. In some embodiments, the expansion medium comprises noggin. In some embodiments, the expansion medium comprises recombinant human noggin, humanized noggin, or murine noggin. In further embodiments, the expansion medium comprises noggin at a concentration of about 10-200 ng / ml, about 50-150 ng / ml, or about 75-125 ng / ml. In some embodiments, the concentration of noggin is about 75 ng / ml, about 100 ng / ml, or about 125 ng / ml.

[0047] In some embodiments, the expansion medium comprises an ALK5 inhibitor at a concentration of 0.2 micro M or higher. In some embodiments, the expansion medium contains an ALK5 inhibitor selected from A83-01, LY-36494, GW788388, RepSox, SB-431542, SB-505124, SB-525334, and SJN-2511, or a combination thereof. In some embodiments, the concentration of the ALK5 inhibitor in the expansion medium is about 0.2-2 micro M. In some embodiments, the expansion medium comprises an ALK5 inhibitor at a concentration of 100-900 nM, 200-800 nM, 300-700 nM, or 400-600 nM. In a further embodiment, the expansion medium contains the ALK5 inhibitor A83-01. In some embodiments, the expansion medium comprises A83-01 at a concentration of 0.2-2 micro M. In some embodiments, the expansion medium comprises A83-01 at a concentration of 100-900 nM, 200-800 nM, 300-700 nM, or 400-600 nM. In some embodiments, the expansion medium comprises A83-01 at a concentration of about 300, 400, 450, 500, 550 or 600 nM.

[0048] In some embodiments, the expansion medium comprises EGF at a concentration of 10 ng / ml or higher. In some embodiments, the EGF contained in the expansion medium is recombinant human, bovine, or murine EGF. In further embodiments, the EGF contained in the expansion medium is recombinant human EGF. In further embodiments, the expansion medium has an EGF concentration of about 10-100 ng / ml, about 25-75 ng / ml, or about 40-60 ng / ml. In some embodiments, the expansion medium has an EGF concentration of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 ng / ml.

[0049] In some embodiments, the expansion medium comprises gastrin or a suitable gastrin substitute at a concentration of 5 nM or higher. The gastrin contained in the expansion medium can be gastrin or a suitable gastrin substitute such as [Leu15]-Gastrin. In some embodiments, the expansion medium comprises [Leu15]-Gastrin I human. In further embodiments, the expansion medium contains gastrin at a concentration of about 5-15 nM. In some embodiments, the expansion medium contains [Leu15]-Gastrin I human at a concentration of about 5-15 nM.

[0050] In some embodiments, the expansion medium comprises ROCK inhibitor at a concentration of 5 micro M or higher. In some embodiments, the expansion medium contains a ROCK inhibitor selected from Y-27632, H-1152, SR 3677, thiazovivin, HA1077, HA1100 hydrochloride, and GSK-429286, or a combination thereof. In some embodiments, the expansion medium contains the ROCK inhibitor Y-27632. In some embodiments, the expansion medium contains a ROCK inhibitor at a concentration of about 5-100 micro M. In some embodiments, the ROCK inhibitor is Y-27632. In some embodiments, the expansion medium contains Y-27632 at a concentration of about 5-100 micro M.

[0051] In some embodiments, the expansion medium comprises Wnt pathway signaling agonist(s) at a concentration of 25 ng / ml or higher. In some embodiments, the Wnt pathway signaling agonist is one or more Wnt proteins. In some embodiments, the Wnt protein is recombinant human, bovine, or murine Wnt protein. In some embodiments, the expansion medium contains a Wnt protein selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16, or a combination thereof. In some embodiments, the expansion medium contains Wnt3a, Wnt5, or Wnt6a. In particular embodiments, the expansion medium contains a soluble Wnt protein such as Wnt3a. In some embodiments, the Wnt protein is in a complex with afamin. In further embodiments, the expansion medium contains Afamin / Wnt3a CM. In some embodiments, the concentration of the Wnt protein in the expansion medium is about 25 ng / ml to 150 ng / ml. In some embodiments, the concentration of Wnt protein contained in the expansion medium is 30 -100 ng / ml.

[0052] In some embodiments, the expansion medium comprises the Wnt pathway signaling agonist R-spondin. In some embodiments, the expansion medium contains an R-spondin selected from R-spondin-1, R-spondin-2, R-spondin-3 and R-spondin-4, or fragments containing furin domains, and / or combinations thereof. In some embodiments, the R-spondin contained in the expansion medium is recombinant human, bovine, or murine R-spondin. In further embodiments, the R-spondin contained in the expansion medium is recombinant human R-spondin. In particular embodiments, the expansion medium contains R-spondin-1. In some embodiments, the expansion medium contains R-spondin-1 at a concentration of about 0.5-1.5 micro g / ml. In some embodiments, the expansion medium contains recombinant human R-spondin-1 at a concentration of about 0.5-1.5 micro g / ml. In some embodiments, the expansion medium contains R-spondin-1 at a concentration of about 0.5-1.5 micro g / ml. In some embodiments, the expansion medium contains recombinant human R-spondin-1 at a concentration of about 0.5-1.5 micro g / ml.

[0053] In further embodiments, the expansion medium comprises more than one Wnt pathway signaling agonist. In some embodiments, the expansion medium contains a Wnt protein and an R-spondin. In some embodiments, the expansion medium contains a Wnt protein at a concentration of about 25 ng / ml to 150 ng / ml and an R-spondin at a concentration of about 0.5-1.5 micro g / ml. In further embodiments, the expansion medium contains Wnt3a protein at a concentration of about 25 ng / ml to 150 ng / ml and an R-spondin-1 at a concentration of about 0.5-1.5 micro g / ml.

[0054] In some embodiments, the expansion medium comprises bFGF (FGF2) at a concentration of 25 ng / ml or higher. In some embodiments, the bFGF contained in the expansion medium is recombinant human, bovine, or murine bFGF. In further embodiments, the bFGF contained in the expansion medium is recombinant human bFGF. In some embodiments, the expansion medium contains bFGF at a concentration of about 25-250 ng / ml (e.g., 50 ng / ml). In some embodiments, the expansion medium comprises bFGF at a concentration of about 25 ng / ml, about 50 ng / ml, about 75 ng / ml, or about 100 ng / ml. In a further embodiment, the expansion medium comprises bFGF at a concentration of about 50 ng / ml.

[0055] In some embodiments, the expansion medium comprises IGF-1 at a concentration of 50 ng / ml or higher. In some embodiments, the IGF-1 contained in the expansion medium is recombinant human, bovine, or murine IGF-1. In further embodiments, the IGF-1 contained in the expansion medium is recombinant human IGF-1. In some embodiments, the expansion medium contains IGF-1 at a concentration of about 50-250 ng / ml. In some embodiments, the expansion medium comprises IGF-1 at a concentration of about 50 ng / ml, about 75 ng / ml, about 100 ng / ml, about 125 ng / ml, or about 150 ng / ml. In a further embodiment, the expansion medium comprises IGF-1 at a concentration of about 100 ng / ml.

[0056] In some embodiments, the expansion medium comprises Advanced DMEM / F-12, 10 mM HEPES, and penicillin-streptomycin. In some embodiments, the expansion medium further comprises 2 mM GlutaMAXTM-I. In some embodiments, the expansion medium comprises 1 x B-27 supplement (Thermo Fisher Scientific) and 0.75-1.5 mM (e.g., 1.25 mM) N-acetylcysteine and 1-3 mM (e.g., 2 mM) L-alanyl-L-glutamine. In some embodiments, the expansion medium does not contain N2supplement.

[0057] In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 1 mg / ml or more. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 2 mg / ml or more, 3 mg / ml or more, 4 mg / ml or more, or 5 mg / ml or more. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 1-10 mg / ml. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 2-5 mg / mlAlbuMAX (registered trademark) I Lipid-Rich BSA. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of about 3, 3.5, 4, 4.5 or 5 mg / ml. In some embodiments, the expansion medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of about 4 mg / ml. AlbuMAX (registered trademark) is supplied by Thermo Fisher Scientific Inc. and can be easily obtained by those skilled in the art through commercial channels.

[0058] Cell differentiation In some embodiments, the monolayer of expanded cells is cultured in a differentiation medium that comprises LPA, sphingolipid, albumin, gastrin, a BMP signaling inhibitor, an ALK5 inhibitor, and EGF.

[0059] In some embodiments, the differentiation medium comprises LPA at a concentration of 2 micro M or higher. In some embodiments, the differentiation medium comprises LPA at a concentration of about 2-50, 4-40, 5-30, 5-20, or 7-15 micro M. In some embodiments, the differentiation medium comprises LPA at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0060] In some embodiments, the differentiation medium comprises a saturated fatty acid selected from palmitic acid and stearic acid, or a combination thereof. In some embodiments, the differentiation medium comprises palmitic acid and stearic acid at a concentration of 2 micro M or more. In some embodiments, the differentiation medium comprises a saturated fatty acid selected from palmitic acid and stearic acid, or a combination thereof at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the differentiation medium comprises one or more saturated fatty acids at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0061] In some embodiments, the differentiation medium comprises fatty acids at a total concentration of 30, 40, 50, 60, 70 micro M or higher. In some embodiments, the differentiation medium comprises fatty acids at a total concentration of 50 micro M or higher.

[0062] In some embodiments, the differentiation medium comprises an unsaturated fatty acid selected from palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof. In some embodiments, the differentiation medium comprises one or more unsaturated fatty acids at a concentration of 2 micro M or more. In some embodiments, the differentiation medium comprises an unsaturated fatty acid selected from palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the differentiation medium comprises one or more saturated fatty acids at a concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0063] In some embodiments, the differentiation medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a total concentration of 2 micro M or more. In some embodiments, the differentiation medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the differentiation medium comprises a member selected from palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, and alpha-linolenic acid, at a total concentration of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0064] In some embodiments, the differentiation medium comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof. In some embodiments, the differentiation medium comprises LPC, LPE, LS, or LPG, at a concentration of 2 micro M or higher. In some embodiments, the differentiation medium comprises LPC, LPE, LS, and LPG, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M. In some embodiments, the differentiation medium comprises LPC and / or LS, at a concentration of 2 micro M or higher. In some embodiments, the differentiation medium comprises LPC and / or LS, at a concentration of about 2-50, 2-40, 2-30, 2-20, or 2-15 micro M.

[0065] In some embodiments, the differentiation medium comprises sphingolipid at a concentration of 1 micro M or higher. In some embodiments, the concentration of sphingolipid in the differentiation medium is from about 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 micro M. In further embodiments, the differentiation medium comprises sphingosine-1-phosphate (S1P). In some embodiments, the differentiation medium comprises S1P at a concentration of 1 micro M or higher. In further embodiments, the differentiation medium comprises S1P at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 micro M.

[0066] In some embodiments, the differentiation medium comprises albumin at a concentration of 1 mg / mL or higher. The albumin contained in the differentiation medium may be purified from a natural source (e.g., human serum or bovine serum) or recombinantly produced. In some embodiments, the differentiation medium comprises bovine serum albumin (BSA). In some embodiments, the differentiation medium comprises human serum albumin (HSA). In some embodiments, the albumin is recombinantly produced, for example, in plants (e.g., rice), bacteria (e.g., E. coli), or yeast (e.g., Pichia pastoris, Saccharomyces cerevisiae). In some embodiments, the differentiation medium comprises recombinant bovine serum albumin (rBSA). In some embodiments, the differentiation medium comprises recombinant human serum albumin (rHSA). In some embodiments, the albumin is purified from animal serum (e.g., bovine or human). In some embodiments, the albumin is purified from bovine serum. Albumin is the main fatty acid binding protein in extracellular fluids. In some embodiments, the albumin is purified from bovine serum using a procedure (e.g., chromatography) that does not disrupt or eliminate the albumin-fatty acid complexes present in the serum (i.e., the purified albumin contains fatty acids). In further embodiments, the differentiation medium comprises albumin at a concentration 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. In some embodiments, the concentration of albumin in the differentiation medium is from about 1-10, or 2-5 mg / ml. In some embodiments, the differentiation medium comprises bovine serum albumin at a concentration of from about 1-10, or 2-5 mg / ml. In some embodiments, the differentiation medium comprises human serum albumin at a concentration of from about 1-10, or 2-5 mg / ml. In some embodiments, the differentiation medium comprises albumin and fatty acids isolated together from bovine serum or human serum at a total concentration of about 1-10, or 2-5 mg / ml.

[0067] In some embodiments, the differentiation medium comprises fatty acids at a total concentration of 30, 40, 50, 60, 70 micro M or higher. In some embodiments, the expansion medium comprises fatty acids at a total concentration of 50 micro M or higher.

[0068] In some embodiments, the differentiation medium comprises a PPAR-delta agonist. In some embodiments, the differentiation medium comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. In further embodiments, the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid) at a concentration of 1 micro M or higher, or 10 micro M or higher.

[0069] In some embodiments, the differentiation medium comprises a GPR40 / FFAR1 agonist. In some embodiments, the differentiation medium comprises a GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. In further embodiments, the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl] methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid) at a concentration of 100 nM or higher, or 1 micro M or higher.

[0070] In some embodiments, the differentiation medium comprises a BMP signaling inhibitor at a concentration of 1 ng / mL or higher. In some embodiments, the BMP signaling inhibitor is selected from noggin, gremlin, DAN, a DAN-like protein containing a DAN cysteine knot domain, cerberus, chordin, a chordin-like protein containing a chordin domain, follistatin, a follistatin-related protein containing a follistatin domain, alpha-2 macroglobulin, and sclerostin / SOST, or a combination thereof. In some embodiments, the BMP signaling inhibitor is a recombinant protein (e.g., a recombinant human, bovine or murine BMP inhibitor polypeptide). In some embodiments, the concentration of the BMP signaling inhibitor in the differentiation medium is about 10-200 ng / ml. In some embodiments, the differentiation medium comprises noggin. In some embodiments, the differentiation medium comprises recombinant human noggin, humanized noggin, or murine noggin. In further embodiments, the differentiation medium comprises noggin at a concentration of about 10-200 ng / ml, about 50-150 ng / ml, or about 75-125 ng / ml. In some embodiments, the concentration of noggin is about 75 ng / ml, about 100 ng / ml, or about 125 ng / ml.

[0071] In some embodiments, the differentiation medium comprises an ALK5 inhibitor at a concentration of 0.2 micro M or higher. In some embodiments, the differentiation medium contains an ALK5 inhibitor selected from A83-01, LY-36494, GW788388, RepSox, SB-431542, SB-505124, SB-525334, and SJN-2511, or a combination thereof. In some embodiments, the concentration of the ALK5 inhibitor in the differentiation medium is about 0.2-2 micro M. In some embodiments, the differentiation medium comprises an ALK5 inhibitor at a concentration of 100-900 nM, 200-800 nM, 300-700 nM, or 400-600 nM. In a further embodiment, the differentiation medium contains the ALK5 inhibitor A83-01. In some embodiments, the differentiation medium comprises A83-01 at a concentration of 0.2-2 micro M. In some embodiments, the differentiation medium comprises A83-01 at a concentration of 100-900 nM, 200-800 nM, 300-700 nM, or 400-600 nM. In further embodiments, the differentiation medium comprises A83-01 at a concentration of about 300, 400, 450, 500, 550 or 600 nM.

[0072] In some embodiments, the differentiation medium comprises EGF at a concentration of 10 ng / ml or higher. In some embodiments, the EGF contained in the differentiation medium is recombinant human, bovine, or murine EGF. In further embodiments, the EGF contained in the differentiation medium is recombinant human EGF. In some embodiments, the differentiation medium has an EGF concentration of about 10-100 ng / ml, about 25-75 ng / ml, or about 40-60 ng / ml. In some embodiments, the differentiation medium has an EGF concentration of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or 75 ng / ml.

[0073] In some embodiments, the differentiation medium comprises gastrin or a suitable gastrin substitute at a concentration of 5 nM or higher. The gastrin contained in the differentiation medium can be gastrin or a suitable substitute such as [Leu15]-Gastrin. In some embodiments, the differentiation medium comprises [Leu15]-Gastrin I human. In further embodiments, the differentiation medium contains gastrin at a concentration of about 5-15 nM. In some embodiments, the differentiation medium contains [Leu15]-Gastrin I human at a concentration of about 5-15 nM.

[0074] In some embodiments, the differentiation medium comprises Advanced DMEM / F-12, 10 mM HEPES, and penicillin-streptomycin. In some embodiments, the differentiation medium further comprises 2 mM GlutaMAXTM-I. In some embodiments, the differentiation medium comprises 1 x B-27 supplement (Thermo Fisher Scientific) and 0.75-1.5 mM (e.g., 1.25 mM) N-acetylcysteine and 1-3 mM (e.g., 2 M) L-alanyl-L-glutamine. In some embodiments, the differentiation medium does not contain N2 supplement.

[0075] In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 1 mg / ml or more. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 2 mg / ml or more, 3 mg / ml or more, 4 mg / ml or more, or 5 mg / ml or more. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 1-10 mg / ml. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of 2-5 mg / mlAlbuMAX (registered trademark) I Lipid-Rich BSA. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of about 3, 3.5, 4, 4.5 or 5 mg / ml. In some embodiments, the differentiation medium contains AlbuMAX (registered trademark) I Lipid-Rich BSA at a concentration of about 4 mg / ml. AlbuMAX (registered trademark) is supplied by Thermo Fisher Scientific Inc. and can be easily obtained by those skilled in the art through commercial channels.

[0076] In particular embodiments, the differentiation medium used according to the provided methods does not contain Wnt protein (e.g., Wnt3a), R-spondin (e.g., R-spondin1), nicotinamide, SB202190, IGF-1 and / or FGF2.

[0077] Culturing According to the provided methods, a cell derived from an intestinal organoid is cultured in cell expansion medium to form a monolayer of expanded cells.

[0078] In some embodiments, culturing step (a) comprises culturing the cell and resulting cells in expansion medium at 37 degrees C in an atmosphere comprising between 4-10% CO2 (e.g., 5% CO2) for 3 days or more. In some embodiments, the cell and resulting cells in culturing step (a) are cultured in expansion medium at 37 degrees C in an atmosphere comprising 5%-10% CO2, for 3, 4, 5, 6, 7, 8, 9 or 10 days or more to form a monolayer of expanded cells. In some embodiments, the cell and resulting cells are cultured in the expansion medium at 37 degrees C in an atmosphere comprising about 5% CO2, for 3-7, 3-6, or 3-5 days to form a monolayer of expanded cells. In further embodiments, the cell and resulting cells are cultured in the expansion medium at 37 degrees C in an atmosphere comprising about 5% CO2 for about 4 days to form a monolayer of expanded cells.

[0079] The monolayer of expanded cells formed according to step (a) of the provided methods is cultured according to step (b) in differentiation medium to form a monolayer of intestinal epithelial-like cells. In some embodiments, the monolayer of expanded cells is cultured in differentiation medium at 37 degrees C in an atmosphere comprising between 4-10% CO2 (e.g., 5% CO2) for 5 days or more. In some embodiments, the monolayer of expanded cells is cultured in differentiation medium at 37 degrees C in an atmosphere comprising 5%-10% CO2, for 5, 6, 7, 8, 9 or 10 days or more. In some embodiments, the monolayer of expanded cells is cultured in differentiation medium at 37 degrees C in an atmosphere comprising about 5% CO2, for 5-8 or 5-7 days (e.g., 6 days).

[0080] In some embodiments, culturing step (b) comprises culturing the monolayer of expanded cells in in a modified differentiation medium, wherein the modified differentiation medium is differentiation medium that does not contain EGF, for about 1 or about 2 days of the culturing of step (b) (e.g., about 1 or about 2 days of the 5-8 days of culturing according to step (b)).

[0081] In some embodiments, the culturing of step (b) according to the provided methods further comprises culturing the monolayer of cells in differentiation medium that additionally includes vitamin D3 or an analog thereof (e.g., VD3) and / or rifampicin during the last about 1.5 -2.5 days (e.g., about 2 days) of step (b). In some embodiments, the monolayer is cultured in the differentiation medium that additionally includes vitamin D or an analog thereof at 37 degrees C in an atmosphere comprising between 4-10% CO2 (e.g., 5% CO2) for about 1, or about 2 days, to form a treated monolayer of intestinal epithelial-like cells.

[0082] Assessment of Barrier Function In some embodiments, the provided methods further include (d) evaluating a barrier function of the monolayer of cells formed according to step (a) and / or (b) of the provided methods. In the disclosure, the “barrier function” refers to the role of physical barriers formed by intestinal epithelial cells. In intestinal epithelial cells with a barrier function, tight junctions are formed between cells, which close the cell-cell gap and limit the permeation of substances. This barrier function is extremely important for intestinal tissues in preventing unwanted substances and bacteria from entering the body and is also an important indicator in assessing drug absorption. In some embodiments, the barrier function of the monolayer formed in step (a) and / or (b) of the provided methods is evaluated by measurement of a transepithelial electrical resistance (TEER) value, by a technique to evaluate the permeability of Lucifer Yellow, a substance that passes through intercellular spaces, or by a compound permeation test using fasted state simulated intestinal fluid (FaSSIF, artificial bile juice). TEER can be determined using techniques known in the art. In some embodiments, TEER is measured using a Millicell ERS-2 system (Merck) and calculated according to the manufacturer’s instructions. Likewise, resistance of the monolayers formed in step (a) and / or (b) to artificial bile juice can routinely be assessed using techniques known in the art. In some embodiments, the monolayer of intestinal epithelial-like cells formed in step (b) of the provided methods has an increased barrier function compared to the monolayer of expanded cells formed in step (a).

[0083] In some embodiments, the treated monolayer of intestinal epithelial-like cells formed in step (b) that has been cultured in differentiation medium containing vitamin D3 or an analog thereof, and / or rifampicin (e.g., for about 2 days) has an increased barrier function compared to the cells of the monolayer of expanded cells formed in step (a).

[0084] In some embodiments, the monolayer of intestinal epithelial-like cells formed in step (b) of the provided methods has a 10-fold or more, 9-fold or more, 8-fold or more, 7-fold or more, 6-fold or more, 5-fold or more, 4-fold or more, 3-fold or more, 2-fold or more, 1.9-fold or more, 1.8-fold or more, 1.7-fold or more, 1.6-fold or more, or 1.5-fold or more increased transepithelial electrical resistance (TEER) value compared to that of the monolayer of expanded cells formed in step (a).

[0085] In some embodiments, treated monolayer of intestinal epithelial-like cells has a 10-fold or more, 9-fold or more, 8-fold or more, 7-fold or more, 6-fold or more, 5-fold or more, 4-fold or more, 3-fold or more, 2-fold or more, 1.9-fold or more, 1.8-fold or more, 1.7-fold or more, 1.6-fold or more, or 1.5-fold or more increased transepithelial electrical resistance (TEER) value compared to that of the monolayer of expanded cells formed in step (a).

[0086] In some embodiments, the monolayer of intestinal epithelial-like cells formed in step (b) of the provided methods has a transepithelial electrical resistance (TEER) value of 500 omega x cm2or higher, 600 omega x cm2or higher, 700 omega x cm2or higher, 800 omega x cm2or higher, 900 omega x cm2or higher, 1000 omega x cm2or higher, 1100 omega x cm2or higher, 1200 omega x cm2or higher, 1300 omega x cm2or higher, 1400 omega x cm2or higher, 1500 omega x cm2or higher, 1600 omega x cm2or higher, 1700 omega x cm2or higher, 1800 omega x cm2or higher, 1900 omega x cm2or higher, or 2000 omega x cm2or higher.

[0087] In some embodiments, the treated monolayer of intestinal epithelial-like cells has a transepithelial electrical resistance (TEER) value of 500 omega x cm2or higher, 600 omega x cm2or higher, 700 omega x cm2or higher, 800 omega x cm2or higher, 900 omega x cm2or higher, 1000 omega x cm2or higher, 1100 omega x cm2or higher, 1200 omega x cm2or higher, 1300 omega x cm2or higher, 1400 omega x cm2or higher, 1500 omega x cm2or higher, 1600 omega x cm2or higher, 1700 omega x cm2or higher, 1800 omega x cm2or higher, 1900 omega x cm2or higher, or 2000 omega x cm2or higher

[0088] In some embodiments, the monolayer of intestinal epithelial-like cells formed in step (b) of the provided methods has an increased expression of at least one gene selected from the group consisting of ABCG2, CES2, CYP2D6, UGT1A1, and UGT1A10 compared to that of the cells of the monolayer of expanded cells formed in step (a) of the provided methods.

[0089] In some embodiments, the treated monolayer of intestinal epithelial-like cells formed in step (b) of the provided methods has an increased expression of at least one gene selected from the group consisting of ABCG2, CES2, CYP2D6, UGT1A1, and UGT1A10 compared to that of the cells of the monolayer of expanded cells formed in step (a) of the provided methods.

[0090] In some embodiments, the disclosure provides the monolayer of intestinal epithelial-like cells formed according to the provided methods.

[0091] In some embodiments, the disclosure provides the treated monolayer of intestinal epithelial-like cells formed according to the provided methods.Exemplary methods of use

[0092] In some embodiments, the disclosure provides a method of screening an agent for the ability to modify a barrier function of an intestinal epithelial cell, comprising contacting a monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells obtained by the provided methods with a test agent. In some embodiments, the method further comprises evaluating a barrier function of the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells after the monolayer or the treated monolayer is contacted with the test agent. In further embodiments, the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells that is contacted with the test agent is a monolayer of duodenal epithelial-like cells.

[0093] In further embodiments, the disclosure provides a method of selecting a test agent that increases or decreases a barrier function of the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells formed according to the provided methods compared to a control.

[0094] In some embodiments, the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract. In some embodiments, the test agents may be isolated from natural products, chemically or biochemically synthesized, or prepared by genetic engineering.

[0095] In some embodiments, the above test agents may be suitably labeled as needed. Labels include, for example, radioactive labels and fluorescent labels. In some embodiments, mixtures of a plurality of the above test agents can be used.

[0096] In some embodiments, contacting can be performed by adding a test agent to the culture solution of the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells. When the test agent is a protein, for example, a vector containing DNA encoding the protein can be introduced into the cell.

[0097] In some embodiments, the barrier function of the monolayer of intestinal epithelial-like cells or the barrier function of the treated monolayer of intestinal epithelial-like cells is evaluated after the monolayer is contacted by the test agent. In further embodiments, the barrier function of the monolayer contacted by the test agent is evaluated by measurement of a transepithelial electrical resistance (TEER) value, by a technique to evaluate the permeability of Lucifer Yellow, a substance that passes through intercellular spaces, or by a compound permeation test using artificial bile juice.

[0098] In some embodiments, the disclosure provides a method for evaluating pharmacokinetics and / or toxicity of an agent in an intestinal epithelial cell, comprising contacting a monolayer of intestinal epithelial-like cells obtained by the provided methods with a test agent and detecting a change in expression of a protein in a cell or cells of the monolayer of intestinal epithelial-like cells after the cell(s) are contacted with the agent. In further embodiments, the change is detected in the expression of a protein selected from the group consisting of ALPI, ABCB1, ABCC2, ABCG2, SLC15A1, SLCO2B1, CES2, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, UGT1A1, UGT1A10, UGT2B7, and UGT2B17. In some embodiments, the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract. In some embodiments, the test agents may be isolated from natural products, chemically or biochemically synthesized, or prepared by genetic engineering.

[0099] In some embodiments, the above test agents may be suitably labeled as needed. Labels include, for example, radioactive labels and fluorescent labels. In some embodiments, mixtures of a plurality of the above test agents can be used.

[0100] In some embodiments, contacting can be performed by adding a test agent to the culture solution of the monolayer of intestinal epithelial-like cells. When the test agent is a protein, for example, a vector containing DNA encoding the protein can be introduced into the cell.

[0101] In further embodiments, the monolayer of intestinal epithelial-like cells that is contacted with the test agent is a monolayer of duodenal epithelial-like cells.

[0102] In some embodiments, the disclosure provides a method for evaluating pharmacokinetics and / or toxicity of an agent in an intestinal epithelial cell, comprising contacting a treated monolayer of intestinal epithelial-like cells obtained by the provided methods with a test agent and detecting a change in expression of a protein in a cell or cells of the treated monolayer of intestinal epithelial-like cells after the cell(s) are contacted with the agent. In further embodiments, the change is detected in the expression of a protein selected from the group consisting of ALPI, ABCB1, ABCC2, ABCG2, SLC15A1, SLCO2B1, CES2, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, UGT1A1, UGT1A10, UGT2B7, and UGT2B17. In some embodiments, the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract. In some embodiments, the test agents may be isolated from natural products, chemically or biochemically synthesized, or prepared by genetic engineering.

[0103] In some embodiments, the above test agents may be suitably labeled as needed. Labels include, for example, radioactive labels and fluorescent labels. In some embodiments, mixtures of a plurality of the above test agents can be used.

[0104] In some embodiments, contacting can be performed by adding a test agent to the culture solution of the treated monolayer of intestinal epithelial-like cells. When the test agent is a protein, for example, a vector containing DNA encoding the protein can be introduced into the cell.

[0105] In further embodiments, the treated monolayer of intestinal epithelial-like cells that is contacted with the test agent is a treated monolayer of duodenal epithelial-like cells.Kits

[0106] In some embodiments, the disclosure provides a kit for producing an intestinal organoid-derived monolayer, wherein the kit comprises, LPA, a sphingolipid, and albumin. In some embodiments, the kit comprises a solution containing LPA at a total concentration of 2 micro M or higher. In some embodiments, the kit comprises a solution containing LPA at a concentration of about 2-50, 5-40, 5-30, 5-20, or 7-15 micro M. In some embodiments, the kit comprises a solution containing sphingolipid at a concentration of 1 micro M or higher. In some embodiments, the kit comprises sphingosine-1-phosphate (S1P). In some embodiments, the kit comprises a S1P at a concentration of 1 micro M or higher. In some embodiments, the kit comprises recombinant or naturally produced bovine serum albumin or human serum albumin. In some embodiments, the kit comprises a solution containing albumin at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher.

[0107] In some embodiments, the kit comprises a PPAR-delta agonist. In some embodiments, the kit comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. In some embodiments, the kit comprises the PPAR-delta agonist GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid) at a concentration of 1 micro M or higher, or 10 micro M or higher.

[0108] In some embodiments, the kit comprises the GPR40 / FFAR1 agonist. In some embodiments, the kit comprises the GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. In some embodiments, the kit comprises the GPR40 / FFAR1 agonist TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl] methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid) at a concentration of 100 nM or higher, or 1 micro M or higher.

[0109] In some embodiments, the kit further comprises an extracellular matrix. In some embodiments, the kit comprises one or more C-terminal fragments of laminin E8 isoforms. In some embodiments, the extracellular matrix comprises the E8 fragment of laminin-511. In some embodiments, the extracellular matrix further comprises fibronectin. In some embodiments, the extracellular matrix comprises the E8 fragment of laminin-511 and fibronectin. In some embodiments, the extracellular matrix comprises iMatrix-511 and fibronectin.

[0110] In some embodiments, the kit further comprises gastrin or a gastrin analog, EGF, a BMP signaling inhibitor, an ALK5 inhibitor, or a combination thereof.

[0111] In some embodiments, the kit comprises gastrin or a gastrin analog. In some embodiments, the kit comprises recombinant human gastrin or murine gastrin. In some embodiments, the kit comprises [Leu15]-Gastrin I human.

[0112] In some embodiments, the EGF contained in the kit is recombinant human, bovine, or murine EGF.

[0113] In some embodiments, the kit further comprises a BMP signaling inhibitor. In some embodiments, the kit comprises a BMP signaling inhibitor selected from noggin, DAN, and a DAN-like protein such as, cerberus and gremlin. In some embodiments, the BMP signalling inhibitor is recombinant human, bovine, or murine noggin, DAN, and a DAN-like protein such as, cerberus and gremlin.

[0114] In some embodiments, the kit further comprises an ALK5 inhibitor. In some embodiments, the ALK5 inhibitor is a member selected from A83-01, LY-36494, GW788388, RepSox, SB-431542, SB-505124, SB-525334, and SJN-2511.

[0115] In some embodiments, the kit further comprises a Rho-Associated Kinase (ROCK) inhibitor. In some embodiments, the kit comprises a ROCK inhibitor selected from Y-27632, H-1152, SR 3677, thiazovivin, HA1077, HA1100 hydrochloride, and GSK-429286.

[0116] In some embodiments, the kit comprises one or more Wnt signaling agonists. In some embodiments, the kit comprises a Wnt protein selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt16. In some embodiments, the Wnt protein is recombinant human, bovine, or murine Wnt protein. In some embodiments, the kit comprises Wnt3a. In further embodiments, the kit comprises Afamin / Wnt3a CM. In some embodiments, the kit comprises R-spondin selected from R-spondin-1, R-spondin-2, R-spondin-3 and R-spondin-4, or fragments containing furin domains, and / or combinations thereof. In some embodiments, the R-spondin contained in the expansion medium is recombinant human, bovine, or murine R-spondin.

[0117] In some embodiments, the kit further comprises bFGF and / or IGF-I. In some embodiments, the kit comprises recombinant human, bovine or murine bFGF and / or IGF-1.

[0118] In some embodiments, the kit further comprises a fatty acid selected from the group consisting of palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof.

[0119] In some embodiments, the kit further comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof.

[0120] In some embodiments, the kit further comprises vitamin D3 or an analog thereof (e.g., VD3) and / or rifampicin.

[0121] In some embodiments, the kit further comprises a cell derived from an intestine or an intestine-derived organoid. In some embodiments, the cell derived from an intestine or the intestine-derived organoid is derived from a human. In some embodiments, the cell derived from an intestine or the intestine-derived organoid is derived from a small intestine or a large intestine. In some embodiments, the small intestine is a duodenum, a jejunum, or an ileum. In further embodiments, the cell derived from an intestine is a duodenum-derived crypt cell. In further embodiments, the intestine-derived organoid is a duodenal organoid.

[0122] In some embodiments, each component of the kit can be packaged in separate containers. Alternatively, each component of the kit can be mixed and packaged in the same container. In some embodiments, one or more components of the kit can be provided as a solution, e.g., an aqueous solution, or a sterile aqueous solution. Alternatively, the components of the kit may be provided in a dried or lyophilized form. In some embodiments, the kit of the disclosure may also include instructions outlining how to use the kit.

[0123] Additional embodiments provided herein include: [F01] A method for producing an intestinal epithelial-like cell with an increased barrier function, comprising (a) culturing a cell derived from an intestinal organoid in a culture medium comprising at least one of AlbuMAX (registered trademark), albumin, fatty acids, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [F02] The method of [F01], wherein the culture medium comprises AlbuMAX (registered trademark) at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. [F03] The method of [F01] or [F02], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher. [F04] The method of any one of [F01] to [F03], wherein the culture medium comprises fatty acids at a total concentration of 50 micro M or higher. [F05] The method of any one of [F01] to [F04], wherein the fatty acids include at least one selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [F06] The method of any one of [F01] to [F05], wherein the culture medium comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. [F07] The method of any one of [F01] to [F06], wherein the culture medium comprises the PPAR-delta agonist GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [F08] The method of any one of [F01] to [F07], wherein the culture medium comprises the GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. [F09] The method of any one of [F01] to [F08], wherein the culture medium comprises the GPR40 / FFAR1 agonist TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl] methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [F10] The method of any one of [F01] to [F09], wherein the culture medium comprises lipids at a concentration of 1 micro M or higher, or 2 micro M or higher. [F11] The method of any one of [F01] to [F10], wherein the culture medium comprises a lysophospholipid or a sphingolipid. [F12] The method of [F11], wherein the culture medium comprises at least one lysophospholipid selected from: lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG). [F13] The method of any one of [F01] to [F12], wherein the culture medium comprises sphingosine-1-phosphate. [F14] The method of any one of [F01] to [F13], wherein the culture medium comprises LPA, LPC, LPE, LS, or LPG, at a concentration of 2 micro M or higher. [F15] The method of any one of [F01] to [F14], wherein the culture medium comprises a sphingolipid such as sphingosine-1-phosphate, at a concentration of 1 micro M or higher. [F16] The method of any one of [F01] to [F15], wherein the culture medium comprises albumin and lysophosphatidic acid. [F17] The method of [F16], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and lysophosphatidic acid at a concentration of 2 micro M or higher. [F18] The method of any one of [F01] to [F17], wherein the culture medium comprises albumin and sphingosine-1-phosphate. [F19] The method of [F18], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [F20] The method of any one of [F01] to [F19], wherein the culture medium comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [F21] The method of [F20], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, lysophosphatidic acid at a concentration of 2 micro M or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [F22] The method of any one of [F01] to [F21], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [F23] The method of [F22], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [F24] The method of [F23], wherein the intestine-derived cell is derived from a human. [F25] The method of [F23] or [F24], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [F26] The method of [F25], wherein the small intestine is a duodenum, a jejunum, or an ileum. [F27] The method of any one of [F23] to [F26], wherein the intestine-derived cell is a duodenum-derived crypt cell. [F28] The method of any one of [F01] to [F27], wherein the intestinal organoid is a duodenal organoid. [F29] The method of any one of [F01] to [F28], wherein the intestinal epithelial-like cell is a duodenal epithelial-like cell. [F30] The method of any one of [F01] to [F29], wherein after step (a), the method further comprises (b) evaluating a barrier function of the cell. [F31] The method of [F30], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [F32] The method of any one of [F01] to [F31], wherein the culture medium comprises an expansion medium and / or a differentiation medium.

[0124] Additional embodiments provided herein include: [G01] A method for culturing a cell derived from an intestinal organoid, comprising (a) culturing a cell derived from an intestinal organoid in a culture medium comprising at least one of AlbuMAX (registered trademark), albumin, fatty acids, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [G02] The method of [G01], wherein the culture medium comprises AlbuMAX (registered trademark) at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. [G03] The method of [G01] or [G02], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher. [G04] The method of any one of [G01] to [G03], wherein the culture medium comprises fatty acids at a total concentration of 50 micro M or higher. [G05] The method of any one of [G01] to [G04], wherein the fatty acids include at least one selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [G06] The method of any one of [G01] to [G05], wherein the culture medium comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. [G07] The method of any one of [G01] to [G06], wherein the culture medium comprises the PPAR-delta agonist GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [G08] The method of any one of [G01] to [G07], wherein the culture medium comprises a GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. [G09] The method of any one of [G01] to [G08], wherein the culture medium comprises the GPR40 / FFAR1 agonist TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [G10] The method of any one of [G01] to [G09], wherein the culture medium comprises lipids at a concentration of 1 micro M or higher, or 2 micro M or higher. [G11] The method of any one of [G01] to [G10], wherein the culture medium comprises a lysophospholipid or a sphingolipid. [G12] The method of [G11], wherein the culture medium comprises at least one lysophospholipid selected from: LPA, LPC, LPE, LS, and LPG. [G13] The method of any one of [G01] to [G12], wherein the culture medium comprises sphingosine-1-phosphate. [G14] The method of any one of [G01] to [G13], wherein the culture medium comprises LPA, LPC, LPE, LS, or LPG, at a concentration of 2 micro M or higher. [G15] The method of any one of [G01] to [G14], wherein the culture medium comprises a sphingolipid such as sphingosine-1-phosphate, at a concentration of 1 micro M or higher. [G16] The method of any one of [G01] to [G15], wherein the culture medium comprises albumin and lysophosphatidic acid. [G17] The method of [G16], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and lysophosphatidic acid at a concentration of 2 micro M or higher. [G18] The method of any one of [G01] to [G17], wherein the culture medium comprises albumin and sphingosine-1-phosphate. [G19] The method of [G18], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [G20] The method of any one of [G01] to [G19], wherein the culture medium comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [G21] The method of [G20], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, lysophosphatidic acid at a concentration of 2 micro M or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [G22] The method of any one of [G01] to [G21], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [G23] The method of [G22], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [G24] The method of [G23], wherein the intestine-derived cell is derived from a human. [G25] The method of [G23] or [G24], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [G26] The method of [G25], wherein the small intestine is a duodenum, a jejunum, or an ileum. [G27] The method of any one of [G23] to [G26], wherein the intestine-derived cell is a duodenum-derived crypt cell. [G28] The method of any one of [G01] to [G27], wherein the intestinal organoid is a duodenal organoid. [G29] The method of any one of [G01] to [G28], wherein after step (a), the method further comprises (b) evaluating a barrier function of the cell. [G30] The method of [G29], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [G31] The method of any one of [G01] to [G30], wherein the culture medium comprises an expansion medium and / or a differentiation medium.

[0125] Additional embodiments provided herein include: [H01] A method for increasing a barrier function of a cell derived from an intestinal organoid, comprising (a) culturing a cell derived from an intestinal organoid in a culture medium comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-deltaagonist, a GPR40 / FFAR1 agonist, and a lipid. [H02] The method of [H01], wherein the culture medium comprises AlbuMAX (registered trademark) at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher. [H03] The method of [H01] or [H02], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher. [H04] The method of any one of [H01] to [H03], wherein the culture medium comprises a fatty acid at a concentration of 50 micro M or higher. [H05] The method of any one of [H01] to [H04], wherein the fatty acid is selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [H06] The method of any one of [H01] to [H05], wherein the culture medium comprises a PPAR-delta agonist at a concentration of 1 micro M or higher, or 10 micro M or higher. [H07] The method of any one of [H01] to [H06], wherein the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [H08] The method of any one of [H01] to [H07], wherein the culture medium comprises a GPR40 / FFAR1 agonist at a concentration of 100 nM or higher, or 1 micro M or higher. [H09] The method of any one of [H01] to [H08], wherein the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl] phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [H10] The method of any one of [H01] to [H09], wherein the culture medium comprises a lipid at a concentration of 1 micro M or higher, or 2 micro M or higher. [H11] The method of any one of [H01] to [H10], wherein the lipid is a lysophospholipid or a sphingolipid. [H12] The method of [H11], wherein the lysophospholipid is lysophosphatidic acid. [H13] The method of any one of [H01] to [H12], wherein the sphingolipid is sphingosine-1-phosphate. [H14] The method of any one of [H01 to [H13], wherein the culture medium comprises lysophosphatidic acid at a concentration of 2 micro M or higher. [H15] The method of any one of [H01] to [H14], wherein the culture medium comprises sphingosine-1-phosphate at a concentration of 1 micro M or higher. [H16] The method of any one of [H01] to [H15], wherein the culture medium comprises albumin and lysophosphatidic acid. [H17] The method of [H16], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and lysophosphatidic acid at a concentration of 2 micro M or higher. [H18] The method of any one of [H01] to [H17], wherein the culture medium comprises albumin and sphingosine-1-phosphate. [H19] The method of [H18], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [H20] The method of any one of [H01] to [H19], wherein the culture medium comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [H21] The method of [H20], wherein the culture medium comprises albumin at a concentration of 4 mg / mL or higher, lysophosphatidic acid at a concentration of 2 micro M or higher, and sphingosine-1-phosphate at a concentration of 1 micro M or higher. [H22] The method of any one of [H01] to [H21], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [H23] The method of [H22], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [H24] The method of [H23], wherein the intestine-derived cell is derived from a human. [H25] The method of [H23] or [H24], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [H26] The method of [H25], wherein the small intestine is a duodenum, a jejunum, or an ileum. [H27] The method of any one of [H23] to [H26], wherein the intestine-derived cell is a duodenum-derived crypt cell. [H28] The method of any one of [H01] to [H27], wherein the intestinal organoid is a duodenal organoid. [H29] The method of any one of [H01] to [H28], wherein after step (a), the method further comprises (b) evaluating a barrier function of the cell. [H30] The method of [H29], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [H31] The method of any one of [H01] to [H30], wherein the culture medium comprises an expansion medium and / or a differentiation medium.

[0126] Additional embodiments provided herein include: [I01] An intestinal epithelial-like cell with an increased barrier function obtained by the method of any one of [F01] to [F32], [G01] to [G31], and [H01] to [H31]. [I02] The intestinal epithelial-like cell of [I01], which has increased expression of at least one gene selected from the group consisting of ABCG2, CES2, CYP2D6, UGT1A1, and UGT1A10 compared to that prior to culturing in a culture medium comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [I03] The intestinal epithelial-like cell of [I01] or [I02], which has a 10-fold or more, 9-fold or more, 8-fold or more, 7-fold or more, 6-fold or more, 5-fold or more, 4-fold or more, 3-fold or more, 2-fold or more, 1.9-fold or more, 1.8-fold or more, 1.7-fold or more, 1.6-fold or more, or 1.5-fold or more increased transepithelial electrical resistance (TEER) value compared to that prior to culturing in a culture medium comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [I04] The intestinal epithelial-like cell of any one of [I01] to [I03], which has a transepithelial electrical resistance (TEER) value of 500 omega x cm2or higher, 600 omega x cm2or higher, 700 omega x cm2or higher, 800 omega x cm2or higher, 900 omega x cm2or higher, 1000 omega x cm2or higher, 1100 omega x cm2or higher, 1200 omega x cm2or higher, 1300 omega x cm2or higher, 1400 omega x cm2or higher, 1500 omega x cm2or higher, 1600 omega x cm2or higher, 1700 omega x cm2or higher, 1800 omega x cm2or higher, 1900 omega x cm2or higher, or 2000 omega x cm2or higher. [I05] The intestinal epithelial-like cell of any one of [I01] to [I04], which is a duodenal epithelial-like cell.

[0127] Additional embodiments provided herein include: [J01] A method of screening for an agent for modifying a barrier function of an intestinal epithelial cell, comprising contacting the intestinal epithelial-like cell obtained by the method of any one of [F01] to [F32], [G01] to [G31], and [H01] to [H31] with a test agent. [J02] The method of [J01], which further comprises evaluating a barrier function of the intestinal epithelial-like cell. [J03] The method of [J01] or [J02], which further comprises selecting a test agent that increased and / or decreased a barrier function of the intestinal epithelial-like cell compared to a control. [J04] The method of any one of [J01] to [J03], wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice. [J05] The method of any one of [J01] to [J04], wherein the intestinal epithelial-like cell is a duodenal epithelial-like cell. [J06] The method of any one of [J01] to [J05], wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

[0128] Additional embodiments provided herein include: [K01] A method for evaluating pharmacokinetics and / or toxicity of an agent in an intestinal epithelial cell, comprising contacting the intestinal epithelial-like cell obtained by the method of any one of [F01] to [F32], [G01] to [G31], and [H01] to [H31] with a test agent. [K02] The method of [K01], which further comprises detecting a change in expression of a protein in the intestinal epithelial-like cell. [K03] The method of [K02], wherein the protein is selected from the group consisting of ALPI, ABCB1, ABCC2, ABCG2, SLC15A1, SLCO2B1, CES2, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, UGT1A1, UGT1A10, UGT2B7, and UGT2B17. [K04] The method of any one of [K01] to [K03], wherein the intestinal epithelial-like cell is a duodenal epithelial-like cell. [K05] The method of any one of [K01] to [K04], wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

[0129] Additional embodiments provided herein include: [L01] A kit for producing an intestinal epithelial-like cell with an increased barrier function, comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [L02] The kit of [L01], which comprises 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher of AlbuMAX (registered trademark). [L03] The kit of [L01] or [L02], which comprises 4 mg / mL or higher of albumin. [L04] The kit of any one of [L01] to [L03], which comprises 50 micro M or higher of a fatty acid. [L05] The kit of any one of [L01] to [L04], wherein the fatty acid is selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [L06] The kit of any one of [L01] to [L05], which comprises 1 micro M or higher or 10 micro M or higher of a PPAR-delta agonist. [L07] The kit of any one of [L01] to [L06], wherein the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [L08] The kit of any one of [L01] to [L07], which comprises 100 nM or higher or 1 micro M or higher of a GPR40 / FFAR1 agonist. [L09] The kit of any one of [L01] to [L08], wherein the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [L10] The kit of any one of [L01] to [L09], which comprises 1 micro M or higher or 2 micro M or higher of a lipid. [L11] The kit of any one of [L01] to [L10], wherein the lipid is a lysophospholipid or a sphingolipid. [L12] The kit of [L11], wherein the lysophospholipid is lysophosphatidic acid. [L13] The kit of [L11] or [L12], wherein the sphingolipid is sphingosine-1-phosphate. [L14] The kit of [L12] or [L13], which comprises 2 micro M or higher of lysophosphatidic acid. [L15] The kit of [L13] or [L14], which comprises 1 micro M or higher of sphingosine-1-phosphate. [L16] The kit of any one of [L12] to [L15], which comprises albumin and lysophosphatidic acid. [L17] The kit of [L16], which comprises 4 mg / mL or higher of albumin, and 2 micro M or higher of lysophosphatidic acid. [L18] The kit of any one of [L13] to [L17], which comprises albumin and sphingosine-1-phosphate. [L19] The kit of [L18], which comprises 4 mg / mL or higher of albumin, and 1 micro M or higher of sphingosine-1-phosphate. [L20] The kit of any one of [L13] to [L19], which comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [L21] The kit of [L20], which comprises 4 mg / mL or higher of albumin, 2 micro M or higher of lysophosphatidic acid, and 1 micro M or higher or sphingosine-1-phosphate. [L22] The kit of any one of [L01] to [L21], which further comprises a cell derived from an intestinal organoid. [L23] The kit of any one of [L01] to [L22], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [L24] The kit of [L23], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [L25] The kit of [L24], wherein the intestine-derived cell is derived from a human. [L26] The kit of [L24] or [L25], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [L27] The kit of [L26], wherein the small intestine is a duodenum, a jejunum, or an ileum. [L28] The kit of any one of [L24] to [L27], wherein the intestine-derived cell is a duodenum-derived crypt cell. [L29] The kit of any one of [L22] to [L28], wherein the intestinal organoid is a duodenal organoid. [L30] The kit of any one of [L01] to [L29], wherein the intestinal epithelial-like cell is a duodenal epithelial-like cell. [L31] The kit of any one of [L01] to [L30], which further comprises a culture medium. [L32] The kit of [L31], wherein the culture medium comprises an expansion medium and / or a differentiation medium.

[0130] Additional embodiments provided herein include: [M01] A kit for culturing a cell derived from an intestinal organoid, comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [M02] The kit of [M01], which comprises 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher of AlbuMAX (registered trademark). [M03] The kit of [M01] or [M02], which comprises 4 mg / mL or higher of albumin. [M04] The kit of any one of [M01] to [M03], which comprises 50 micro M or higher of a fatty acid. [M05] The kit of any one of [M01] to [M04], wherein the fatty acid is selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [M06] The kit of any one of [M01] to [M05], which comprises 1 micro M or higher or 10 micro M or higher of a PPAR-delta agonist. [M07] The kit of any one of [M01] to [M06], wherein the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [M08] The kit of any one of [M01] to [M07], which comprises 100 nM or higher or 1 micro M or higher of a GPR40 / FFAR1 agonist. [M09] The kit of any one of [M01] to [M08], wherein the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [M10] The kit of any one of [M01] to [M09], which comprises 1 micro M or higher or 2 micro M or higher of a lipid. [M11] The kit of any one of [M01] to [M10], wherein the lipid is a lysophospholipid or a sphingolipid. [M12] The kit of [M11], wherein the lysophospholipid is lysophosphatidic acid. [M13] The kit of [M11] or [M12], wherein the sphingolipid is sphingosine-1-phosphate. [M14] The kit of [M12] or [M13], which comprises 2 micro M or higher of lysophosphatidic acid. [M15] The kit of [M13] or [M14], which comprises 1 micro M or higher of sphingosine-1-phosphate. [M16] The kit of any one of [M12] to [M15], which comprises albumin and lysophosphatidic acid. [M17] The kit of [M16], which comprises 4 mg / mL or higher of albumin, and 2 micro M or higher of lysophosphatidic acid. [M18] The kit of any one of [M13] to [M17], which comprises albumin and sphingosine-1-phosphate. [M19] The kit of [M18], which comprises 4 mg / mL or higher of albumin, and 1 micro M or higher of sphingosine-1-phosphate. [M20] The kit of any one of [M13] to [M19], which comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [M21] The kit of [M20], which comprises 4 mg / mL or higher of albumin, 2 micro M or higher of lysophosphatidic acid, and 1 micro M or higher or sphingosine-1-phosphate. [M22] The kit of any one of [M01] to [M21], which further comprises a cell derived from an intestinal organoid. [M23] The kit of any one of [M01] to [M22], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [M24] The kit of [M23], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [M25] The kit of [M24], wherein the intestine-derived cell is derived from a human. [M26] The kit of [M24] or [M25], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [M27] The kit of [M26], wherein the small intestine is a duodenum, a jejunum, or an ileum. [M28] The kit of any one of [M24] to [M27], wherein the intestine-derived cell is a duodenum-derived crypt cell. [M29] The kit of any one of [M22] to [M28], wherein the intestinal organoid is a duodenal organoid. [M30] The kit of any one of [M01] to [M29], which further comprises a culture medium. [M31] The kit of [M30], wherein the culture medium comprises an expansion medium and / or a differentiation medium.

[0131] Additional embodiments provided herein include: [N01] A kit for increasing a barrier function of a cell derived from an intestinal organoid, comprising at least one of AlbuMAX (registered trademark), albumin, a fatty acid, a PPAR-delta agonist, a GPR40 / FFAR1 agonist, and a lipid. [N02] The kit of [N01], which comprises 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher of AlbuMAX (registered trademark). [N03] The kit of [N01] or [N02], which comprises 4 mg / mL or higher of albumin. [N04] The kit of any one of [N01] to [N03], which comprises 50 micro M or higher of a fatty acid. [N05] The kit of any one of [N01] to [N04], wherein the fatty acid is selected from the group consisting of palmitoleic acid, stearic acid, oleic acid, linolic acid, and alpha-linolenic acid. [N06] The kit of any one of [N01] to [N05], which comprises 1 micro M or higher or 10 micro M or higher of a PPAR-delta agonist. [N07] The kit of any one of [N01] to [N06], wherein the PPAR-delta agonist is GW501516 (endurobol, 2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid). [N08] The kit of any one of [N01] to [N07], which comprises 100 nM or higher or 1 micro M or higher of a GPR40 / FFAR1 agonist. [N09] The kit of any one of [N01] to [N08], wherein the GPR40 / FFAR1 agonist is TAK-875 (fasiglifam, 2-[(3S)-6-[[3-[2,6-dimethyl-4-(3-methylsulfonylpropoxy)phenyl]phenyl]methoxy]-2,3-dihydro-1-benzofuran-3-yl]acetic acid). [N10] The kit of any one of [N01] to [N09], which comprises 1 micro M or higher or 2 micro M or higher of a lipid. [N11] The kit of any one of [N01] to [N10], wherein the lipid is a lysophospholipid or a sphingolipid. [N12] The kit of [N11], wherein the lysophospholipid is lysophosphatidic acid. [N13] The kit of [N11] or [N12], wherein the sphingolipid is sphingosine-1-phosphate. [N14] The kit of [N12] or [N13], which comprises 2 micro M or higher of lysophosphatidic acid. [N15] The kit of [N13] or [N14], which comprises 1 micro M or higher of sphingosine-1-phosphate. [N16] The kit of any one of [N12] to [N15], which comprises albumin and lysophosphatidic acid. [N17] The kit of [N16], which comprises 4 mg / mL or higher of albumin, and 2 micro M or higher of lysophosphatidic acid. [N18] The kit of any one of [N13] to [N17], which comprises albumin and sphingosine-1-phosphate. [N19] The kit of [N18], which comprises 4 mg / mL or higher of albumin, and 1 micro M or higher of sphingosine-1-phosphate. [N20] The kit of any one of [N13] to [N19], which comprises albumin, lysophosphatidic acid, and sphingosine-1-phosphate. [N21] The kit of [N20], which comprises 4 mg / mL or higher of albumin, 2 micro M or higher of lysophosphatidic acid, and 1 micro M or higher or sphingosine-1-phosphate. [N22] The kit of any one of [N01] to [N21], which further comprises a cell derived from an intestinal organoid. [N23] The kit of any one of [N01] to [N22], wherein the cell derived from the intestinal organoid is in the form of a monolayer membrane. [N24] The kit of [N23], wherein the monolayer membrane is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; (ii) separating the intestinal organoid into single cells; and (iii) culturing the single cell. [N25] The kit of [N24], wherein the intestine-derived cell is derived from a human. [N26] The kit of [N24] or [N25], wherein the intestine-derived cell is derived from a small intestine or a large intestine. [N27] The kit of [N26], wherein the small intestine is a duodenum, a jejunum, or an ileum. [N28] The kit of any one of [N24] to [N27], wherein the intestine-derived cell is a duodenum-derived crypt cell. [N29] The kit of any one of [N22] to [N28], wherein the intestinal organoid is a duodenal organoid. [N30] The kit of any one of [N01] to [N29], which further comprises a culture medium. [N31] The kit of [N30], wherein the culture medium comprises an expansion medium and / or a differentiation medium. All prior art references cited herein are incorporated by reference into the present specification.

[0132] The provided methods will be explained in further detail below by way of examples, but these examples do not limit the scope of the invention.

[0133] [Example 1] Development of a monolayer model using intestinal organoids Based on a previous report, crypts were isolated from the adjacent normal duodenum of surgical specimens of cancer, and duodenal organoids were established (Gastroenterology (2011) 141, 1762). Specifically, fragmented crypts were embedded in Matrigel (Corning), and cultured in organoid establishment medium (Advanced DMEM / F-12, Antibiotic-Antimycotic (Thermo Fisher Scientific), Primocin (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 2 mM GlutaMAX-I (Thermo Fisher Scientific), 1 x B-27 Supplement (Thermo Fisher Scientific), 2 x N-2 Supplement (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine, 10 nM Gastrin (Tocris), 10 mM Nicotinamide (Sigma), 10% Afamin / Wnt3a CM (JSR) or 50 ng / mL recombinant human Wnt3a (R&D systems), 1 mg / mL recombinant human R-spondin-1 (Peprotech), 100 ng / mL animal-free recombinant human EGF (Peprotech), 100 ng / mL recombinant human noggin (Peprotech), 500 nM A83-01 (Tocris), 10 mM SB202190 (Sigma), 1 mM Prostaglandin E2 (Sigma), 100 ng / mL recombinant human FGF-10 (Peprotech), 1 ng / mL recombinant human FGF-basic (Peprotech), and 10.5 mM Y-27632 (Sigma)) to establish organoids. The established organoids were cultured and passaged using IntestiCultTMOrganoid Growth Medium (Human) (STEMCELL Technologies). Passage was performed once a week. Specifically, Gentle Cell Dissociation Reagent (STEMCELL Technologies) was added to disrupt the Matrigel dome, and cells were collected and then incubated for 10 min. After centrifugation, the cells were suspended in ice-cold DMEM with 1% BSA, and pipetted repeatedly for fragmentation. The organoid fragments were diluted at an appropriate ratio and embedded in Matrigel. After a culture medium was added, the fragments were cultured at 37°C.

[0134] Next, the intestinal organoids were used to develop a monolayer model using Transwell (registered trademark). First, a Transwell (registered trademark) 0.4 micro m pore polyester membrane insert (Corning, 3470 and 3378) was coated by adding a 19:1 mixture of iMatrix-511 silk (Nippi) and Fibronectin bovine plasma (F1141: Sigma) and incubating at 37°C for 2 hours. Intestinal organoids were collected in the same manner as described above, fragmented by pipetting, and separated into single cells by treatment with TrypLE select (10x) (Thermo Fisher Scientific) diluted 4-fold with PBS. The cells were seeded onto Transwell (registered trademark) at 105cells / well. The cells were cultured for 4 days in Expansion Medium (Advanced DMEM / F-12, 10mM HEPES, 2 mM GlutaMAX-I, Penicillin-Streptomycin (Thermo Fisher Scientific), 1 x B-27 Supplement, 1 mM N-acetylcysteine, 10 nM [Leu15]-Gastrin I human (G9145; Sigma), 20% Afamin / Wnt3a CM, 50 ng / mL mouse EGF recombinant protein, 100 ng / mL recombinant murine noggin, 1 mg / mL recombinant human R-Spondin-1, 500 nM A83-01, 100 mg / mL recombinant human IGF-I (590906; BioLegend), 50 mg / mL human heat stable bFGF recombinant protein (PHG0369; Thermo Fisher Scientific), 10 mM Y-27632). The medium was then changed to Differentiation Medium (Advanced DMEM / F-12, 10mM HEPES, 2 mM GlutaMAX-I, Penicillin-Streptomycin, 1 x B-27 Supplement, 1 mM N-acetylcysteine, 10 nM [Leu15]-Gastrin I human, 100 ng / mL recombinant murine noggin, and 500 nM A83-01, 50 ng / mL mouse EGF recombinant protein), and the cells were further cultured for 6 days.

[0135] [Example 2] Increase of the barrier function of the monolayer model by addition of AlbuMAX(registered trademark) Seven intestinal organoid lines from different donors were compared in terms of proliferation properties on Transwell (registered trademark). Following 4-day culture in Expansion Medium after seeding, viable cells on Transwell (registered trademark) were stained with MTT (23547-76; Nacalai Tesque). As a result, cell confluency was found to vary among the organoid lines (FIG. 1). To explore culture conditions to allow stable proliferation on Transwell (registered trademark), a series of studies were carried out on culture medium additives. As a result, addition of AlbuMAX (registered trademark) I Lipid-Rich BSA (11020-039; Thermo Fisher Scientific), which is lipid rich albumin, to Expansion Medium at 4 mg / mL was found to accelerate monolayer formation on Transwell (registered trademark) for all organoids (FIG. 1). To evaluate the proliferation properties of each cell population, EdU incorporation was assayed using Click-iTTMPlus EdU cell proliferation kit (C10638; Thermo Fisher Scientific). As shown in FIG. 2, EdU-positive cells were increased by addition of AlbuMAX (registered trademark), indicating that cell proliferation became active.

[0136] To evaluate the barrier function of the cells on Transwell (registered trademark), transepithelial electrical resistance (TEER) was measured using Millicell ERS-2 system (Merck). As shown in FIG. 3, the barrier function was found to be increased by addition of AlbuMAX (registered trademark). Next, a lucifer yellow permeation experiment was performed to see whether the developed monolayer structure can be maintained in FaSSIF (FFF02; Biorelevant), a simulated intestinal fluid of the living body. Specifically, 200 mM lucifer yellow (L0144; Sigma) was added, and 2 hours later, the fluorescence in the lower compartment of Transwell (registered trademark) was measured to evaluate the permeation of lucifer yellow. The result showed that, while the cells from donors 2 and 4 did not form a barrier function in the absence of AlbuMAX (registered trademark), the cells from all donors exhibited a robust barrier function in the presence of AlbuMAX (registered trademark) (FIG. 4). Next, gene expression was evaluated in the cells cultured in Differentiation Medium for 6 days. Specifically, RNA was extracted using TRIzol (Thermo Fisher Scientific) and purified using RNeasy mini kit (74104; QIAGEN). cDNA was synthesized using Superscript III First-strand synthesis system for RT-PCR (18080-051; Thermo Fisher Scientific), and quantitative PCR was performed using TaqManTMfast advanced master mix for qPCR (4444557; Thermo Fisher Scientific). The TaqMan probes used are shown in Table 1. The results of analysis revealed that the expression of the drug transporter ABCG2 and the drug metabolizing enzymes CES2, CYP2D6, UGT1A1, and UGT1A10 was significantly increased under the AlbdeffuMAX (registered trademark)-added conditions (FIG. 5).

[0137]

[0138] [Example 3] Mechanism Analysis of the effect of addition of AlbuMAX(registered trademark) Evaluation of the dose-response of AlbuMAX (registered trademark) addition showed that the barrier function improved even at 1 mg / mL, and became higher at 4 mg / mL (FIG. 6).

[0139] Since AlbuMAX (registered trademark) is lipid rich albumin, an analysis was performed to investigate which component has an improving effect on the barrier function. Addition of 4 mg / mL BSA (A7030; Sigma) also increased the barrier function, but not as much as AlbuMAX (registered trademark) (FIG. 7). Next, various fatty acids were added. As a result, a range of fatty acids showed a barrier function-improving effect on their own (FIG. 8). A similar effect was observed with GW501516, which is an agonist for PPAR-delta, an intranuclear receptor for ligands such as unsaturated fatty acids (FIG. 9). Improvement of the barrier function was also observed with addition of TAK-875, which is an agonist for GPR40 / FFAR1, a fatty acid signal receptor (FIG. 10). Similar barrier function improvement was also observed when a lysophospholipid (oleoyl-L-alpha-lysophosphatidic acid; LPA) or sphingosine 1-phosphate (S1P), a sphingolipid, was added (FIG. 11).

[0140] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0141] The provided methods have been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0142] The foregoing description of the specific embodiments will so fully reveal the general nature of the provided methods that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the provided methods. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0143] The embodiments of the methods and compositions provided above are described in detail with reference to actual examples and illustrated examples with the aim of helping clear understanding. However, the description and illustration in the present specification should not be interpreted as limiting the scope of the present disclosure. The disclosure of all patent literatures and scientific literatures cited herein is explicitly incorporated herein by reference in its entirety. In particular, the disclosure of Tanaka et al., “A robust and reproducible human intestinal organoid derived monolayer model for analyzing drug absorption” Science Advances (38 pages) is incorporated herein by reference in its entirety.

[0144] The method provided in the disclosure is useful for producing intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function in high yield. In particular, the intestinal epithelial-like cells (intestinal epithelial-like monolayers) with an increased barrier function obtained in this disclosure have superior tight junction function and greatly increased barrier function compared to intestinal epithelial-like cells obtained by conventional methods. Such intestinal epithelial-like cells (intestinal epithelial-like monolayers) are useful for efficient evaluation of drug kinetics and / or toxicity in the intestinal tract. The intestinal epithelial-like cells (intestinal epithelial-like monolayers) obtained by the provided method are also useful in the evaluation of absorption, metabolism, and excretion of drug candidate compounds in the intestinal tract.

Claims

1. A method for preparing an intestinal organoid-derived monolayer, comprising, (a) culturing a cell derived from an intestinal organoid on an extracellular matrix in a cell culture expansion medium that comprises lysophosphatidic acid (LPA), sphingolipid, albumin, a BMP signaling inhibitor, an ALK5 inhibitor, a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, gastrin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF / FGF2), and IGF-I (insulin-like growth factor), to form a monolayer of expanded cells; (b) culturing the monolayer of expanded cells in a differentiation medium comprising LPA, sphingolipid, albumin, gastrin, a BMP signaling inhibitor, an ALK5 inhibitor, and EGF to form a monolayer of intestinal epithelial-like cells; optionally, (c) culturing the monolayer of intestinal epithelial-like cells in a medium containing the differentiation medium and vitamin D3 or an analog thereof (e.g., 1alpha, 25-dihydroxy-vitamin D3 (VD3)) and / or rifampicin, to form a treated monolayer of intestinal epithelial-like cells.

2. The method of claim 1, wherein (b) further comprises culturing the monolayer up to 1 or 2 days in the differentiation medium without EGF.

3. The method of claim 1 or 2, wherein the extracellular matrix comprises one or more C-terminal fragments of laminin E8 isoforms (e.g., iMatrix-511) and fibronectin.

4. The method of any one of claims 1-3, wherein the culturing of (a), (b), and / or (c) is conducted in a Transwell (registered trademark).

5. The method of any one of claims 1-4, wherein the expansion medium and / or differentiation medium comprises LPA at a total concentration of 2 micro M or higher.

6. The method of any one of claims 1-5, wherein the expansion medium and / or differentiation medium comprises a member selected from palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof.

7. The method of any one of claims 1-6, wherein the expansion medium and / or differentiation medium comprises sphingolipid at a concentration of 1 micro M or higher, or from about 1-5 micro M.

8. The method of any one of claims 1-7, wherein the expansion medium and / or differentiation medium comprises sphingosine-1-phosphate (S1P), optionally at a concentration of 1 micro M or higher.

9. The method of any one of claims 1-8, wherein the albumin in the expansion medium and / or differentiation medium is bovine serum albumin or human serum albumin isolated from biological serum, or recombinant bovine serum albumin or human serum albumin.

10. The method of any one of claims 1-9, wherein the expansion medium and / or differentiation medium comprises albumin at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher, or from about 1 mg / mL to about 10 mg / mL.

11. The method of any one of claims 1-10, wherein the expansion medium and / or differentiation medium comprises albumin at a concentration of 4 mg / mL or higher, lysophosphatidic acid at a concentration of 2 micro M or higher, and sphingosine-1-phosphate at a concentration of 10 nM or higher.

12. The method of any one of claims 1-11, wherein the expansion medium and / or differentiation medium further comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof.

13. The method of any one of claims 1-12, wherein the expansion medium and / or differentiation medium comprises LPC, LPE, LS, and / or LPG, at a concentration of 2 micro M or higher.

14. The method of any one of claims 1-13, wherein the expansion medium and / or differentiation medium comprises a BMP signaling inhibitor selected from noggin, DAN, and a DAN-like protein such as, cerberus and gremlin, or a combination thereof.

15. The method of any one of claims 1-14, wherein the expansion medium and / or differentiation medium comprises a BMP signaling inhibitor at a concentration of about 10 to about 200 ng / ml.

16. The method of any one of claims 1-15, wherein the expansion medium and / or differentiation medium comprises noggin, optionally, at a concentration of about 10-200 ng / ml (e.g., 100 ng / ml).

17. The method of any one of claims 1-16, wherein the ALK5 inhibitor is A83-01.

18. The method of any one of claims 1-17, wherein the expansion medium and / or differentiation medium comprises an ALK5 inhibitor at a concentration of about 0.2-2 micro M.

19. The method of any one of claims 1-18, wherein the expansion medium and / or differentiation medium comprises recombinant human, bovine, or murine EGF.

20. The method of any one of claims 1-19, wherein the expansion medium and / or differentiation medium comprises EGF at a concentration of about 10-100 ng / ml (e.g., about 40-60 ng / ml or 50 ng / ml).

21. The method of any one of claims 1-20, wherein the expansion medium and / or differentiation medium comprises gastrin (e.g., [Leu15]-Gastrin I human) at a concentration of about 5-15 nM.

22. The method of any one of claims 1-21, wherein the expansion medium comprises the ROCK inhibitor Y-27632.

23. The method of any one of claims 1-22, wherein the expansion medium comprises a ROCK inhibitor at a concentration of about 5-100 micro M.

24. The method of any one of claims 1-23, wherein the expansion medium comprises a Wnt pathway signaling agonist at a concentration of about 25 ng / ml to about 150 ng / ml.

25. The method of any one of claims 1-24, wherein the expansion medium comprises a Wnt protein (e.g., Wnt3a).

26. The method of any one of claims 1- 25, wherein the expansion medium comprises R-spondin (e.g., R-spondin-1, R-spondin-2, R-spondin-3 or R-spondin-4), optionally at a concentration of 0.5-1.5 micro g / ml.

27. The method of any one of claims 1-26, wherein Wnt pathway signaling agonist is Afamin / Wnt3a CM and / or R-spondin-1.

28. The method of any one of claims 1-27, wherein the expansion medium comprises recombinant human, bovine or murine bFGF (FGF2).

29. The method of any one of claims 1-28, wherein the expansion medium comprises bFGF at a concentration of about 25-500 ng / ml (e.g., 50 ng / ml).

30. The method of any one of claims 1-29, wherein the expansion medium comprises recombinant human, bovine, or murine IGF-1.

31. The method of any one of claims 1-30, wherein the expansion medium comprises IGF-1 at a concentration of about 50-500 ng / ml (e.g., 100 ng / ml).

32. The method of any one of claims 1-31, wherein the differentiation medium does not contain Wnt protein (e.g., Wnt3a), R-spondin (e.g., R-spondin1), nicotinamide, SB202190; IGF-1 and / or FGF2.

33. The method of any one of claims 1-32, wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is obtained by a method comprising: (i) culturing an intestine-derived cell to obtain an intestinal organoid; and (ii) separating the intestinal organoid into single cells.

34. The method of claim 33, wherein the intestinal organoid is a duodenal organoid.

35. The method of any one of claims 1- 34, wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is derived from a human.

36. The method of any one of claims 1-35, wherein the cultured cell (the cell derived from an intestinal organoid) of (a) is derived from a small intestine or a large intestine.

37. The method of claim 36, wherein the small intestine is a duodenum, a jejunum, or an ileum.

38. The method of anyone of claims 33-37, wherein the intestine-derived cell is a duodenum-derived crypt cell.

39. The method of any one of claims 1- 38, wherein the culturing of (a) takes place over about 3-5 days (e.g., about 4 days) at 37 degrees C and 5% CO2and the culturing of (b) takes place over about 5-7 days (e.g., about 6 days) at 37 degrees C and 5% CO2.

40. The method of any one of claims 1- 39, wherein up to about 1 day or about 2 days of the culturing of (b) is performed in differentiation medium that does not contain EGF.

41. The method of claim 1 or 40, wherein VD3 and / or rifampicin is added to the differentiation medium during the last about 1.5 -2.5 days (e.g., about 2 days) of the culturing of (b).

42. The method of any one of claims 1-41, which further comprises (d) evaluating a barrier function of the cell.

43. The method of claim 42, wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice.

44. The method of any one of claims 1-43, wherein the monolayer of intestinal epithelial-like cells formed in (b) has an increased barrier function compared to the monolayer of expanded cells formed in (a).

45. The method of any one of claims 1-44, wherein cells of the monolayer of intestinal epithelial-like cells formed in (b) have an increased expression of at least one gene selected from the group consisting of ABCG2, CES2, CYP2D6, UGT1A1, and UGT1A10 compared to cells of the monolayer of expanded cells formed in (a).

46. The method of any one of claims 1-45, wherein the monolayer of intestinal epithelial-like cells formed in (b) has a 10-fold or more, 9-fold or more, 8-fold or more, 7-fold or more, 6-fold or more, 5-fold or more, 4-fold or more, 3-fold or more, 2-fold or more, 1.9-fold or more, 1.8-fold or more, 1.7-fold or more, 1.6-fold or more, or 1.5-fold or more increased transepithelial electrical resistance (TEER) value compared to that the monolayer of expanded cells formed in (a).

47. The method of any one of claims 1-46, wherein the monolayer of intestinal epithelial-like cells formed in (b) has a transepithelial electrical resistance (TEER) value of 500 omega x cm2or higher, 600 omega x cm2or higher, 700 omega x cm2or higher, 800 omega x cm2or higher, 900 omega x cm2or higher, 1000 omega x cm2or higher, 1100 omega x cm2or higher, 1200 omega x cm2or higher, 1300 omega x cm2or higher, 1400 omega x cm2or higher, 1500 omega x cm2or higher, 1600 omega x cm2or higher, 1700 omega x cm2or higher, 1800 omega x cm2or higher, 1900 omega x cm2or higher, or 2000 omega x cm2or higher.

48. The method of any one of claims 1-47, wherein the monolayer of intestinal epithelial-like cells formed in (b) is a monolayer of duodenal epithelial-like cells.

49. The monolayer of intestinal epithelial-like cells formed according to the method of any one of claims 1- 48.

50. The treated monolayer of intestinal epithelial-like cells formed according to the method of any one of claims 1- 48.

51. A method of screening an agent for the ability to modify a barrier function of an intestinal epithelial cell, comprising contacting the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells obtained by the method of any one of claims 1- 48, the monolayer of claim 49, or the treated monolayer of claim 50 with a test agent.

52. The method of claim 51, which further comprises evaluating a barrier function of the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells after the monolayer is contacted with the test agent.

53. The method of claim 51 or 52, which further comprises selecting a test agent that increases or decreases a barrier function of the monolayer of intestinal epithelial-like cells compared to a control.

54. The method of any one of claims 51-53, wherein the barrier function is evaluated by measurement of a transepithelial electrical resistance (TEER) value or by a compound permeation test using artificial bile juice.

55. The method of any one of claims 51-54, wherein the monolayer of intestinal epithelial-like cells is a monolayer of duodenal epithelial-like cells.

56. The method of any one of claims 51-55, wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

57. A method for evaluating pharmacokinetics and / or toxicity of an agent in an intestinal epithelial cell, comprising contacting the monolayer of intestinal epithelial-like cells or the treated monolayer of intestinal epithelial-like cells obtained by the method of any one of claims 1- 48, the monolayer of claim 49, or the treated monolayer of claim 50 with a test agent.

58. The method of claim 57, which further comprises detecting a change in expression of a protein in cells of the monolayer of intestinal epithelial-like cell or the treated monolayer of intestinal epithelial-like cells after the cells are contacted with the test agent.

59. The method of claim 58, wherein a change is detected in the expression of a protein selected from the group consisting of ALPI, ABCB1, ABCC2, ABCG2, SLC15A1, SLCO2B1, CES2, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, UGT1A1, UGT1A10, UGT2B7, and UGT2B17.

60. The method of any one of claims 57-59, wherein the monolayer of intestinal epithelial-like cells is a monolayer of duodenal epithelial-like cells.

61. The method of any one of claims 57-60, wherein the test agent is selected from the group consisting of a natural compound, an organic compound, an inorganic compound, a protein, an antibody, a peptide, an amino acid, a nucleic acid, a compound library, an expression product of a gene library, a cell extract, a cell culture supernatant, a fermentative microbial product, a marine organism extract, a plant extract, a prokaryotic cell extract, an eukaryotic single cell extract, and an animal cell extract.

62. A kit for producing an intestinal organoid-derived monolayer, comprising, lysophosphatidic acid (LPA), a sphingolipid, and albumin.

63. The kit of claim 62, which further comprises an extracellular matrix.

64. The kit of claim 63, wherein the extracellular matrix comprises C-terminal fragments of laminin E8 isoforms (e.g., iMatrix-511) and fibronectin.

65. The kit of any one of claims 62-64, which further comprises gastrin, EGF, a BMP signaling inhibitor, an ALK5 inhibitor, or a combination thereof.

66. The kit of any one of claims 62-65, which further comprises a Rho-Associated Kinase (ROCK) inhibitor, one or more Wnt pathway signaling agonists, or a combination thereof.

67. The kit of any one of claims 62-66, which further comprises bFGF and / or IGF-I.

68. The kit of any one of claims 62-67, wherein the kit comprises a member selected from palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and alpha-linolenic acid, or a combination thereof.

69. The kit of any one of claims 62-68, which comprises a solution containing LPA at a total concentration of 2 micro M or higher.

70. The kit of any one of claims 62-69, which comprises sphingosine-1-phosphate (S1P).

71. The kit of any one of claims 62-70, which comprises a solution containing sphingolipid at a concentration of 1 micro M or higher.

72. The kit of any one of claims 62-71, which comprises bovine serum albumin or human serum albumin isolated from biological serum, or recombinant bovine serum albumin or human serum albumin.

73. The kit of any one of claims 62-72, which comprises a solution containing albumin at a concentration of 1 mg / mL or higher, 2 mg / mL or higher, 3 mg / mL or higher, or 4 mg / mL or higher.

74. The kit of any one of claims 62-73, which comprises a solution containing lysophosphatidic acid at a concentration of 2 micro M or higher, sphingolipid at a concentration of 1 micro M or higher and albumin at a concentration of 4 mg / mL or higher.

75. The kit of any one of claims 62-74, which comprises a lysophospholipid selected from lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LS), lysophosphatidylinositol (LPI), and lysophosphatidylglycerol (LPG), or a combination thereof.

76. The kit of any one of claims 62-75, which contains a solution comprising LPC, LPE, LS, and / or LPG, at a concentration of 2 micro M or higher.

77. The kit of any one of claims 62-76, which comprises a BMP signaling inhibitor selected from noggin, DAN, and a DAN-like protein such as, cerberus and gremlin, or a combination thereof.

78. The kit of any one of claims 62-77, which contains a solution comprising a BMP signaling inhibitor at a concentration of about 10 to about 200 ng / ml.

79. The kit of any one of claims 62-78, which comprises noggin.

80. The kit of any one of claims 62-79, which comprises the ALK5 inhibitor A83-01.

81. The kit of any one of claims 62-80, which comprises ALK5 inhibitor at a concentration of about 0.2-2 micro M.

82. The kit of any one of claims 62-81, which comprises recombinant human, bovine, or murine EGF.

83. The kit of any one of claims 62-82, which contains a solution comprising EGF at a concentration of about 10-100 ng / ml.

84. The kit of any one of claims 62-83, which comprises the ROCK inhibitor Y-27632.

85. The kit of any one of claims 62-84, which contains a solution comprising a ROCK inhibitor at a concentration of at a concentration of about 5-100 micro M.

86. The kit of any one of claims 62-85, which contains a solution comprising a Wnt pathway signaling agonist that is a Wnt protein selected from Wnt3a, Wnt5, and Wnt6a.

87. The kit of any one of claims 62-86, which comprises Wnt pathway signaling agonist R-spondin (e.g., R-spondin-1, R-spondin-2, R-spondin-3, R-spondin-4, or a combination thereof).

88. The kit of any one of claims 62-87, which comprises the Wnt pathway signaling agonist Afamin / Wnt3a CM and / or R-spondin-1.

89. The kit of any one of claims 62-88, which comprises recombinant human, bovine, or murine bFGF (FGF2).

90. The kit of any one of clams 62-89, which contains a solution comprising bFGF at a concentration of about 25-250 ng / ml.

91. The kit of any one of claims 62-90, which comprises recombinant human, bovine, or murine IGF-1.

92. The kit of any one of claims 62-91, which contains a solution comprising IGF-1 at a concentration of at a concentration of about 50-250 ng / ml.

93. The kit of any one of claims 62-82, which further comprises vitamin D3 or an analog thereof (e.g.,VD3) and / or rifampicin.

94. The kit of any one of claims 62-93, which further comprises a cell derived from an intestine or an intestine-derived organoid.

95. The kit of claim 94, wherein the cell derived from an intestine or intestine-derived organoid is derived from a human.

96. The kit of claim 94 or 95, wherein the cell derived from an intestine or intestine-derived organoid is derived from a small intestine or a large intestine.

97. The kit of claim 96, wherein the small intestine is a duodenum, a jejunum, or an ileum.

98. The kit of any one of claims 94-97, wherein the cell derived from an intestine is a duodenum-derived crypt cell.

99. The kit of any one of claims 94-98, wherein the intestine-derived organoid is a duodenal organoid.

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