Method for producing cells having high intestinal epithelial cell-like function and morphology similar to small intestine from intestinal cancer-derived cells, and use thereof

Abstract

The purpose of the present invention is to provide a method for producing intestinal epithelial cells having functions close to those of a living organism from cells derived from intestinal cancer. Another purpose of the present invention is to provide a method for inducing differentiation from intestinal cancer-derived cells into intestinal epithelial cells having functions close to those of a living organism. Also provided is a culture medium that can be used in these methods.　Provided are: a method for producing intestinal epithelial cells, the method comprising air-liquid interface culture of cells derived from intestinal cancer in the presence of at least three or more selected from the group consisting of epidermal growth factors, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators, where the expression level of the CES1 gene is lower and the expression level of the CES2 gene is higher than when culture is performed in the presence of the at least three or more without performing air-liquid interface culture; and the use of the method.

Description

Method for producing cells with high intestinal epithelial cell-like function and morphology from intestinal cancer-derived cells, and use thereof

[0001] The present invention relates to a method for producing cells having high intestinal epithelial cell-like function and morphology, similar to small intestine cells, from intestinal cancer-derived cells, and to uses thereof.

[0002] The intestinal tract plays a variety of roles in the body, including food absorption, drug absorption, metabolism, and excretion, defense against pathogens, and immunity. Furthermore, the intestinal tract has an irregular structure composed of multiple cell types, and the rapid differentiation and maturation of intestinal epithelial stem cells, as well as the maintenance and proliferation of these cells, are associated with various diseases. For these reasons, the intestinal tract is deeply connected to individual health, disease, and drug discovery, making it important to evaluate the intestinal tract in medical and pharmaceutical research.

[0003] Clinical trials and the use of human samples would be ideal for assessing the intestinal tract, but ethical and supply issues remain. While in vivo experiments using laboratory animals such as monkeys and mice are also an option, species differences mean that the results cannot be directly applied to humans. Furthermore, social trends call for alternatives to animal testing from the perspective of animal protection. For these reasons, research and development is underway to establish a human intestinal assessment system that allows for appropriate in vitro evaluation.

[0004] One example of a human in vitro intestinal evaluation system is a three-dimensional tissue structure (intestinal organoid) that mimics intestinal tissue. Intestinal organoids are known to be produced by a specific differentiation induction method. For example, Patent Document 1 describes a method for producing intestinal organoids from pluripotent stem cells, including: (1) differentiating pluripotent stem cells into endoderm-like cells; (2) differentiating the endoderm-like cells obtained in step (1) into intestinal epithelial stem cell-like cells; (3) culturing the intestinal epithelial stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor, and ROCK inhibitor; (4) culturing the cells after step (3) to form spheroids; and (5) differentiating the spheroids formed in step (4) to form intestinal organoids, which includes culturing in the presence of epidermal growth factor, BMP inhibitor, and Wnt signal activator.

[0005] Furthermore, it is known that by utilizing the high proliferation ability of the intestinal epithelial stem cells contained in intestinal organoids, it is possible to culture intestinal cells with a crypt-villi-like uneven structure. Patent Document 2 describes a method for producing intestinal cells from pluripotent stem cells, including: (1) differentiating pluripotent stem cells into endoderm-like cells; (2) differentiating the endoderm-like cells obtained in step (1) into intestinal epithelial stem cell-like cells; (3) culturing the intestinal epithelial stem cell-like cells obtained in step (2) in the presence of epidermal growth factor, fibroblast growth factor, TGFβ receptor inhibitor, GSK-3β inhibitor, and ROCK inhibitor; (4) culturing the cells obtained in step (3) to form spheroids; (5) differentiating the spheroids formed in step (4) to form intestinal organoids, which includes culturing in the presence of epidermal growth factor, BMP inhibitor, and Wnt signal activator; and (6) culturing the cells constituting the intestinal organoids formed in step (5) in the presence of epidermal growth factor, cAMP signal activator, TGFβ receptor inhibitor, and Wnt signal activator in a gas-liquid phase.

[0006] As a method for obtaining an in vitro evaluation system, Patent Document 3 describes a method comprising: culturing stem cells capable of differentiating into mucus-producing cells on the upper surface of a cell support structure having both an upper surface and a lower surface until at least a portion of the upper surface of the cell support structure is covered by the stem cells; and further culturing the stem cells to produce a cell monolayer comprising mucus-producing cells, the cell monolayer having a basal side and a luminal side, wherein the mucus-producing cells of the cell monolayer establish a mucus layer on the luminal side of the cell monolayer, thereby producing a live cell construct comprising a cell monolayer comprising mucus-producing cells and a mucus layer.

[0007] Patent Literature 4 describes a method for producing small intestinal epithelial-like cells, including (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D; and (ii) obtaining small intestinal epithelial-like cells. It is also described that this method may further include differentiating pluripotent stem cells into endoderm cells and differentiating the endoderm cells into small intestinal epithelial progenitor cells. Non-Patent Literature 1 describes the formation and proliferation of epithelial organoids from mouse colon and human small intestine and colon. Patent Literature 5 describes a bioprinted artificial three-dimensional in vivo intestinal tissue model, including (i) a layer of intestinal stromal tissue containing myofibroblasts for forming an artificial three-dimensional in vivo intestinal tissue model, and (ii) a layer of intestinal epithelial cells on the layer of intestinal stromal tissue. Patent Document 6 describes a method for producing a product resulting from the metabolism and / or modification of a substance, comprising placing a sac-shaped cell structure containing small intestinal epithelial cells and having a villous layer on its outer surface, and a liquid containing the substance so that the liquid contacts the villous layer, and allowing the product to accumulate inside the cell structure. Patent Document 7 describes a cell culture medium for organoid culture, characterized by containing at least two selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), and epiregulin (EREG), and at least one of the following components i) to iii): i) Wnt agonists, ii) bone morphogenetic protein (BMP) inhibitors, and iii) transforming growth factor-β (TGF-β) inhibitors.

[0008] International Publication WO2020 / 091020 International Publication WO2022 / 168908 International Publication WO2020 / 102682 International Publication WO2020 / 262492 International Publication WO2018 / 089743 JP 2020-162434 International Publication WO2017 / 199811

[0009] Toshiro Sato et al., “Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium” Gastroenterology. 2011 Nov;141(5):1762-72. doi: 10.1053 / j.gastro.2011.07.050.

[0010] However, in human in vitro intestinal evaluation systems that use pluripotent stem cells such as iPS cells, various problems can arise, such as difficulty in stabilizing quality due to differences in differentiation ability depending on the pluripotent stem cell strain; specialized techniques are required to culture iPS cells and intestinal organoids, making cell properties unstable between researchers and facilities; many factors are required for maintenance culture and differentiation induction, resulting in high costs; and the supply of intestinal cells derived from the same lot of iPS cells is limited, making it difficult to establish a global standard.

[0011] The present invention aims to provide a method for producing intestinal epithelial cells having a three-dimensional structure and function similar to that of an in vivo cell from intestinal cancer-derived cells without using pluripotent stem cells. Alternatively, the present invention aims to provide a method for inducing differentiation of intestinal cancer-derived cells into intestinal epithelial cells having a three-dimensional structure and function similar to that of an in vivo cell. Alternatively, the present invention aims to provide a culture medium that can be used in these methods.

[0012] That is, the present invention relates to the following: [1] A method for producing intestinal epithelial cells from intestinal cancer-derived cells, comprising: culturing the intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more members selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator, wherein the expression level of the CES1 gene in the intestinal epithelial cells is lower and the expression level of the CES2 gene is higher than when the intestinal epithelial cells are cultured in the presence of the at least three or more members selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator without air-liquid interface culture. [2] A method for inducing differentiation of intestinal cancer-derived cells into intestinal epithelial cells, comprising: culturing the intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more members selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator, wherein the expression level of the CES1 gene in the intestinal epithelial cells is lower and the expression level of the CES2 gene is higher than when the intestinal epithelial cells are cultured in the presence of the at least three or more members without air-liquid interface culture. [3] A cell culture method comprising air-liquid interface culturing of intestinal cancer-derived cells in the presence of at least three or more selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. [4] The method according to any one of [1] to [3], in which the intestinal epithelial cells are capable of forming villus structures, crypt structures, or both. [5] The production method according to any one of [1] to [4], in which the intestinal epithelial cells are capable of forming a cell layer with a lower transepithelial electrical resistance than when cultured in the absence of at least three or more selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. [6] The production method according to [5], in which air-liquid interface culturing of intestinal cancer-derived cells in the presence of a ROCK inhibitor in addition to epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator.[7] The method according to any one of [1] to [6], wherein the intestinal cancer-derived cells are cultured at the air-liquid interface in the presence of at least three or more agents selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator for one day or longer. [8] The method according to any one of [1] to [7], comprising culturing the intestinal cancer-derived cells at the air-liquid interface in the presence of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. [9] The method according to any one of [1] to [8], wherein the intestinal cancer-derived cells are selected from Caco-2 cells, HT-29 cells, LS-174T cells, T84 cells, SW-480 cells, HCT-116 cells, DLD-1 cells, and LoVo cells.

[10] The method according to any one of [1] to [9], wherein the TGFβ receptor inhibitor is A-83-01, the Wnt signal enhancer is CHIR99021, and the cAMP signal activator is forskolin.

[11] The method according to any one of [1] to

[10] , comprising culturing intestinal cancer-derived cells on a gelatin-coated culture surface.

[12] The method according to any one of [1] to

[11] , comprising culturing intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator, as well as one or more selected from the group consisting of a gamma-secretase inhibitor, a B27 supplement, and an N2 supplement.

[13] A culture medium for use in the method according to any one of [1] to

[12] , comprising at least three or more selected from the group consisting of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator.

[14] A kit comprising the medium according to

[13] and cells derived from intestinal cancer.

[0013] The present invention also relates to the following:

[15] Intestinal epithelial cells obtained by the production method according to any one of [1] to

[14] .

[16] The cells according to

[15] , which are capable of forming a cell layer having a lower transepithelial electrical resistance value than when cultured in the absence of at least three or more elements selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator.

[17] A method for evaluating the pharmacokinetics or toxicity of a test substance, using the intestinal epithelial cells according to

[15] or

[16] .

[18] The method according to

[17] , wherein the pharmacokinetics is the absorbability or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, or induction of drug transporters of the test substance.

[19] The method according to

[17] or

[18] , comprising the following steps (i) to (iii): (i) preparing a cell layer composed of intestinal epithelial cells obtained by the production method according to claim 1 in [1] or a cell layer composed of intestinal epithelial cells according to

[15] or

[16] ; (ii) contacting the cell layer with a test substance; (iii) quantifying the test substance that has permeated the cell layer, and evaluating the absorbability or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[20] The method according to

[17] or

[18] , comprising the following steps (I) and (II): (I) contacting a test substance with a cell layer composed of intestinal epithelial cells obtained by the production method according to claim 1 or the intestinal epithelial cells according to

[15] or

[16] ; (II) measuring and evaluating the metabolism or absorption, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[0014] According to one aspect of the present invention, intestinal epithelial cells having specific functions similar to those in vivo can be obtained from intestinal cancer-derived cells. According to another aspect, differentiation of intestinal cancer-derived cells into intestinal epithelial cells having specific functions similar to those in vivo can be promoted.

[0015] ALI (air-liquid interface culture) and M5 medium influence the three-dimensional structure and mRNA expression of Caco-2 cells. A) Micrographs of Caco-2 cells cultured on cell culture inserts on days 3 and 15 are shown. The Caco-2 medium group was cultured without differentiation inducers, while the M5 medium group was cultured with all differentiation inducers. B) Transepithelial electrical resistance (TEER) values ​​were measured immediately before medium changes from day 3 to day 15, the end of culture. C) Gene expression levels of cells harvested on day 15 were assessed by real-time RT qPCR. Values ​​are shown relative to Caco-2 cells cultured in LL (liquid phase) culture. (Mean + SD n = 3) Effects of differentiation inducers on Caco-2 cells during ALI culture. A, B) Caco-2 cells were cultured on cell culture inserts for 22 days, and ALI was performed from day 3 until the end of culture. TEER values ​​were measured from day 6 onward, immediately before medium changes (n = 3). C, D) Micrographs taken at the end of 22 days of culture. Dead cells accumulated within the insert were washed with PBS before imaging. The effect of differentiation-inducing factors on gene expression in Caco-2 cells during ALI culture. Caco-2 cells were seeded onto cell culture inserts and cultured for 22 days. ALI was performed from day 3 until the end of culture. After washing to remove dead cells, all live cells were collected and subjected to real-time RT qPCR. Caco-2 cells in the graph represent cells cultured in Caco-2 medium in LL, and ASI represents human small intestinal tissue. All values ​​are relative to Caco-2 cells. Hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control for normalization. (n = 3) The effect of medium and air-liquid interface culture on barrier function. Lucifer yellow permeability assays were performed to assess intestinal barrier function. After the culture was completed, the cells were washed, Lucifer yellow was added to the insert, and the cells were incubated at 37°C for 30 minutes. After that, the entire volume of the buffer solution under the insert was collected, and the fluorescence intensity was measured using a multiplate reader. The transmittance coefficient of Lucifer yellow (P app) was calculated. n = 3. Changes in the three-dimensional structure and gene expression of Caco-2 cells under the new culture conditions. A) Bright-field images taken by optical microscope on days 11 and 21 after the start of culture. Dead cells accumulated within the insert were washed with PBS before imaging. B) Cross-sectional images of cells stained with HE staining on day 21 after the start of culture. C) Caco-2 cells were seeded on cell culture inserts and cultured for 21 days. Dead cells were removed by washing with PBS, and all cells were harvested and subjected to RT-qPCR. ASI indicates adult small intestine tissue (ASI). All mRNA levels in each sample are relative to ASI. Hypoxanthine phosphoribosyltransferase (HPRT) was used as an endogenous control for normalization. (n = 1 or 3) Comparison of Caco-2 cell function between conventional and new groups. A) TEER was measured immediately before medium change from day 11 to day 21, the end of culture. (Mean ± SD n = 8) B) LY permeation test was performed on day 21 of culture. (Mean ± SD n = 1 or 3) C) Immunofluorescence staining results on day 21 of culture. D) Activity evaluation of drug-metabolizing enzyme CYP3A4 / 5. Midazolam was used as the substrate drug, and ketoconazole was used as the inhibitor (inh) (Mean ± SD n = 3 or 4).

[0016] The present invention is described below. The features of the present invention described below can be combined in any combination. In addition, when "including" or "having" is used below, it means that other features may be included, and each of these features can be freely replaced with "consisting of" or "consisting only of," etc. "consisting of" or "consisting only of" means that no other unspecified features are included. In addition, when "in the present invention" is used below, it does not refer to a specific embodiment unless otherwise specified.

[0017] [Production Method of the Present Invention] In one aspect, the present invention relates to a method for producing intestinal epithelial cells with specific functions from intestinal cancer-derived cells (hereinafter, sometimes referred to as the production method of the present invention). The production method of the present invention comprises culturing intestinal cancer-derived cells at an air-liquid interface in the presence of specific factors. According to the production method of the present invention, intestinal epithelial cells with specific functions similar to those in vivo can be produced from intestinal cancer-derived cells.

[0018] <Cells derived from intestinal cancer> Cells derived from intestinal cancer that can be used in the production method of the present invention are typically established cell lines derived from intestinal cancer. In one embodiment, the cells derived from intestinal cancer are established cell lines derived from colorectal cancer. In another embodiment, the cells derived from intestinal cancer are selected from established cell lines derived from colon cancer and established cell lines derived from rectal cancer. In another embodiment, the cells derived from intestinal cancer are established cell lines derived from intestinal adenocarcinoma (intestinal adenocarcinoma).

[0019] In a preferred embodiment, the intestinal cancer-derived cells are selected from Caco-2 cells, HT-29 cells, LS-174T cells, T84 cells, SW-480 cells, HCT-116 cells, DLD-1 cells, and LoVo cells. In a more preferred embodiment, the intestinal cancer-derived cells are selected from Caco-2 cells and HT-29 cells. In one embodiment, the intestinal cancer-derived cells are Caco-2 cells. The intestinal cancer-derived cells may be obtained by any method known to those skilled in the art. Caco-2 cells can be obtained, for example, from the RIKEN BioResource Center (cell number: RCB0988). HT-29 cells, LS-174T cells, HCT-116 cells, and LoVo cells can be obtained, for example, from the American Type Culture Collection (ATCC) (ATCC numbers: ATCC HTB-38 (HT-29 cells), ATCC CL-188 (LS-174T cells), ATCC CCL-247 (HCT-116 cells), and ATCC CCL-229 (LoVo cells)). T84 cells and SW-480 cells can be obtained, for example, from the European Collection of Cell Cultures (ECACC) (ECACC strain numbers: 88021101 (T84 cells) and 87092801 (SW-480 cells)). DLD-1 cells can be obtained, for example, from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health, and Nutrition (cell number: JCRB9094).

[0020] <Factors> The production method of the present invention involves culturing intestinal cancer-derived cells in the presence of a predetermined factor. "In the presence of a predetermined factor" is synonymous with a culture system containing a predetermined factor. In the present invention, a culture system refers to a system that may include a culture medium, a culture medium, etc. The culture medium may be one commonly used in the art, such as Dulbecco's modified Eagle's medium (DMEM). In one embodiment, culturing in the presence of a predetermined factor refers to culturing in a medium to which a predetermined factor has been added.

[0021] In one embodiment, the production method of the present invention comprises culturing intestinal cancer-derived cells in the presence of at least three or more selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. In another embodiment, the production method of the present invention comprises culturing intestinal cancer-derived cells in the presence of at least three or more selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator, as well as a γ-secretase inhibitor. In another embodiment, the production method of the present invention comprises culturing intestinal cancer-derived cells in the presence of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. In another embodiment, the production method of the present invention comprises culturing intestinal cancer-derived cells in the presence of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, cAMP signal activator, and γ-secretase inhibitor.

[0022] In some embodiments, the production methods of the present invention comprise culturing intestinal cancer-derived cells in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, and a ROCK inhibitor. In some embodiments, the production methods of the present invention comprise culturing intestinal cancer-derived cells in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, a ROCK inhibitor, and a γ-secretase inhibitor. In some embodiments, the production methods of the present invention comprise culturing intestinal cancer-derived cells in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, and a ROCK inhibitor, followed by culturing in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a cAMP signal activator, and a ROCK inhibitor. In one embodiment, the method comprises culturing cells derived from intestinal cancer in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, a ROCK inhibitor, and a γ-secretase inhibitor, followed by culturing in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a cAMP signal activator, a ROCK inhibitor, and a γ-secretase inhibitor.

[0023] (Epidermal Growth Factor) Epidermal growth factor (EGF, sometimes referred to as epidermal growth factor or epidermal cell proliferation factor) is a polypeptide that acts as a ligand for EGF receptors expressed in epithelial cells, promoting the proliferation of epithelial cells. In the present invention, EGF preferably has a human amino acid sequence, but is not particularly limited thereto. EGF may be produced from human or animal cells by genetic recombination technology, or may be a peptide compound. Peptide compounds are chemically synthesized peptide compounds that may have activity equivalent to or greater than that of biologically derived peptides. Peptide compounds are also sometimes referred to as alternative peptide compounds. Some peptide compounds will be developed in the future, and it is naturally expected that they will be used by those skilled in the art.

[0024] The concentration of EGF added to the culture system may be, for example, 1 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, more preferably 30 ng / mL to 500 ng / mL, and even more preferably 50 ng / mL to 150 ng / mL. Note that, when the concentration of each factor is mentioned in the present invention, it means that the concentration condition needs to be met at a certain point during the culture period, but does not mean that the concentration condition needs to be met throughout the culture period (the same applies hereinafter).

[0025] (TGFβ Receptor Inhibitors) TGFβ (transforming growth factor β) receptor inhibitors are not particularly limited as long as they are substances (typically compounds) having TGFβ receptor inhibitory activity, but are preferably substances that exhibit inhibitory activity against one or more of the TGFβ receptors ALK4, ALK5, and ALK7. TGFβ receptor inhibitory activity can be assessed by methods known to those skilled in the art. In the case of ALK5, for example, the method may involve confirming whether the inhibitor has activity to inhibit the action of TGFβ using a commercially available kit containing a TGFβR1 peptide substrate (e.g., TGFβR1 Kinase Enzyme System, Promega). Examples of TGFβ receptor inhibitors include A-83-01, SB431542, SB-505124, SB525334, D4476, ALK5 inhibitor, LY2157299, LY364947, GW788388, RepSox, etc. The TGFβ receptor inhibitor is preferably A-83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide). Alternatively, a peptide compound having TGFβ inhibitory activity may be used instead of the TGFβ receptor inhibitor. The peptide compound having TGFβ inhibitory activity may be a TGFβ inhibitory peptide, i.e., one that binds to TGFβ and inhibits the binding of TGFβ to the TGFβ receptor, or a TGFβ receptor inhibitory peptide, i.e., one that binds to the TGFβ receptor and inhibits the binding of TGFβ to the TGFβ receptor. Examples of TGFβ inhibitory peptides include PG-002 (PeptiGrowth), a TGFβ1 inhibitory peptide.

[0026] The concentration of the TGFβ receptor inhibitor added to the culture system (in the case of A-83-01) may be, for example, 0.005 μM to 50 μM, preferably 0.05 μM to 5 μM, and more preferably 0.1 μM to 1 μM. When using a compound other than A-83-01, the concentration can be determined by one skilled in the art based on the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and A-83-01. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments similar to those described in the Examples below.

[0027] (Wnt Signal Enhancement Factor) The Wnt signal enhancement factor can be any substance (typically a compound) that enhances Wnt signaling in cells. Wnt signal enhancement activity can be evaluated by methods known to those skilled in the art, such as detecting a signal resulting from enhanced Wnt signaling by a reporter assay using a commercially available kit (e.g., Leading Light (registered trademark) Wnt Reporter Assay Starter Kit, Cosmo Bio). The Wnt signal enhancement factor can be, for example, one or more selected from a GSK-3 inhibitor, a Wnt signal activator, and a Wnt agonist, and is preferably a GSK-3 inhibitor.

[0028] Any substance (typically a compound) having GSK (glycogen synthase kinase)-3 (preferably GSK-3β) inhibitory activity can be used as the GSK-3 inhibitor. It is known that inhibition of GSK-3 in cells enhances Wnt signaling. GSK-3 inhibitory activity can be assessed by methods known to those skilled in the art, such as by confirming the inhibitory activity of GSK-3 using a commercially available kit (e.g., AlphaScreen SureFire phospho-GSK3 assay kit, PerkinElmer). The preferred GSK-3 inhibitor is CHIR99021 (6-{2-{2-[4-(2,4-dichloro-phenyl)-5-(5-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino]-ethylamino}-nicotinonitrile).

[0029] The concentration of the GSK-3 inhibitor (in the case of CHIR99021) added to the culture system may be, for example, 0.03 μM to 300 μM, preferably 0.3 μM to 30 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 μM to 5 μM. When using a GSK-3 inhibitor other than CHIR99021, the concentration added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the GSK-3 inhibitor used and CHIR99021. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0030] Any substance (typically a compound) that activates Wnt signaling in cells can be used as the Wnt signaling activator. Wnt signaling activation can be evaluated by methods known to those skilled in the art, and the methods may be similar to the methods for evaluating Wnt signaling enhancement activity described above. Examples of Wnt signaling activators include R-spondin 1.

[0031] The concentration of the Wnt signaling activator (in the case of R-spondin 1) added to the culture system may be, for example, 10 ng / mL to 1000 ng / mL, preferably 50 ng / mL to 500 ng / mL. When using a Wnt signaling activator other than R-spondin 1, the concentration to be added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the Wnt signaling activator used and R-spondin 1. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0032] Any substance (typically a compound) that binds to a Wnt receptor (such as Frizzled, LRP6, Ror1, Ror2, or Ryk) and enhances intracellular Wnt signaling can be used as a Wnt agonist. The activity of a Wnt agonist can be assessed by methods known to those skilled in the art, such as detecting signals resulting from enhanced Wnt signaling by a reporter assay using a commercially available kit (e.g., Leading Light® Wnt Reporter Assay Starter Kit, Cosmo Bio). Examples of Wnt agonists include Wnt3a, Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, and Wnt16.

[0033] The concentration of the Wnt agonist (in the case of Wnt3a) added to the culture system may be, for example, 100 ng / mL to 1000 ng / mL, preferably 250 ng / mL to 500 ng / mL. When using a Wnt agonist other than Wnt3a, the concentration to be added to the culture system can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties (particularly activity) between the Wnt agonist and Wnt3a used. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0034] (cAMP Signal Activator) The cAMP signal activator may be one or more selected from the group consisting of cAMP derivatives, cAMP degrading enzyme inhibitors and cAMP activators, and is preferably a cAMP activator.

[0035] As cAMP derivatives, for example, PKA activators (such as 8-Br-cAMP (8-Bromoadenosine-3',5'-cyclic monophosphate sodium salt, CAS Number: 76939-46-3), 6-Bnz-cAMP (N6-Benzoyladenosine-3',5'-cyclic monophosphate sodium salt, CAS Number: 1135306-29-4), cAMPS-Rp ((R)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number: 151837-09-1), cAMPS-Sp ((S)-Adenosine, cyclic 3',5'-(hydrogenphosphorothioate)triethylammonium salt, CAS Number: 93602-66-5), Dibutyryl-cAMP (N6,O2'-Dibutyryl adenosine 3',5'-cyclic monophosphate sodium salt, CAS Number: 16980-89-5), 8-Cl-cAMP (8-Chloroadenosine- 3', 5'- cyclic monophosphate salt, CAS Number: 124705-03-9)), Epac activators (Rp-8-Br-cAMPS (8-Bromoadenosine 3',5'-cyclic Monophosphothioate, Rp-Isomer. sodium salt, CAS Number: 129735-00-8), 8-CPT-cAMP (8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate, CAS Number: 93882-12-3), 8-pCPT-2'-O-Me-cAMP (8-(4-Chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium, CASNumber: 634207-53-7), etc.) can be used.

[0036] An example of the concentration of a cAMP derivative added to a culture system (in the case of 8-Br-cAMP) is 0.01 mM to 30 mM, preferably 0.05 mM to 10 mM, and more preferably 0.1 mM to 5 mM. When using an exemplified compound, i.e., a compound other than 8-Br-cAMP, the concentration can be determined by one skilled in the art within the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and the properties of the exemplified compound (8-Br-cAMP). Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0037] cAMP degrading enzyme inhibitors include IBMX (3-isobutyl-1-methylxanthine) (MIX), Theophylline, Papaverine, Pentoxifylline (Trental), KS-505 , 8-Methoxymethyl-IBMX, Vinpocetine (TCV-3B), EHNA, Trequinsin (HL-725), Lixazinone (RS-82856), (LY-186126), Cilostamide (OPC3689), Bemoradan (RWJ-22867), Anergrelide (BL4162A), Indolidan (LY195115), Cilostazol (OPC-13013), Milrinone (WIN47203), Siguazodan (SKF-94836), 5-Methyl-imazodan (CI 930), SKF-95654, Pirilobendan (UD-CG 115BS), Enoximone (MDL 17043), Imazodan (CL 914), SKF-94120, Vesnarinone (OPC 8212), Rolipram (Ro-20-1724), (ZK-62711), Denbufyll'ine, Zaprinast (M&B-22, 948), Dipyridamole, Zaprinast (M&B-22, 948), Dipyridamole, Zardaverine, AH-21-132, Sulmazol (AR-L 115 BS), and the like.

[0038] An example of the concentration of a cAMP inhibitor added to a culture system (in the case of IBMX) is 0.01 mM to 10 mM, preferably 0.05 mM to 5 mM, and more preferably 0.1 mM to 1 mM. When using an exemplified compound, i.e., a compound different from IBMX, the concentration can be determined by one skilled in the art within the above concentration range, taking into consideration the differences (particularly differences in activity) between the properties of the compound used and the properties of the exemplified compound (IBMX). Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0039] Examples of cAMP activators that can be used include forskolin, indomethacin, NKH477 (colforsin daropate), cell-derived toxin proteins (pertussis toxin, cholera toxin), PACAP-27, PACAP-38, and SKF83822.

[0040] An example of the concentration of a cAMP activator added to a culture system (in the case of forskolin) is 0.1 μM to 1 M, preferably 1 μM to 100 μM, more preferably 3 μM to 50 μM, and even more preferably 5 μM to 15 μM. When using an exemplified compound, i.e., a compound other than forskolin, the concentration can be determined by a person skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of the exemplified compound (forskolin) (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments similar to those described in the Examples below.

[0041] (γ-Secretase Inhibitor) Any substance (typically a compound) having γ-secretase inhibitory activity can be used as the γ-secretase inhibitor. γ-Secretase inhibitory activity can be evaluated by methods known to those skilled in the art. For example, the method may involve using a commercially available kit (e.g., γ-Secretase Activity Kit, R&D Systems) to confirm whether the substance has the activity of inhibiting γ-secretase activity. The γ-secretase inhibitor may be, for example, DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester) (hereinafter sometimes referred to as DAPT), dibenzazepine, etc., and preferably DAPT.

[0042] The concentration of a gamma-secretase inhibitor (in the case of DAPT) added to a culture system is, for example, 0.03 μM to 300 μM, preferably 0.3 μM to 30 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 μM to 5 μM. When using a gamma-secretase inhibitor other than DAPT, the concentration added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) of the gamma-secretase inhibitor used and DAPT. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0043] (ROCK Inhibitor) Any substance that inhibits Rho kinase (Rho-associated protein kinase) can be used as a ROCK (Rho kinase) inhibitor. ROCK inhibitory activity can be assessed by methods known to those skilled in the art. For example, a commercially available kit (e.g., ROCK Activity Assay Kit, Cell Biolabs) may be used to confirm whether a substance has the activity to inhibit ROCK activity. The preferred ROCK inhibitor is Y27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide·2HCl).

[0044] The concentration of a ROCK inhibitor (in the case of Y27632) added to a culture system is, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 1 to 20 μM. When using a ROCK inhibitor other than Y27632, the concentration of the inhibitor added to the culture system can be determined by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the ROCK inhibitor used and Y27632. Furthermore, whether the determined concentration range is appropriate can be confirmed by preliminary experiments.

[0045] (B27 Supplement) The production method of the present invention may also include culturing intestinal cancer-derived cells in the presence of a B27 supplement in addition to the aforementioned factors. B27 supplements are available from Life Technologies and other companies and contain vitamins (biotin, DL-α-tocopherol acetate, DL-α-tocopherol, vitamin A), proteins (bovine serum albumin fraction V fatty acid-free, catalase, human insulin, transferrin, superoxide dismutase), and other components (corticosterone, D-galactose, ethanolamine hydrochloride, glutathione, L-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine dihydrochloride, sodium selenite, and triiodo-L-thyronine). The B27 supplement may be added to the culture system at a concentration of, for example, 0.01-30%, preferably 0.1-20%, more preferably 0.5-10%, and even more preferably 1-5%.

[0046] (N2 Supplement) The production method of the present invention may include culturing intestinal cancer-derived cells in the presence of an N2 supplement in addition to the above-mentioned factors. B27 supplement is available from Life Technologies, Inc. and contains proteins (human transferrin, insulin) and other components (progesterone, putrescine, selenite). The concentration of B27 supplement added to the culture system may be, for example, 0.01 to 20%, preferably 0.05 to 10%, and more preferably 0.1 to 5%.

[0047] (Other Factors) The production method of the present invention may include culturing intestinal cancer-derived cells in the presence of other factors suitable for the production method of the present invention, in addition to the above-mentioned factors. The other factors may be, for example, serum, amino acids, antibiotics, etc. The serum may be, for example, mammalian serum, preferably fetal bovine serum (FBS). Alternatively, serum substitutes (e.g., Knockout Serum Replacement (KSR)) can be used instead of serum. The serum or serum substitute may be added to the culture system at a concentration of, for example, 0.1-20%, preferably 0.5-15%. The amino acids are preferably essential amino acids such as L-glutamine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, and L-serine, or L-alanyl-L-glutamine. The amino acid may be added to the culture system at a concentration of, for example, 0.1-10%, preferably 0.5-5%. The antibiotic may be, for example, penicillin, streptomycin, ampicillin, puromycin, gentamicin, etc., and is preferably penicillin or streptomycin. The concentration of penicillin added to the culture system may be, for example, 10 to 500 units / mL, and preferably 50 to 150 units / mL. The concentration of streptomycin added to the culture system may be, for example, 10 to 500 μg / mL, and preferably 50 to 150 μg / mL.

[0048] <Air-Liquid Interface Culture> "Air-Liquid Interface Culture" (ALI; also referred to as air-liquid interface culture or air-liquid culture) refers to culture in which the entire cell surface (excluding the portion in contact with or adhered to the culture surface, the portion in contact with or adhered to other cells, etc.) is not in contact with the medium (liquid phase), but rather a portion of the cell surface is in contact with the gas phase. As long as the culture is performed in this state, the culture form, such as the culture vessel used, is not particularly limited. ALI can be performed by any method, for example, by removing the liquid medium from the top surface of the cultured cells. In the present invention, when referring to a state in contact with the gas phase, it is possible to ignore a state in which the liquid medium remains between the cell surface and the gas phase after removal of the liquid medium, or a state in which the medium permeates from the liquid phase to the gas phase, i.e., a state in which an unintended small amount of liquid medium is present between the cell surface and the gas phase.

[0049] A concept opposite to ALI is "liquid culture" (LL: Liquid-Liquid Culture, also known as liquid-liquid culture or liquid phase culture). LL refers to culturing cells in a liquid culture system without contact with gas. An example of an LL method is a method in which cells are seeded in a culture vessel equipped with a cell-adhesive culture surface and then cultured in a liquid medium after adhesion of the cells. However, this method is not particularly limited as long as it is a method in which cells are cultured in a liquid culture system without contact with gas. The liquid medium may be any suitable liquid medium for the production method of the present invention, such as DMEM.

[0050] The production method of the present invention may include liquid culture of cells derived from intestinal cancer. In one embodiment, the production method of the present invention comprises liquid culture of cells derived from intestinal cancer, followed by air-liquid interface culture.

[0051] <Culture Surface> In the production method of the present invention, the culture surface is not particularly limited, and for example, a culture vessel equipped with a cell-adhesive culture surface can be used. The cell-adhesive culture surface may be a culture surface coated by a method commonly used in the art, such as a culture surface coated with 0.001 to 10% (w / v) gelatin. Alternatively, the cell-adhesive culture surface may be coated with a basement membrane component (e.g., laminin, type IV collagen, entactin, heparan sulfate proteoglycan, vitronectin, fibronectin) or a fragment thereof. A specific example of a basement membrane component is laminin-511 or its E8 fragment. Regarding the E8 fragment of laminin-511, a highly purified recombinant form (recombinant human laminin-511-E8 protein) is commercially available (product name: iMatrix-511, manufactured by Nippi Corporation and sold by Matrixome Co., Ltd.).

[0052] As described above, the production method of the present invention allows for the culture of intestinal cancer-derived cells on a culture surface coated with gelatin. On the other hand, methods using pluripotent stem cells often require the use of a culture surface coated with a basement membrane component. Therefore, the production method of the present invention may be superior to methods using pluripotent stem cells in terms of the ease of obtaining the materials used and cost.

[0053] The culture vessel used in the production method of the present invention is not particularly limited and may be, for example, a cell culture insert, a dish, a flask, a multi-well plate, etc. Culture can also be performed on an organ-on-a-chip (microphysiological system, MPS: biomimetic system).

[0054] <Culture Period> In the production method of the present invention, the period for culturing intestinal cancer-derived cells at the air-liquid interface in the presence of a predetermined factor can be appropriately set depending on the type of factor used, and may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more, preferably 8 days or more, 9 days or more, or 10 days or more, and more preferably 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days or more, or 18 days or more. The upper limit of this period is not particularly limited as long as the problem of the present invention is solved, and may be, for example, 100 days or less, 90 days or less, 80 days or less, 70 days or less, 60 days or less, or 50 days or less. The above-mentioned lower and upper limits may be arbitrarily combined to express the period (range).

[0055] When the production method of the present invention includes liquid culture of cells derived from intestinal cancer, the period of liquid culture is not particularly limited as long as the problem of the present invention is solved, and may be, for example, 5 minutes or more, 0.5 hours or more, 1 hour or more, 5 hours or more, 10 hours or more, 12 hours or more, 1 day or more, or 2 days or more, or, for example, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, or 3 days or less. The above-mentioned lower limits and upper limits may be arbitrarily combined to represent a period (range).

[0056] <Other Culture Conditions> Other culture conditions (such as culture temperature) can be those generally used in the culture of animal cells. That is, for example, the cells can be cultured in an environment of 37°C and 5% CO2. The basal medium is not particularly limited, but preferably, a basal medium suitable for the culture of epithelial cells (for example, a mixed medium of DMEM and Ham's F12 medium, DMEM / F12) can be used.

[0057] In the production method of the present invention, medium exchange or passaging can be carried out as appropriate. The frequency of such exchange or passaging is not particularly limited as long as the object of the present invention is achieved, and may be, for example, once every 2 to 3 days. Furthermore, the method of medium exchange or passaging is not particularly limited as long as the object of the present invention is achieved, and may be any method known to those skilled in the art.

[0058] <Intestinal Epithelial Cells Obtained by the Present Invention> (Villi Structure, Crypt Structure) According to certain embodiments of the production method of the present invention, intestinal epithelial cells capable of forming villus structures, crypt structures, or both can be obtained. In the present invention, intestinal epithelial cells refer to intestinal epithelial cells and cells with similar properties. Forming a villus structure refers to the surface of a cell layer composed of multiple cells having multiple protrusions (villi) with a length of 1 millimeter or less. Forming a crypt structure refers to the surface of a cell layer having multiple tubular depressions (crypts) with a length of several hundred micrometers or less. Villi and crypts contribute to nutrient and drug absorption, digestive juice secretion, intestinal immunity, and other functions in the intestinal tract (mainly the small intestine). Whether intestinal epithelial cells form a villus structure, a crypt structure, or both can be confirmed by any appropriate method known to those skilled in the art. For example, this method may involve observing the surface of the cell layer with an appropriate microscope and examining the presence or absence of an uneven structure.

[0059] (Gene Expression) In a certain embodiment, the intestinal epithelial cells obtained by the production method of the present invention have an expression pattern of intestinal epithelial cell markers similar to that in a living body. Examples of intestinal epithelial cell markers include carboxylesterase (CES), ATP-binding cassette transporter B1 / multidrug resistance protein 1 (ABCB1 / MDR1), ATP-binding cassette transporter G2 / breast cancer resistance protein (ABCG2 / BCRP), cytochrome P450 3A4 (CYP3A4), cytochrome P450 2C9 (CYP2C9), cytochrome P450 2C19 (CYP2C19), cytochrome P450 2D6 (CYP2D6), fatty acid binding protein 2 (FABP), pregnane X receptor (PXR), solute carrier (SLC) family member 5A1 / sodium-coupled glucose transporter 1 (SLC5A1 / SGLT1), solute carrier (SLC) family member 15A1 / peptide transporter 1 (SLC15A1 / PEPT1), and solute carrier (SLC) family member 20A1 / peptide transporter 2 (SLC20A1 / PEPT2). Examples of intestinal epithelial cell markers include organic anion transporter 2B1 (SLCO2B1 / OATP2B1), sucrase-isomaltase (SI), uridine diphosphate-glucuronosyltransferase 1A1 (UGT1A1), uridine diphosphate-glucuronosyltransferase 1A4 (UGT1A4), villin, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), mucin 2 (MUC2), zonula occludens-1 (ZO-1), and lysozyme (LYZ). Expression of intestinal epithelial cell markers can be evaluated by methods known to those skilled in the art, and the method may include, for example, quantifying gene expression of the intestinal epithelial cell marker by real-time RT-PCR.

[0060] To obtain a cell population consisting only of the target cells (in vivo-like highly functional intestinal epithelial cells) or a cell population containing the target cells at a high ratio (high purity), the cell population after culture may be optionally selected and separated using a cell surface marker characteristic of in vivo-like highly functional intestinal epithelial cells as an index.

[0061] One indicator of intestinal epithelial cells obtained by the production method of the present invention is the expression level of the CES gene in intestinal epithelial cells. In one embodiment, intestinal epithelial cells obtained by the production method of the present invention, the expression level of the CES1 gene is lower and the expression level of the CES2 gene is higher than in any of the following cases A1) to A3): A1) when cultured in the presence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator without ALI; A2) when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator; A3) when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator without ALI.

[0062] Such an expression pattern of the CES gene indicates that the intestinal epithelial cells obtained by the production method of the present invention are similar to intestinal epithelial cells in vivo. Quantification and comparison of the expression levels of the target genes can be performed by an appropriate method known to those skilled in the art. For example, the method may use real-time RT-PCR, as described above.

[0063] In one aspect, in intestinal epithelial cells obtained by the production method of the present invention, the expression level of the MUC2 gene is higher than when ALI is not performed and / or when culture is not performed in the presence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. In another aspect, intestinal epithelial cells obtained by the production method of the present invention, the expression level of the CYP3A4 gene is higher than when ALI is not performed and / or when culture is not performed in the presence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. These gene expression patterns indicate that the intestinal epithelial cells obtained by the production method of the present invention are similar to in vivo intestinal epithelial cells.

[0064] (Barrier Function) In one embodiment, the intestinal epithelial cells obtained by the production method of the present invention have a transepithelial electrical resistance (TEER) value of 400 Ω·cm, as measured, for example, by Millicell-ERS2. 2 A confluent cell layer with a pore size of 400 Ω·cm or less can be formed. In this case, cells may be cultured using a cell culture insert with a pore size of 0.4 μm. TEER is the electrical resistance generated by tight junctions between cells and is used as an indicator of the barrier function of the cell layer. TEER values ​​can be measured by methods known to those skilled in the art, such as using a Millicell ERS-2 as shown in this example, following the instructions provided with the device. In the present invention, a confluent cell layer refers to a state in which the seeded cells are in close contact and cover 90% or more of the culture surface. When a confluent cell layer is formed using intestinal epithelial cells obtained by the production method of the present invention, the TEER value is 400 Ω·cm or less. 2 Preferably, the resistance is 390 Ω·cm or less. 2 Below, 380 Ω・cm 2 Below, 370 Ω・cm 2 Below, 360 Ω・cm 2 Below, 350 Ω・cm 2 Below, 300 Ω・cm 2 Below, 250 Ω・cm 2 Below, 200 Ω・cm 2 Below, 150 Ω・cm 2 or less, or 100 Ω·cm 2 Alternatively, the intestinal epithelial cells obtained by the production method of the present invention can form a cell layer with a lower TEER value than when cultured in the absence of at least three or more elements selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. A TEER value within this range indicates that the intestinal epithelial cells obtained by the production method of the present invention have a barrier function similar to that of the intestinal tract in vivo.

[0065] Barrier function can also be evaluated by analyzing the permeability of tight junctions formed by intestinal epithelial cells. Permeability can be analyzed, for example, by measuring the fluorescence intensity of Lucifer yellow that has permeated from the apical side to the basolateral side of the cells over a certain period of time. In one embodiment, the Lucifer yellow permeability of intestinal epithelial cells obtained by the production method of the present invention is 1×10 －６ cm / sec or less, preferably 0.9 × 10 －６ In one embodiment, the intestinal epithelial cells obtained by the production method of the present invention have a higher Lucifer yellow permeability than when cultured in the absence of at least three or more elements selected from the group consisting of EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator.

[0066] (Other) In some embodiments, intestinal epithelial cells obtained by the production method of the present invention exhibit higher expression levels of MUC2 or PEPT1 protein than when cultured without ALI and in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. Protein expression levels can be assessed by appropriate methods known to those skilled in the art. Such methods may include, for example, immunoassays, Western blotting, or the like, and may also include visualization of protein expression by techniques such as immunofluorescence staining.

[0067] In some embodiments, intestinal epithelial cells obtained by the production method of the present invention exhibit at least one of CYP3A4 activity and CYP3A5 activity higher than when cultured without ALI and in the absence of at least three or more elements selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. CYP3A4 and CYP3A5 belong to the cytochrome P450 family, a type of drug-metabolizing enzyme, and higher activity indicates better small intestinal function. CYP3A4 and CYP3A5 activity can be assessed by appropriate methods known to those skilled in the art. The method may, for example, involve applying substrates for CYP3A4 and CYP3A5 (e.g., midazolam) to the cells and examining whether the metabolites of the substrates are increased.

[0068] In summary, the intestinal epithelial cells obtained by the present invention have one or more of the following properties a) to f): a) they are able to form villus structures, crypt structures, or both; b) they have a lower expression level of the CES1 gene and a higher expression level of the CES2 gene than in any of the cases A1) to A3) below; c) they have a higher expression level of the MUC2 gene than in any of the cases A1) to A3) below; d) they have a higher expression level of the CYP3A4 gene than in any of the cases A1) to A3) below; e) they are able to form a cell layer with a lower TEER value than when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. f) they have a higher Lucifer yellow permeability than when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. A1) When ALI is not performed and the cells are cultured in the presence of at least three or more substances selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators; A2) When ALI is performed but the cells are cultured in the absence of at least three or more substances selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators; A3) When ALI is not performed and the cells are cultured in the absence of at least three or more substances selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators.

[0069] As described above, the production method of the present invention allows for the production of intestinal epithelial cells capable of forming villus structures, crypt structures, or both from intestinal cancer-derived cells. The production method of the present invention has the following main advantages. First, there are many intestinal cancer-derived cell lines that are inexpensive, stable, and abundant, and many that are used as global standards and have accumulated a vast amount of data. Therefore, the use of intestinal cancer-derived cells offers supply and cost advantages. Furthermore, unlike intestinal cancer-derived cells such as Caco-2 cells cultured by conventional methods, the intestinal epithelial cells obtained by the production method of the present invention have metabolic enzyme expression levels and expression patterns similar to those of living organisms. Furthermore, while conventional methods for producing intestinal cells from pluripotent stem cells require the formation of intestinal organoids and the use of cells that constitute the intestinal organoids, the production method of the present invention does not require the step of forming intestinal organoids. Therefore, in some embodiments, the production method of the present invention does not involve the formation of intestinal organoids.

[0070] [Induction Method of the Present Invention] In one aspect, the present invention relates to a method for inducing differentiation of intestinal cancer-derived cells into intestinal epithelial cells with specific functions similar to those in vivo (hereinafter, sometimes referred to as the induction method of the present invention). The induction method of the present invention can promote differentiation of intestinal cancer-derived cells into intestinal epithelial cells with one or more of the following characteristics a) to f): a) capable of forming a villus structure, a crypt structure, or both; b) lower CES1 gene expression level and higher CES2 gene expression level than in any of the following A1) to A3); c) higher MUC2 gene expression level than in any of the following A1) to A3); d) higher CYP3A4 gene expression level than in any of the following A1) to A3); e) capable of forming a cell layer with a lower TEER value than in culture in the absence of at least three or more elements selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. f) Lucifer yellow permeability is greater than that when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. A1) When cultured in the presence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators without ALI; A2) When cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators with ALI; A3) When cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators without ALI.

[0071] The induction method of the present invention can include any combination of the features described above in the section [Production method of the present invention]. Note that the induction method of the present invention does not necessarily require that intestinal epithelial cells having one or more of the characteristics a) to f) be obtained.

[0072] [Culture method of the present invention] In one aspect, the present invention relates to a cell culture method (hereinafter, sometimes referred to as the culture method of the present invention) comprising culturing intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more elements selected from the group consisting of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator. According to the culture method of the present invention, intestinal epithelial cells having one or more of the following properties a) to e) can be obtained from intestinal cancer-derived cells, or differentiation into intestinal epithelial cells having one or more of the following properties a) to f) can be promoted: a) A villus structure, a crypt structure, or both can be formed; b) The expression level of the CES1 gene is lower and the expression level of the CES2 gene is higher than in any of the following A1) to A3); c) The expression level of the MUC2 gene is higher than in any of the following A1) to A3); d) The expression level of the CYP3A4 gene is higher than in any of the following A1) to A3); e) A cell layer having a lower TEER value can be formed than in the case of culture in the absence of at least three or more members selected from the group consisting of EGF, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator. f) Lucifer yellow permeability is greater than that when cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators. A1) When cultured in the presence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators without ALI; A2) When cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators with ALI; A3) When cultured in the absence of at least three or more selected from the group consisting of EGF, TGFβ receptor inhibitors, Wnt signal enhancers, and cAMP signal activators without ALI.

[0073] For each feature of the culture method of the present invention, reference can be made to the items explained in the above section [Production method of the present invention].

[0074] [Culture Medium of the Present Invention] In one aspect, the present invention relates to a culture medium (hereinafter sometimes referred to as the culture medium of the present invention) for use in the production method of the present invention, the induction method of the present invention, or the culture method of the present invention.

[0075] In one embodiment, the medium of the present invention comprises at least three or more selected from the group consisting of EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator. In one embodiment, the medium of the present invention comprises at least three or more selected from the group consisting of EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator, and a γ-secretase inhibitor. In one embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator. In one embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, and a γ-secretase inhibitor. In one embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, and a ROCK inhibitor. In one embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a Wnt signal enhancer, a cAMP signal activator, a ROCK inhibitor, and a γ-secretase inhibitor. In one embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a cAMP signal activator, and a ROCK inhibitor. In another embodiment, the medium of the present invention comprises EGF, a TGFβ receptor inhibitor, a cAMP signal activator, a ROCK inhibitor, and a γ-secretase inhibitor.

[0076] The concentration of EGF in the medium of the present invention may be, for example, 1 ng / mL to 10 μg / mL, preferably 10 ng / mL to 1 μg / mL, more preferably 30 ng / mL to 500 ng / mL, and even more preferably 50 ng / mL to 150 ng / mL.

[0077] The TGFβ receptor inhibitor in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the TGFβ receptor inhibitor in the medium of the present invention (in the case of A-83-01) may be, for example, 0.005 μM to 50 μM, preferably 0.05 μM to 5 μM, and more preferably 0.1 μM to 1 μM. When using a compound different from A-83-01, the concentration of the compound added can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of A-83-01 (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments based on the examples described below.

[0078] The Wnt signaling enhancer in the medium of the present invention may be one or more selected from the group consisting of a GSK-3 inhibitor, a Wnt signaling activator, and a Wnt agonist, and is preferably a GSK-3 inhibitor. The Wnt signaling enhancer can be selected as described above in the section on the production method of the present invention.

[0079] The GSK-3 inhibitor in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the GSK-3 inhibitor (in the case of CHIR99021) added to the culture system is, for example, 0.03 μM to 300 μM, preferably 0.3 μM to 30 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 μM to 5 μM. Note that when a GSK-3 inhibitor other than CHIR99021 is used, the concentration added to the culture system can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the GSK-3 inhibitor used and CHIR99021. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0080] The Wnt signaling activator in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the Wnt signaling activator (in the case of R-spondin 1) added to the culture system can be, for example, 10 ng / mL to 1000 ng / mL, preferably 50 ng / mL to 500 ng / mL. When using a Wnt signaling activator other than R-spondin 1, the concentration to be added to the culture system can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties (particularly activity) between the Wnt signaling activator used and R-spondin 1. Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0081] The Wnt agonist in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the Wnt agonist (in the case of Wnt3a) added to the culture system can be, for example, 100 ng / mL to 1000 ng / mL, preferably 250 ng / mL to 500 ng / mL. When using a Wnt agonist other than Wnt3a, the concentration to be added to the culture system can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties (particularly activity) between the Wnt3a and R-spondin 1 used. Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0082] The cAMP signal activator in the medium of the present invention may be one or more selected from the group consisting of cAMP derivatives, cAMP degrading enzyme inhibitors, and cAMP activators, and is preferably a cAMP activator. The cAMP signal activator can be selected as described above in the section on the production method of the present invention.

[0083] The cAMP derivative in the medium of the present invention can be selected as described above in the section on the preparation method of the present invention. The concentration of the cAMP derivative in the medium of the present invention (for 8-Br-cAMP) is, for example, 0.01 mM to 30 mM, preferably 0.05 mM to 10 mM, and more preferably 0.1 mM to 5 mM. When using an exemplified compound, i.e., a compound other than 8-Br-cAMP, the concentration in the medium can be determined by one skilled in the art based on the above concentration range, taking into account the differences (particularly activity) between the properties of the compound used and the properties of the exemplified compound (8-Br-cAMP). Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments.

[0084] The cAMP inhibitor in the medium of the present invention can be selected as described above in the section on the production method of the present invention. An example of the concentration of the cAMP inhibitor in the medium of the present invention (in the case of IBMX) is 0.01 mM to 10 mM, preferably 0.05 mM to 5 mM, and more preferably 0.1 mM to 1 mM. When using an exemplified compound, i.e., a compound different from IBMX, the concentration in the medium can be set according to the above concentration range by one skilled in the art, taking into consideration the differences in the properties of the compound used and the properties of the exemplified compound (IBMX) (particularly differences in activity). Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0085] The cAMP activator in the medium of the present invention can be selected as described above in the section on the preparation method of the present invention. The concentration of the cAMP activator in the medium of the present invention (forskolin) can be, for example, 0.1 μM to 1 M, preferably 1 μM to 100 μM, more preferably 3 μM to 50 μM, and even more preferably 5 μM to 15 μM. When using an exemplified compound, i.e., a compound other than forskolin, the concentration in the medium can be determined by one skilled in the art based on the above concentration range, taking into account the differences in the properties of the compound used and the properties of the exemplified compound (forskolin) (particularly differences in activity). Furthermore, whether the selected concentration range is appropriate can be confirmed by preliminary experiments similar to those described in the Examples below.

[0086] The gamma-secretase inhibitor in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the gamma-secretase inhibitor (in the case of DAPT) in the medium of the present invention is, for example, 0.03 μM to 300 μM, preferably 0.3 μM to 30 μM, more preferably 0.5 μM to 10 μM, and even more preferably 1 μM to 5 μM. When using a gamma-secretase inhibitor other than DAPT, the concentration in the medium can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the gamma-secretase inhibitor used and DAPT. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0087] The ROCK inhibitor in the medium of the present invention can be selected as described above in the section on the production method of the present invention. The concentration of the ROCK inhibitor (in the case of Y27632) in the medium of the present invention is, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 1 to 20 μM. When using a ROCK inhibitor other than Y27632, the concentration in the medium can be set by one skilled in the art in accordance with the above concentration range, taking into account the differences in the properties (particularly activity) between the ROCK inhibitor used and Y27632. Furthermore, whether the set concentration range is appropriate can be confirmed by preliminary experiments.

[0088] In one embodiment, the medium of the present invention further comprises a B27 supplement. The concentration of the B27 supplement in the medium of the present invention may be, for example, 0.01 to 30%, preferably 0.1 to 20%, more preferably 0.5 to 10%, and even more preferably 1 to 5%.

[0089] In one embodiment, the medium of the present invention further comprises an N2 supplement. The concentration of the N2 supplement in the medium of the present invention may be, for example, 0.01 to 20%, preferably 0.05 to 10%, and more preferably 0.1 to 5%.

[0090] The medium of the present invention may further contain other factors suitable for the production method or induction method of the present invention. These other factors may be, for example, serum, amino acids, or antibiotics. The serum may be, for example, mammalian serum, preferably fetal bovine serum (FBS). Alternatively, serum substitutes (e.g., Knockout Serum Replacement (KSR)) may be used instead of serum. The concentration of serum or serum substitute in the medium of the present invention may be 0.1-20%, preferably 0.5-15%. The amino acids are preferably essential amino acids such as L-glutamine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-glycine, L-proline, and L-serine, or L-alanyl-L-glutamine. The concentration of amino acids in the medium of the present invention may be, for example, 0.1-10%, preferably 0.5-5%. The antibiotic may be, for example, penicillin, streptomycin, ampicillin, puromycin, gentamicin, etc., and is preferably penicillin or streptomycin. The concentration of penicillin in the medium of the present invention may be, for example, 10 to 500 units / mL, and preferably 50 to 150 units / mL. The concentration of streptomycin in the medium of the present invention may be, for example, 10 to 500 μg / mL, and preferably 50 to 150 μg / mL.

[0091] The culture of the present invention is preferably a liquid medium. Furthermore, the medium of the present invention may have the same composition as a known basal medium in addition to the above-mentioned factors. The composition may be the same as that of a basal medium such as DMEM, DMEM / F12, or Advanced-DMEM / F12, and is preferably the same as that of Advanced-DMEM / F12.

[0092] [Kit] In one aspect, the present invention relates to a kit comprising the medium of the present invention and cells derived from intestinal cancer (hereinafter, sometimes referred to as the kit of the present invention). The cells derived from intestinal cancer contained in the kit of the present invention are not particularly limited and can be selected as described above in the section on the production method of the present invention. Furthermore, the number of cells derived from intestinal cancer contained in the kit of the present invention is not particularly limited and can be, for example, 1 x 10 4 ~1×10 8 cells, 1 × 10 5 ~1×10 7 The intestinal cancer-derived cells contained in the kit of the present invention may be frozen.

[0093] The kit of the present invention may include a container containing the culture medium of the present invention and a container containing cells derived from intestinal cancer. These containers are not particularly limited as long as they are suitable for storing the culture medium or cells. The kit of the present invention may also include an instruction manual describing operating instructions such as the culture method.

[0094] [Uses of Intestinal Epithelial Cells] (First Use) As a first use of the intestinal epithelial cells obtained by the production method of the present invention, various assays are provided. The intestinal epithelial cells obtained by the production method of the present invention can be used in a model system of the intestinal tract, particularly the small intestine, and are useful for evaluating pharmacokinetics (absorption, metabolism, etc.) and toxicity in the intestinal tract, particularly the small intestine. In one embodiment, a method for evaluating the pharmacokinetics or toxicity of a test substance using intestinal epithelial cells obtained by the production method of the present invention is provided.

[0095] Specifically, intestinal epithelial cells obtained by the production method of the present invention can be used to test the absorbability or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, toxicity, etc. of a test substance. That is, in one aspect, the present invention provides a method for evaluating the absorbability or membrane permeability, metabolism, excretion, drug interactions, pharmacokinetics such as induction of drug-metabolizing enzymes and induction of drug transporters, toxicity, etc. of a test substance as one use of intestinal epithelial cells. This method includes the steps of: (i) preparing a cell layer composed of intestinal epithelial cells obtained by the production method of the present invention; (ii) contacting the cell layer with a test substance; and (iii) quantifying the test substance that has permeated the cell layer and evaluating the absorbability or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance. The absorbability of a test substance can also be evaluated by a method according to another aspect including steps (I) and (II) described below.

[0096] In step (i), intestinal epithelial cells are typically cultured on a semipermeable membrane (porous membrane) to form a cell layer. Specifically, for example, a culture vessel equipped with a cell culture insert (e.g., Transwell (registered trademark) provided by Corning Incorporated) is used, and cells are seeded and cultured in the cell culture insert to obtain a cell layer composed of intestinal epithelial cells.

[0097] The "contact" in step (ii) is typically carried out by adding the test substance to the medium. The timing of adding the test substance is not particularly limited. Therefore, the test substance may be added at a certain time point after starting culture in a medium not containing the test substance, or the culture may be started in a medium containing the test substance in advance.

[0098] Organic or inorganic compounds of various molecular sizes can be used as test substances. Examples of organic compounds include nucleic acids, peptides, proteins, lipids (simple lipids, complex lipids (phosphoglycerides, sphingolipids, glycosylglycerides, cerebrosides, etc.)), prostaglandins, isoprenoids, terpenes, steroids, polyphenols, catechins, and vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, A, D, E, etc.). Existing or potential components of pharmaceuticals, nutritional foods, food additives, pesticides, and cosmetics are also suitable test substances. Plant extracts, cell extracts, culture supernatants, etc. can also be used as test substances. Two or more test substances can be added simultaneously to examine interactions, synergies, and other effects between the test substances. Test substances can be natural or synthetic. In the latter case, efficient assay systems can be constructed using, for example, combinatorial synthesis techniques.

[0099] The period for contacting the test substance can be set arbitrarily. The contact period is, for example, 10 minutes to 3 days, preferably 1 hour to 1 day. The contact may be performed in multiple batches.

[0100] In step (iii), the test substance that has permeated the cell layer is quantified. For example, when a culture vessel equipped with a cell culture insert such as a Transwell® is used, the test substance that has permeated the cell culture insert, i.e., the test substance that has migrated through the cell layer into the upper or lower vessel, is quantified using a measurement method such as mass spectrometry, liquid chromatography, or immunological techniques (e.g., fluorescence immunoassay (FIA) or enzyme immunoassay (EIA)), depending on the test substance. Based on the quantification results (the amount of test substance that has permeated the cell layer) and the amount of test substance used (typically the amount added to the medium), the test substance's absorbability or membrane permeability, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity are determined and evaluated.

[0101] In another aspect, the present invention also provides a method for evaluating the metabolism or absorption of a test substance, which comprises the steps of (I) contacting the test substance with intestinal epithelial cells obtained by the production method of the present invention, and (II) measuring and evaluating the metabolism or absorption, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

[0102] Step (I), i.e., contacting the intestinal epithelial cells with the test substance, can be carried out in the same manner as step (ii) above, except that it is not essential to form a cell layer in advance.

[0103] After step (I), the metabolism or absorption of the test substance, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity are measured and evaluated (step (II)). The metabolism and other properties may be measured and evaluated immediately after step (I), i.e., immediately after contact with the test substance, without any substantial time interval, or after a certain period of time (e.g., 10 minutes to 5 hours) has elapsed. Metabolism can be measured, for example, by detecting metabolites. In this case, the culture medium after step (I) is usually used as a sample, and predicted metabolites are measured qualitatively or quantitatively. An appropriate measurement method may be selected depending on the metabolite; for example, mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)), etc., can be used.

[0104] Typically, when a metabolic product of the test substance is detected, it is determined or evaluated that "the test substance has been metabolized." Furthermore, the metabolic amount of the test substance can be evaluated according to the amount of metabolic product. The metabolic efficiency of the test substance may be calculated based on the detection result of the metabolic product and the amount of the test substance used (typically the amount added to the medium).

[0105] It is also possible to measure the metabolism of a test substance using the expression of drug-metabolizing enzymes (cytochrome P450 (particularly CYP3A4), uridine diphosphate-glucuronosyltransferase (particularly UGT1A8 and UGT1A10), sulfotransferase (particularly SULT1A3, etc.)) in intestinal epithelial cells as an indicator. The expression of drug-metabolizing enzymes can be evaluated at the mRNA level or protein level. For example, an increase in the mRNA level of a drug-metabolizing enzyme can be determined to indicate that the test substance has been metabolized. Similarly, an increase in the activity of a drug-metabolizing enzyme can be determined to indicate that the test substance has been metabolized. As with the case of using metabolites as indicators, quantitative determination and evaluation can also be performed based on the expression level of the drug-metabolizing enzyme.

[0106] To evaluate the absorption of a test substance, for example, the amount of the test substance remaining in the culture medium is measured. Typically, the test substance is quantified using the culture medium after step (I) as a sample. An appropriate measurement method can be selected depending on the test substance. For example, mass spectrometry, liquid chromatography, immunological techniques (e.g., fluorescence immunoassay (FIA) and enzyme immunoassay (EIA)), etc. can be used. Typically, when a decrease in the content of the test substance in the culture medium is observed, it is determined and evaluated that the test substance has been absorbed. In addition, the amount or absorption efficiency of the test substance can be determined and evaluated depending on the degree of decrease. Absorption can also be evaluated by measuring the amount of test substance taken up into or permeated through cells.

[0107] The measurement and evaluation of metabolism and the measurement and evaluation of absorption may be carried out simultaneously or in parallel. Furthermore, each method according to the first use may optionally include a step of obtaining intestinal epithelial cells by the preparation method of the present invention.

[0108] (Second Use) A second use of the intestinal epithelial cells obtained by the production method of the present invention includes culturing microorganisms (bacteria, fungi, viruses, etc.) or parasites using the intestinal epithelial cells or cell sheets obtained by the production method of the present invention. By culturing microorganisms or parasites using the intestinal epithelial cells or cell sheets obtained by the production method of the present invention, the infectivity of the microorganisms or parasites to the intestinal epithelial cells can be analyzed. Furthermore, if infectivity is detected, the maintenance or proliferation ability of the microorganisms or parasites can be confirmed. Furthermore, by culturing microorganisms or parasites using the intestinal epithelial cells or cell sheets obtained by the production method of the present invention, it is possible to obtain microorganisms or parasites that repeatedly proliferate. Furthermore, the infection state of pathogenic microorganisms can be reproduced and used for screening therapeutic drugs, or the effects of substances produced by microorganisms or parasites on intestinal cells can be reproduced and used to screen for substances that are useful or harmful to the small intestine of a living organism.

[0109] Specific examples of microorganisms or parasites include, but are not limited to, Escherichia coli, lactic acid bacteria, bifidobacteria, Vibrio parahaemolyticus, Salmonella, various enterobacteria, influenza virus, rotavirus, adenovirus, astrovirus, Sapporo virus, norovirus, HIV, and coronavirus.

[0110] (Third Use) A third use of the intestinal epithelial cells obtained by the production method of the present invention is a co-culture model using the intestinal epithelial cells obtained by the production method of the present invention. The co-culture model described above can reproduce the structure of biological tissue and analyze interactions with other cells. Furthermore, the co-culture model described above can reproduce the pathology of a disease and screen for therapeutic drugs.

[0111] The cells to be co-cultured are preferably cell types present in the living body of a primate or rodent, particularly cells present around the small intestine. Examples include, but are not limited to, hepatocytes, pancreas, vascular endothelial cells, immune cells, nerve cells, fibroblasts, muscle cells, and interstitial cells. The cells to be co-cultured may also be established cell lines derived from primates or rodents. Examples include, but are not limited to, immortalized cell lines such as HepG2 cells and THP-1 cells, pluripotent stem cells such as ES cells and iPS cells, and cells differentiated therefrom.

[0112] The culture vessel used for co-culture is not particularly limited, and examples include cell culture inserts, dishes, flasks, multi-well plates, etc. Culture can also be performed on an organ-on-a-chip (MPS: biomimetic system).

[0113] Test substances that can be tested in the co-culture model include organic and inorganic compounds of various molecular sizes. Existing or potential components of pharmaceuticals, nutritional foods, etc. are also suitable test substances. Plant extracts, cell extracts, culture supernatants, etc. may also be used as test substances. Two or more of these may also be used in combination.

[0114] The present invention will be further explained below by way of examples, but the present invention is not limited to the specific embodiments shown in the examples.

[0115] [Methods] 1) Maintenance Culture: Caco-2 cells (RCB0988), a human colon cancer cell line, were purchased from the RIKEN BioResource Research Center. For maintenance culture, Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine (L-Glu), 1% non-essential amino acids (NEAA), 100 units / mL penicillin G, and 100 μg / mL streptomycin was used. Cells were passaged every 3–5 days using 0.5% trypsin-ethylenediaminetetraacetic acid (EDTA) at a split ratio of 1:10–1:20.

[0116] 2) Differentiation Induction: Caco-2 cells were detached as described above and seeded onto 0.4 μm pore size cell culture inserts coated with iMatrix 511 diluted to 0.16 μg / mL in PBS(-) or 0.1% gelatin solution. Cells were cultured in differentiation induction medium (M5 medium). M5 medium was Advanced-DMEM / F12 containing 1% GlutaMAX, 2% FBS, 100 units / mL penicillin G, 100 μg / mL streptomycin, and the following culture supplements and differentiation inducers: B27 supplement, N2 supplement, 100 ng / mL epidermal growth factor (EGF), 0.5 μM A-83-01, 3 μM CHIR99021, 10 μM forskolin, and 2.5 μM DAPT (except in Example 2, where M5 medium did not contain DAPT). In Examples 5) and 6), the presence or absence of differentiation-inducing factors in M5 medium was as specified for each experimental condition. Medium was changed every 2-3 days, and cells were cultured in either conventional liquid phase culture (LL) or air-liquid interface (ALI) culture. For ALI culture, the medium on the cell culture insert was removed from day 3 after seeding until the end of culture (days 15-21). Dead cells that accumulated in the insert during culture were washed appropriately with medium that did not contain differentiation-inducing factors.

[0117] 3) Measurement of transepithelial electrical resistance (TEER) value 200 μL of medium warmed to 37°C was placed on the insert, and the TEER value was measured using Millicell-ERS2.

[0118] 4) Extraction of total ribonucleic acid (RNA) After differentiation induction, total RNA was extracted according to the attached manual of Agencourt RNAdvance Tissue.

[0119] 5) Reverse transcription reaction Complementary DNA (cDNA) was synthesized using ReverTra Ace qPCR RT Master Mix according to the attached manual.

[0120] 6) Real-Time RT-PCR: Real-time RT-PCR was performed using the KAPA SYBR Fast qPCR Kit, cDNA as a template, according to the attached manual. Results were normalized using hypoxanthine phosphoribosyltransferase (HPRT) as an endogenous control.

[0121] 7) HE staining Cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes and embedded in paraffin. Sections were attached to glass slides and subjected to HE staining.

[0122] 8) Lucifer yellow permeability test The permeability test buffer was prepared by adding HEPES buffer to HBSS to make a 1% concentration and adjusting the pH to 6.4. After the culture was completed, the cells were washed with the permeability test buffer warmed to 37°C, and 150 μL of the same buffer was added below the insert, and 100 μL of the permeability test buffer containing Lucifer yellow added to a concentration of 110 μM was added to the insert, and 50 μL was immediately collected. After 30 minutes of incubation at 37°C, the entire amount of buffer below the insert was collected. The fluorescence intensity of the collected buffer was measured using a multiplate reader, and the permeability coefficient (P) of Lucifer yellow was calculated. app ) was calculated. n = 3

[0123] Methanol-fixed cells were blocked with 5% FBS and then incubated overnight with primary antibodies (MUC2, Villin1, Claudin7, PEPT1) at 4°C. Then, secondary antibodies were incubated for 1 hour at room temperature, followed by DAPI incubation for 5 minutes at room temperature.

[0124] [Results] Example 1) Effects of culture medium and air-liquid interface culture No difference was observed on the third day of culture, but three-dimensional growth of Caco-2 cells was observed in the ALI group on the 15th day of culture. In particular, the group using a combination of M5 medium and ALI exhibited a significant development of uneven structures (Fig. 1A). Transepithelial electrical resistance (TEER) values, which are an index of the barrier function of the intestinal epithelium, were measured over time. The TEER value significantly decreased in the M5 medium group, reaching 100 Ω cm. 2The TEER value was approximately the same as that of the intestinal tract in vivo (Figure 1B). Gene expression showed a significant increase in MUC2, a marker for goblet cells (Table 1, Figure 1C). Expression of CYP3A4, the most important drug-metabolizing enzyme in the intestinal tract, also increased (Table 1, Figure 1C). The expression pattern of CES1 / 2 in the intestinal tract was high in CES2 and almost no CES1, but in Caco-2 cells, CES1 expression was also high. These results suggest that the combination of M5 medium and ALI brings the CES1 / 2 expression pattern closer to that of the intestinal tract in vivo.

[0125]

[0126] Example 2) Effects of ALI and Differentiation-Inducing Factors, etc. on Caco-2 Cell Structure The effects of the presence or absence of some of the factors, culture supplements, and coating agents used in differentiation induction on Caco-2 cell differentiation were evaluated (Table 2). To the Caco-2 medium group, each of the differentiation-inducing factors contained in M5 medium was added and their effects were examined. Additionally, the presence or absence of culture supplements B27 and N2 used in the culture of iPS cell-derived intestinal cells, and the replacement of the coating agent iMatrix511 silk with gelatin were also examined. To the M5 medium group, each of the factors was removed one by one and their effects were examined. Adding EGF or CHIR99021 to Caco-2 medium and performing ALI culture resulted in a TEER value of 200-300 Ω cm. 2 The TEER value was stable in the group containing all factors, including EGF and CHIR99021 (Fig. 2A). In contrast, in M5 medium, an excessive increase in TEER was observed only in the All- group, which excluded all differentiation-inducing factors (Fig. 2B). Forskolin, A-83-01, and CHIR99021 were suggested to contribute to the formation of the uneven three-dimensional structure (Fig. 2C, D). Furthermore, in the Caco-2 medium group, the absence of EGF also resulted in a decrease in cell thickness. On the other hand, B27, N2, and iMatrix511 were not suggested to be involved in the formation of the three-dimensional structure.

[0127]

[0128] Example 3) Changes in gene expression levels in Caco-2 cells due to differentiation-inducing factors. The expression levels of LGR5 and CYP3A4 were reduced in the group cultured in M5 medium without EGF, suggesting that EGF contributes to the increase in the expression levels of LGR5 and CYP3A4. Furthermore, forskolin increased the expression level of MUC2, and CHIR99021 was suggested to be involved in the expression of CES1 / 2, MDR1, and CYP3A4 (Figure 3).

[0129] Example 4) Effects of culture medium and air-liquid interface culture on barrier function A Lucifer yellow permeability test was performed to evaluate the intestinal barrier function. -6 A permeability of less than 1 × 10 cm / sec is considered to be sufficient for the barrier function. There was no significant difference between LL and ALI, and permeability increased when M5 medium was used (Figure 4). However, the value was 1 × 10 -6 The intestinal barrier function was sufficient because the intestinal barrier function was less than 100 cm / sec.

[0130] Example 5) Detailed Investigation of Medium Components and Differentiation-Inducing Factors. Among the additive factors shown to contribute to functional improvement of Caco-2 cells in the experiments listed in Table 2, we investigated the optimal culture conditions by carefully examining the duration of addition of two factors: CHIR99021, which induces differentiation from stem cell maintenance toward maturation, and DAPT, which is involved in increasing the expression levels of CYP3A4 and MUC2 (Table 3). While no significant 3D structure was observed in the conventional group cultured at ALI in Caco-2 medium, 3D structure was observed from Day 11 in the novel group (Figure 5A). While the conventional group formed a monolayer cell membrane, novel groups 2 and 4 exhibited a concave-convex 3D structure (Figure 5B). Applying novel culture methods 1–4 to Caco-2 cells demonstrated gene expression similar to that observed at ASI (Figure 5C).

[0131]

[0132] Example 6) Functional evaluation of Caco-2 cells using the new culture method. By performing ALI culture using M5 medium and differentiation-inducing factors, the TEER value decreased compared to the conventional group, but it was shown to remain stable (Figure 6A). In the new 1, 2, and 3 groups, there was no significant increase in LY permeation associated with the decrease in TEER value, and the apparent membrane permeability coefficient (Papp) was 1 x 10 -6 The pulmonary flow rate (Vp) was below 1.5 cm / sec, indicating that the barrier function was intact (Figure 6B). Compared to the conventional group, the protein expression of MUC2 and PEPT1 was confirmed in the new 2 group (Figure 6C). A metabolic activity test of the drug-metabolizing enzyme CYP3A4 / 5 revealed increased metabolite production in the new 2 and 3 groups compared to the conventional group, and the addition of an inhibitor reduced metabolite production, indicating improved CYP3A4 / 5 function (Figure 6D).

[0133] The present invention can provide a new intestinal evaluation system that can replace techniques that use human intestinal cells derived from living bodies, which are difficult to obtain, or iPS cells, which are expensive to maintain and require advanced techniques for culturing.

Claims

1. A method for producing intestinal epithelial cells from cells derived from intestinal cancer, comprising: culturing the intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more elements selected from the group consisting of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator; the method results in a lower expression level of the CES1 gene and a higher expression level of the CES2 gene in the intestinal epithelial cells than when the cells are cultured in the presence of the at least three or more elements without air-liquid interface culture.

2. The method of claim 1, wherein the intestinal epithelial cells are capable of forming a villus structure, a crypt structure, or both.

3. The method of claim 1, wherein the intestinal epithelial cells are capable of forming a cell layer having a lower transepithelial electrical resistance value than when cultured in the absence of at least three or more elements selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator.

4. The method of claim 1, wherein the cells derived from intestinal cancer are cultured at the air-liquid interface in the presence of at least three or more substances selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator for one day or more.

5. The method of claim 1, which comprises culturing cells derived from intestinal cancer at the air-liquid interface in the presence of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator.

6. The method of claim 5, which comprises culturing intestinal cancer-derived cells at an air-liquid interface in the presence of an epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator, as well as a ROCK inhibitor.

7. The method of claim 1, wherein the cells derived from intestinal cancer are selected from Caco-2 cells, HT-29 cells, LS-174T cells, T84 cells, SW-480 cells, HCT-116 cells, DLD-1 cells, and LoVo cells.

8. The method of claim 1, wherein the TGFβ receptor inhibitor is A-83-01, the Wnt signal enhancer is CHIR99021, and the cAMP signal activator is forskolin.

9. The method of any one of claims 1, comprising culturing cells derived from intestinal cancer on a culture surface coated with gelatin.

10. The method of claim 1, comprising culturing intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more substances selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator, as well as one or more substances selected from a gamma-secretase inhibitor, a B27 supplement, and an N2 supplement.

11. A method for inducing differentiation of intestinal cancer-derived cells into intestinal epithelial cells, comprising: culturing the intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more elements selected from the group consisting of epidermal growth factor, TGFβ receptor inhibitor, Wnt signal enhancer, and cAMP signal activator; wherein the expression level of the CES1 gene in the intestinal epithelial cells is lower and the expression level of the CES2 gene is higher than when the cells are cultured in the presence of the at least three or more elements without air-liquid interface culture.

12. A cell culture method comprising culturing intestinal cancer-derived cells at an air-liquid interface in the presence of at least three or more substances selected from the group consisting of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator.

13. A culture medium for use in the method according to any one of claims 1 to 12, comprising at least three or more members selected from the group consisting of epidermal growth factor, a TGFβ receptor inhibitor, a Wnt signal enhancer, and a cAMP signal activator.

14. A kit comprising the medium according to claim 12 and cells derived from intestinal cancer.

15. A method for evaluating the pharmacokinetics or toxicity of a test substance, using intestinal epithelial cells obtained by the production method described in claim 1.

16. The method according to claim 15, wherein the pharmacokinetics of the test substance is absorption or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, or induction of drug transporters.

17. A method according to claim 15 or 16, comprising the following steps (i) to (iii): (i) preparing a cell layer composed of intestinal epithelial cells obtained by the method according to claim 1; (ii) contacting the cell layer with a test substance; and (iii) quantifying the test substance that has permeated the cell layer and evaluating the absorbability or membrane permeability, metabolism, excretion, drug interactions, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

18. The method according to claim 15 or 16, comprising the following steps (I) and (II): (I) contacting a test substance with intestinal epithelial cells obtained by the production method according to claim 1; (II) measuring and evaluating the metabolism or absorption, drug interaction, induction of drug-metabolizing enzymes, induction of drug transporters, or toxicity of the test substance.

Images