Method for producing small intestinal epithelial-like cells

Abstract

According to the present invention, a method for producing small intestinal epithelium-like cells is provided, which comprises the steps of: (i) culturing small intestinal epithelium progenitor cells in a culture medium containing active vitamin D; and (ii) acquiring small intestinal epithelium-like cells.

Description

[Technical field]

[0001] The present invention relates to a method for producing small intestinal epithelial-like cells, which are expected to be applied in the field of drug discovery.Furthermore, the present invention relates to a method for producing small intestinal epithelial-like cells, which includes a step of cryopreserving cells during production and re-inducing them after thawing.The present invention also relates to a method for evaluating drug toxicity, a method for testing drug interactions, and a method for testing drug-metabolizing enzyme induction, which use small intestinal epithelial-like cells produced by the above production method. [Background technology]

[0002] Pluripotent stem cells are undifferentiated cells that have the ability to differentiate and self-replicate. Cells differentiated from pluripotent stem cells have the ability to repair damaged tissues, and they are being actively researched due to their usefulness in the field of regenerative medicine. Among pluripotent stem cells, iPS cells (induced pluripotent stem cells) are artificially created pluripotent stem cells that are dedifferentiated by introducing specific transcription factors such as OCT3 / 4, SOX2, KLF4, and C-MYC into somatic cells such as fibroblasts. Cells with differentiation pluripotency can theoretically differentiate into all tissues and organs, including small intestinal epithelial cells.

[0003] Non-Patent Document 1 is a document that reports the preparation of small intestinal tissue from human pluripotent stem cells. Non-Patent Document 1 reports the successful preparation of organoids containing small intestinal epithelial cells, Paneth cells, goblet cells, and intestinal epithelial endocrine cells. Non-Patent Document 2 is a document that reports the successful preparation of small intestinal stem cells capable of long-term self-renewal from human pluripotent stem cells. The small intestinal stem cells prepared in Non-Patent Document 2 can be differentiated into organoids containing all cells present in the small intestine, as in Non-Patent Document 1. Non-Patent Document 3 attempted to differentiate human pluripotent stem cells into small intestinal epithelial-like cells, and succeeded in preparing cells that express small intestinal markers such as SI (Sucrase Isomaltase), SLC15A1 (solute carrier family 15 member 1) / PEPT1 (peptide transporter 1), and LGR5 (Leucine-rich repeat containing G protein-coupled receptor 5). The cells also have the ability to incorporate β-Ala-Lys-AMCA (β-Ala-Lys-N-7-amino-4methylcoumarin-3-acetic acid) dipeptide. However, the expression of the drug metabolizing enzyme CYP3A4 (Cytochrome P450 3A4) in the cells is about 1 / 500 of that in the human small intestine, which is a problem.

[0004] In addition to the above non-patent documents, as shown in Patent Document 1, there have been reports of attempts to differentiate into small intestinal epithelial cells. However, there have been no reports yet of efficient production of small intestinal epithelial cells from human pluripotent stem cells that allow simultaneous evaluation of drug metabolism and substance absorption without using gene transfer technology. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 147975 [Non-patent literature]

[0006] [Non-Patent Document 1] Nature, 2011 Feb 3;470(7332):105-9 [Non-Patent Document 2] Stem Cell Reports, 2014 jun 3;2(6):838-52 [Non-Patent Document 3] Drug Metab Pharmacokinet, 2014;29(1):44-51 Summary of the Invention [Problem to be solved by the invention]

[0007] Primary cultured human small intestinal epithelial cells, which are direct cultures of bioisolated cells, are difficult to obtain and have individual differences. For this reason, Caco-2 cells, a cell line derived from human colon cancer, are currently used as an in vitro absorption evaluation model of the small intestine. However, unlike human small intestinal epithelial cells, Caco-2 cells hardly express the drug metabolizing enzyme CYP3A4, making it impossible to evaluate drug metabolism. In addition, Caco-2 cells are derived from cancer cells, and it is difficult to say that they reflect the metabolic absorption of normal cells. To solve these problems, active development of technology to differentiate pluripotent stem cells into small intestinal epithelial-like cells is underway.

[0008] Small intestinal epithelial-like cells differentiated from pluripotent stem cells by conventional induction methods have significantly lower activity of the drug metabolizing enzyme CYP3A4 even after maturation compared to the small intestine in vivo. The objective of the present invention is to provide small intestinal epithelial-like cells that have properties closer to those of primary cultured human small intestinal epithelial cells without using gene transfer technology. [Means for solving the problem]

[0009] As a result of intensive research aimed at solving the above problems, the inventors discovered that the expression of drug-metabolizing enzymes is increased when small intestinal epithelial progenitor cells are cultured in the presence of active vitamin D during the process of differentiating small intestinal epithelial progenitor cells into small intestinal epithelial-like cells, and thus completed the invention.

[0010] That is, the present invention provides the following. [1] A method for producing small intestinal epithelial-like cells, comprising the following steps: (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D; and (ii) A step of obtaining the small intestinal epithelial-like cells obtained in the step (i). [2] The method described in [1], further comprising a step of differentiating endoderm cells into small intestinal epithelial progenitor cells. [3] The method described in [2], further comprising a step of differentiating the pluripotent stem cells into endoderm cells. [4] The method according to any one of [1] to [3], further comprising the steps of freezing the small intestinal epithelial progenitor cells and thawing the frozen small intestinal epithelial progenitor cells in the step (i). [5] The method described in [4], wherein the freezing step is carried out 5 to 15 days after the start of the step (i). [6] The method described in [4] or [5], wherein the thawed small intestinal epithelial progenitor cells are cultured in a medium further containing one or more substances selected from a Rho kinase inhibitor, a vinca alkaloid antitumor drug, a MEK inhibitor, and cAMP and its derivatives. [7] A method for evaluating drug toxicity and / or a method for evaluating pharmacokinetics, comprising using small intestinal epithelial-like cells produced by the method according to any one of [1] to [6]. [8] A method for testing drug interactions, comprising using small intestinal epithelial-like cells produced by the method according to any one of [1] to [6]. [9] A method for testing induction of a drug-metabolizing enzyme, comprising using small intestinal epithelial-like cells produced by the method according to any one of [1] to [6].

[10] Culture medium containing a TGFβ receptor inhibitor, a GSK inhibitor and active vitamin D.

[11] The medium described in

[10] , wherein the TGFβ receptor inhibitor is SB431542 and the GSK inhibitor is LY2090314.

[12] The medium according to

[10] or

[11] , further comprising one or more substances selected from a Rho kinase inhibitor, a vinca alkaloid antitumor drug, a MEK inhibitor, and cAMP and its derivatives.

[13] The medium described in

[12] , wherein the Rho kinase inhibitor is Y27632, the vinca alkaloid antitumor drug is vinblastine, the MEK inhibitor is PD0325901, and the cAMP derivative is Br-cAMP.

[14] Use of small intestinal epithelial-like cells produced by the method according to any one of [1] to [6] in a drug toxicity evaluation method and / or a pharmacokinetic evaluation method.

[15] Use of small intestinal epithelial-like cells produced by the method described in any one of [1] to [6] in a method for testing drug-drug interactions.

[16] Use of small intestinal epithelial-like cells produced by the method according to any one of [1] to [6] in a drug-metabolizing enzyme induction test method. Effect of the Invention

[0011] The present invention makes it possible to stably provide small intestinal epithelial-like cells capable of evaluating drug metabolism and absorption. Furthermore, the method of the present invention makes it possible to easily re-form a monolayer membrane having tight junction function from frozen small intestinal epithelial-like cells, and to provide small intestinal epithelial-like cells expressing metabolic enzymes in short-term culture. The small intestinal epithelial-like cells obtained by the present invention can be applied to drug discovery research and food nutrition metabolism and absorption tests. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 illustrates the method of the present invention. [Diagram 2] FIG. 2 is a graph showing the gene expression of drug-metabolizing enzymes in cells obtained by the method of the present invention, expressed as relative values. [Diagram 3] FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. [Figure 4] FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. [Diagram 5] FIG. 2 shows a microscopic view of cells obtained by the method of the present invention. [Figure 6] FIG. 2 shows a microscopic view of cells obtained by the method of the present invention. [Figure 7]FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. [Figure 8] FIG. 2 shows a microscopic view of cells obtained by the method of the present invention. [Figure 9] FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. [Figure 10] FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. [Figure 11] FIG. 1 shows the expression levels of marker genes in cells obtained by the method of the present invention, expressed as relative values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, "small intestinal epithelial cells" refers to cells contained in the tissue covering the lumen surface of the small intestine, including absorptive cells, endocrine cells, and crypt cells (including mucus gland cells, serous gland cells, and stem cells) of the small intestine. To distinguish from cells in the body and cells directly obtained from the body, cells having the properties of small intestinal epithelial cells artificially differentiated or induced outside the body (ex vivo, in vitro) are called "small intestinal epithelial-like cells." Examples of markers that serve as indicators of small intestinal epithelial-like cells include intestinal cell marker genes and their products, such as Villin 1, Cdx2 (Caudal related homeobox gene type 2), SI (sucrase-isomaltase), Ifabp, Isx, lactase, Glut2, ZO-1 protein (Zonula (Zona) occludens 1 protein, also known as Tight-junction protein-1 (TJP-1)), the drug metabolizing enzyme CYP3A4, the transporters P-gp, PEPT1, ANPEP, OSTα, and BCRP.

[0014] In the present invention, the term "small intestinal epithelial progenitor cells" refers to cells capable of differentiating into small intestinal epithelial-like cells, and includes small intestinal epithelial stem cells and intestinal epithelial stem cells. Markers that serve as indicators of small intestinal epithelial progenitor cells include intestinal cell marker genes and their products, such as leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), ephrin B2 receptor (EphB2), Musashi-1, CDX2, ALCAM, ASCL2, ITGB1, OLFM4, and SOX9.

[0015] In the present invention, "definitive endoderm cells" refer to endoderm cells, particularly cells that can differentiate into the entire intestinal tract, including the esophagus, stomach, small intestine, and large intestine, as well as organs derived from the intestinal tract, such as the lung, liver, thymus, parathyroid gland, thyroid gland, gallbladder, and pancreas. Specifically, the term refers to cells that are positive for endodermal cell markers E-cadherin (ECD) and CXCR4, or ECD and CD55.

[0016] In the present invention, the term "pluripotent stem cells" refers to cells that have the ability to proliferate under artificially constructed conditions (in vitro), such as in a test tube, and can differentiate into cells of all tissues of the body. In the present invention, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells can be used as pluripotent stem cells.

[0017] The ES cells used in the present invention may be mammalian-derived ES cells, and the type of ES cells is not particularly limited, and may be, for example, somatic cells of primates such as humans and chimpanzees, or rodents such as mice and rats, and preferably human ES cells. In the present invention, mouse ES cell line R1 may be used if derived from mice, and human ES cell lines KhES-1, KhES-2, KhES-3, and H1 and H9 may be used if derived from humans.

[0018] iPS cells are cells with pluripotency (multipotency) and proliferation ability, which are produced by reprogramming somatic cells by introducing reprogramming factors. iPS cells show properties similar to ES cells. The somatic cells used to produce iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the cells is also not particularly limited, but preferably somatic cells of mammals (e.g., primates such as humans and chimpanzees, and rodents such as mice and rats), and particularly preferably human somatic cells, can be used. iPS cells can be produced by various methods reported so far. Cells transformed into iPS cells, that is, cells that have undergone reprogramming (reprogramming), can be selected using the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Crypt as an index. iPS cells can also be provided, for example, from Kyoto University, a national university corporation, or the RIKEN Bioresource Center, an independent administrative institution.

[0019] The present invention will be described in detail below.

[0020] (1) A method for producing small intestinal epithelial-like cells The present invention provides a method for producing small intestinal epithelial-like cells, comprising the steps of: (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D; and (ii) obtaining small intestinal epithelial-like cells;

[0021] The method of obtaining small intestinal epithelial progenitor cells used in the method of the present invention is not particularly limited, and for example, they may be isolated from a living body by a known method, or may be prepared by differentiation from endoderm cells or pluripotent stem cells. For example, JP 2008-206510 A describes a method of obtaining intestinal stem / progenitor cells from the intestine of a living body. The origin is not particularly limited, but preferably mammalian cells (e.g., primates such as humans and chimpanzees, and rodents such as mice and rats), and particularly preferably human cells, can be used.

[0022] When differentiating small intestinal epithelial progenitor cells from pluripotent stem cells, for example, pluripotent stem cells can be differentiated into endoderm cells (definitive endoderm cells), and endoderm cells can be differentiated into small intestinal epithelial progenitor cells. A known method can be used to differentiate pluripotent stem cells into endoderm cells. For example, a medium containing a compound such as a GSK3 inhibitor (e.g., CHIR99021), Activin A, a PI3K inhibitor (e.g., Ly294002), a DNA methylation inhibitor (e.g., 5-Aza-2'-deoxycytidine), or DMSO (dimethyle sulfoxide) in a basic medium can be used, and a medium containing Activin A can be particularly preferably used. The amount of the compound added is not particularly limited as long as it is an amount that allows differentiation from pluripotent stem cells into endoderm cells. In addition, a commercially available kit such as Cellartis (registered trademark) Definitive Endoderm Differentiation Kit (Takara Bio Inc.) can be used. The culture period for differentiation is not particularly limited, but is, for example, 3 to 10 days, and preferably 4 to 8 days.

[0023] A known method can be used to differentiate endoderm cells into small intestinal epithelial precursor cells. For example, a medium containing a compound such as a GSK-3 inhibitor (e.g., LY2090314, BIO) or a γ-secretase inhibitor (e.g., DAPT) as a Notch signal inhibitor in a basal medium can be used. The amount of the compound added is not particularly limited as long as it is an amount that allows differentiation of endoderm cells into small intestinal epithelial precursor cells. For example, when LY2090314 is used, it is added to the medium at a concentration of 1 to 40 nM, preferably 5 to 30 nM. The culture period for differentiation is not particularly limited, but is, for example, 2 to 8 days, preferably 3 to 5 days.

[0024] As the active vitamin D used in step (i) of the present invention, for example, active vitamin D2 or active vitamin D3 (calcitriol) can be used. The concentration of active vitamin D is, for example, 1 nM to 10 μM, preferably 10 nM to 1 μM, and particularly preferably 50 to 500 nM, as a final concentration in the medium. The medium used in step (i) is not particularly limited as long as it is a medium that differentiates small intestinal epithelial progenitor cells into small intestinal epithelial-like cells, and for example, a medium supplemented with a GSK-3 inhibitor (LY2090314, BIO, SB216763, SB415286, CHIR99021, etc.), a Wnt signal agonist (Wnt3a, etc.), a TGFβ receptor inhibitor (for example, an ALK5 inhibitor that blocks Smad signals (SB431542, etc.)), an epidermal growth factor (EGF) (for example, human EGF), and / or a γ-secretase inhibitor (DAPT) can be used. Preferably, a medium containing a GSK-3 inhibitor (preferably LY2090314) and a TGFβ receptor inhibitor (preferably SB431542) added to a basal medium can be used. More preferably, a medium containing a GSK-3 inhibitor (preferably LY2090314), a TGFβ receptor inhibitor (preferably SB431542) and EGF (preferably human EGF) added to a basal medium can be used. The amount of the compound added is not particularly limited as long as it is an amount that allows differentiation from small intestinal epithelial precursor cells to small intestinal epithelial-like cells. The medium is used with the addition of active vitamin D. The culture period for differentiation is not particularly limited, but is, for example, 5 to 30 days, preferably 10 to 25 days. During the culture period, passage and / or medium exchange can be performed as appropriate.

[0025] In the present invention, the basal medium used for the preparation of small intestinal epithelial progenitor cells and step (i) is not particularly limited, and examples thereof include MEM (Earle's Salt), Iscove's modified Dulbecco's medium (IMDM), Dulbecco's modified Eagle's medium (D-MEM), Ham's F-12 medium, RPMI1640 medium, and Glasgow basal medium. Culture conditions (culture temperature, etc.) may be those generally adopted in the culture of animal cells. That is, for example, the cells may be cultured in an environment of 37°C and 5% CO2.

[0026] In the present invention, from the viewpoint of maintaining the proliferation rate and survival rate of the cells, it is preferable to add serum, plasma or a serum substitute (Knockout serum replacement (KSR), B-27 Supplement, etc.) to the medium used for the preparation of small intestinal epithelial progenitor cells and in step (i). As serum, fetal bovine serum, human serum, sheep serum, etc. can be used. The amount of serum or serum substitute added is, for example, 0.1% (v / v) to 10% (v / v).

[0027] In the present invention, feeder cells can be used for the preparation of small intestinal epithelial progenitor cells and the culture in step (i). For example, mitomycin C-treated mouse embryonic fibroblasts (MEFs) can be used. Also, extracellular matrix (ECM) secreted by connective tissue cells can be used. ECM consists of, for example, polysaccharides, water, elastin, and glycoproteins (matrix proteins). Matrix proteins include collagen, entactin (nidogen), fibronectin, and laminin. The ECM can be provided by culturing ECM-producing cells, for example, fibroblasts, and then removing these cells. As ECM-producing cells, chondrocytes producing collagen and proteoglycans, fibroblasts producing type IV collagen, laminin, interstitial procollagen, and fibronectin, and colonic myofibroblasts producing collagen (types I, III, and V), chondroitin sulfate proteoglycans, hyaluronic acid, fibronectin, and tenascin-C can be used. Furthermore, ECM is commercially available, and for example, a basement membrane preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Matrigel™ (BD Biosciences)) or a human recombinant laminin fragment (e.g., iMatrix-511) can be used. The above culture can also be performed in the presence of an isolated matrix protein or a fragment thereof. For example, collagen, fibronectin, vitroctin, laminin, or a fragment thereof can be used for the culture. As the matrix protein or a fragment thereof, for example, an isolated substance or a recombinant substance produced by genetic recombination technology can be used.

[0028] In the present invention, cell culture may be performed using an appropriate container selected in consideration of the number of cells and other conditions. Any type of container for cell culture may be used, such as a plate, a petri dish, a flask, or a bag. In addition, a carrier for serving as a scaffold for cells, such as beads, a membrane, or a hollow fiber, may be used.

[0029] Step (i) of the present invention may further include a step of freezing the small intestinal epithelial progenitor cells and a step of thawing the frozen small intestinal epithelial progenitor cells. Long-term storage of the cells by interrupting the culture and freezing them is useful for supplying small intestinal epithelial-like cells at an appropriate time. The step of freezing the cells is carried out 5 to 15 days, preferably 7 to 13 days, after starting the culture of the small intestinal epithelial progenitor cells.

[0030] The step of freezing cells can be performed by freezing the cells in the medium as is, or by replacing the medium with a cryoprotectant and then freezing. Cell freezing methods are generally broadly divided into slow freezing and rapid freezing (vitrification). Either freezing method may be used in the present invention. The slow freezing method is a method in which cells are suspended in a cryopreservation solution containing glycerin, DMSO, keratin hydrolysate, hydrolyzed gelatin, serum, serum albumin, etc. as a cryoprotectant, and the temperature is lowered by about 1°C per minute to gradually freeze the cells. By slowly cooling, water molecules in the cells are replaced with the cryoprotectant, dehydration is performed, and the growth of ice crystals in and around the cells is suppressed, preventing damage to the cell membrane and intracellular structure, and denaturation and cleavage of proteins. It is not necessary to strictly control the temperature using a programmable freezer, and a simple slow freezing method in which cells are slowly frozen using a polystyrene foam container or a commercially available cell freezing box may be used.

[0031] On the other hand, vitrification is a method in which cells are frozen into a glass-like state by rapid cooling in order to suppress the formation of ice crystals inside and outside the cells due to freezing. A known vitrification method uses a vitrification preservation solution containing cryoprotectants consisting of high concentrations of DMSO, acetamide, propylene glycol, and polyethylene glycol.

[0032] Cryoprotectants are commercially available, such as STEM-CELLBANKER (Nihon Zenyaku Kogyo Co., Ltd.), CryoDefend-Stem Cell (R&D Systems Co., Ltd.), Bambanker (Nihon Genetics Co., Ltd.), and Labobanker (registered trademark) 2 (Juji Field Co., Ltd.), and cells can be frozen according to the respective instructions.

[0033] Frozen cells can be stored in a low-temperature freezer or ultra-low-temperature freezer (usually within the range of -20°C to -150°C) or in liquid nitrogen (usually within the range of -150°C to -196°C).

[0034] If the cells to be frozen are attached to the container wall, they can be released (detached) from the container wall, collected, and suspended in a cryoprotectant. Cells can be detached by, for example, enzymatic treatment with a protease (e.g., trypsin, etc.) and / or mechanical or physical treatment with pipetting or a scraper. Examples of cell detachment agents that can be used include Accutase (registered trademark, Innovative Cell Technologies), TrypLE (registered trademark) Express (Thermo Fisher Scientific), TrypLE (registered trademark) Select (Thermo Fisher Scientific), and TrypZean (Sigma), which are free of mammalian and bacterial components.

[0035] The step of thawing the frozen cells can be carried out by any cell thawing method. For example, a water bath, an incubator, a thermostat, or the like can be used to bring the frozen cells into contact with a solid, liquid, or gaseous medium (e.g., water, gas phase) at a temperature higher than the melting temperature. The frozen cells can also be thawed by immersing them in a medium (e.g., culture solution) at a temperature higher than the freezing temperature. The temperature of the thawing means or immersion medium is not particularly limited as long as it is a temperature at which the cells can be thawed within a desired time, and is, for example, 4 to 50°C, preferably 30°C to 40°C, and more preferably 36 to 38°C. The thawing time is not particularly limited as long as it does not significantly impair the survival rate or function of the cells after thawing, and is, for example, 1 to 10 minutes, and preferably 2 to 5 minutes.

[0036] The thawed small intestinal epithelial progenitor cells are seeded again in a medium, for example, the medium used before freezing. The culture period after thawing is appropriately set according to the total culture period of the small intestinal epithelial progenitor cells and the culture period before freezing, but may be set so that the total culture period is about 5 to 30 days, for example, 2 to 10 days, preferably 4 to 8 days. In one embodiment of the present invention, the thawed small intestinal epithelial progenitor cells can be cultured in a medium containing, in addition to active vitamin D, one or more substances selected from the group consisting of MEK inhibitors, cAMP and its derivatives, Rho kinase inhibitors, and vinca alkaloid antitumor drugs. In a preferred embodiment of the present invention, the thawed small intestinal epithelial progenitor cells can be cultured in a medium containing active vitamin D, TGFβ receptor inhibitors, and GSK inhibitors, and further containing one or more substances selected from the group consisting of MEK inhibitors, cAMP and its derivatives, Rho kinase inhibitors, and vinca alkaloid antitumor drugs.

[0037] Examples of Rho kinase (ROCK) inhibitors include, but are not limited to, Y27632, HA1100, HA1077, thiazovivin, and GSK429286. The Rho kinase inhibitor can be added to the medium during the entire or part of the culture period after thawing of the small intestinal epithelial progenitor cells, and is added to the medium for 1 to 5 days after thawing of the cells, preferably 1 to 3 days after thawing, and particularly preferably 1 day after thawing. The concentration of the Rho kinase inhibitor may be appropriately set, and when using Y27632, it is added to the medium at a final concentration of, for example, 0.1 to 100 μM, preferably 1 to 10 nM.

[0038] Vinca alkaloids are bis-indole alkaloids with anticancer activity isolated from Catharanthus roseus (L.) G. Don and Catharanthus roseus (L.) G. Don cv. Flavus, both plants of the Apocynaceae family. There are four types of Catharanthus roseus alkaloids and their derivatives currently in clinical use: vinblastine (VLB), vincristine (VCR), vindesine (VDS), and vinorelbine (NVB). Vinca alkaloids are cell cycle-specific drugs that act primarily on the G2 phase (late DNA synthesis) of cells. It has been reported that the mechanism of action of vinca alkaloids is to bind to tubulin, inhibit the polymerization of tubulin double minute to form microtubules, and induce depolymerization of microtubules, inhibit the formation of spindles, and arrest the division and proliferation of tumor cells at metaphase of mitosis, thereby exerting an antitumor effect. Vinca alkaloid antitumor drugs can be added to the medium throughout the entire period of culture after thawing of small intestinal epithelial progenitor cells, and are preferably added to the medium for 1 to 6 days after thawing of cells, and particularly preferably for 2 to 4 days after thawing. When vinblastine is used, it is added to the medium at a final concentration of, for example, 0.1 to 1000 nM, preferably 1 to 100 nM.

[0039] As the MEK inhibitor, known MEK inhibitors can be used, for example, trametinib, cobimetinib, selumetinib, refametinib, pimasertib, U0126, MEK162 / ARRY-162, AZD8330 / ARRY-424704, GDC-0973 / Examples of the MEK inhibitor include RG7420, GDC-0623 / RG7421 / XL518, CIF / RG7167 / RO4987655, CK127 / RG7304 / RO5126766, E6201, TAK-733, PD-0325901, AS703988 / MSC2015103B, WX-554, CI-1040 / PD184352, AS703026, PD318088, PD98059, and SL327. The concentration of the MEK inhibitor may be appropriately set. For example, when PD-0325901 is used, it is added to the medium at a final concentration of, for example, 0.1 to 100 μM, preferably 0.5 to 50 nM.

[0040] The cAMP derivative is a compound in which a substituent has been modified to cyclic AMP, and examples thereof include 8-bromo cyclic adenosine monophosphate (8-Br-cAMP, also simply referred to as Br-cAMP), 8-chloro cyclic adenosine monophosphate (8-Cl-cAMP), 8-(4-chlorophenylthio) cyclic adenosine monophosphate (8-CPT-cAMP), and dibutyryl cyclic adenosine monophosphate (DB-cAMP). The concentration of cAMP or a cAMP derivative may be appropriately set. For example, when Br-cAMP is used, it is added to the medium at a final concentration of, for example, 0.1 to 1000 μM, preferably 0.5 to 500 nM, more preferably 1 to 300 nM.

[0041] The step of obtaining small intestinal epithelial-like cells is a step of obtaining cells differentiated into small intestinal epithelium obtained in step (i) or a subsequent step. The cells contained in the cell population obtained in step (i) or a subsequent step may be obtained as small intestinal epithelial-like cells as they are, or they may be obtained as small intestinal epithelial-like cells by isolating, separating and purifying the small intestinal epithelial-like cells using a marker for small intestinal epithelial cells.

[0042] The small intestinal epithelial-like cells obtained by the production method of the present invention are cells obtained by artificially differentiating small intestinal epithelial precursor cells, and are characterized by expressing a drug-metabolizing enzyme or a drug transporter. The small intestinal epithelial-like cells also express Villin and CDX2, which are small intestinal epithelial markers. Furthermore, the expression levels of the drug-metabolizing enzyme CYP3A4 and transporters PEPT1, P-gp, etc. in the small intestinal epithelial-like cells are equal to or higher than those of Caco-2 cells, which are widely used as an intestinal epithelial cell model. The small intestinal epithelial-like cells also form strong tight junctions between cells, which are characteristic of small intestinal epithelial cells, and highly express TEER and ZO-1 protein (Zonula (Zona) occludens1 protein, also known as Tight-junction protein-1 (TJP-1)). The present invention makes it possible to provide stable and excellent small intestinal epithelial-like cells, instead of primary cultured human small intestinal epithelial cells, which are difficult to obtain and have individual differences as a problem.

[0043] The small intestinal epithelial-like cells obtained by the production method of the present invention are a cell population having a positive rate of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% for Villin 1. Small intestinal epithelial-like cells may be further isolated, separated, or purified from this cell population using a marker for small intestinal epithelial cells.

[0044] (2) Culture medium for producing small intestinal epithelial-like cells The present invention provides a medium containing a TGFβ receptor inhibitor, a GSK inhibitor, and active vitamin D. This medium is prepared by adding a TGFβ receptor inhibitor, a GSK inhibitor, and active vitamin D to the basal medium described in (1). The medium of the present invention may further contain EGF, for example, human EGF. The medium of the present invention may further contain one or more substances selected from the group consisting of MEK inhibitors, cAMP and its derivatives, Rho kinase inhibitors, and vinca alkaloid antitumor drugs. The concentration of each component contained in the medium of the present invention can be set to a concentration suitable for producing small intestinal epithelial-like cells. The medium of the present invention can be used to produce small intestinal epithelial-like cells as described in (1). Each component contained in the medium of the present invention is as described in (1).

[0045] (3) Drug evaluation method using small intestinal epithelial-like cells The present invention provides a method for evaluating drug toxicity and a method for evaluating pharmacokinetics, which are characterized by using small intestinal epithelial-like cells obtained by the above-mentioned production method.Furthermore, the present invention provides a method for testing drug interactions and a method for testing drug-metabolizing enzyme induction, which are characterized by using the small intestinal epithelial-like cells.By using the small intestinal epithelial-like cells obtained by the present invention, it becomes possible to perform with higher accuracy and reproducibility the evaluation of drug toxicity / pharmacokinetics of drug candidate compounds, research into drug interactions, and evaluation of drug-metabolizing enzyme induction ability, which have conventionally been performed using primary cultured human small intestinal epithelial cells. EXAMPLES

[0046] The present invention will be specifically described below with reference to examples and experimental examples in order to deepen understanding of the present invention, but these are not intended to limit the scope of the present invention.

[0047] Example 1 Preparation of small intestinal epithelial-like cells expressing CYP3A4 The following medium 1 to medium 3 were prepared. "Medium 1" Human iPS cells were cultured in DEF-CS (Takara Bio) supplemented with bFGF to a final concentration of 30 ng / mL, and maintained in an undifferentiated state.

[0048] "Medium 2" Differentiation into endoderm cells was induced using the Cellartis DEF-CS 500 Culture System (Takara Bio Inc.) according to the attached protocol.

[0049] "Medium 3" For differentiation into endoderm cells and beyond, DMEM-high Glucose medium containing 10% Knockout Serum Replacement (Thermo Fisher Scientific), 100 units / mL penicillin G, 100 μg / mL streptomycin sulfate, 2 mM L-Glutamin, and 1% Non Essential Amino Acid Solution (Thermo Fisher Scientific) was used.

[0050] Endoderm cells were generated using human iPS cells, which are pluripotent stem cells. ChiPSC18 (Takara Bio Inc.) was used as the human iPS cell line. 1.5 × 10 6 The cells were seeded onto a fibronectin-coated T75 flask and precultured under feeder-free conditions using Medium 1 according to the manual attached to the product.

[0051] The human iPS cell line obtained by preculture was induced to differentiate into endoderm cells by culturing for 6 days using medium 2 and a Cellartis (registered trademark) Definitive Endoderm Differentiation Kit, in accordance with the manual attached to the product.

[0052] Endoderm cells, 1 x 10 6 / well(2.5×10 5 cells / cm 2 ) on a 12-well plate (3.8 cm 2 ) and cultured for 4 days in medium 3 containing 20 nM LY2090314 to induce differentiation into small intestinal epithelial progenitor cells.

[0053] Small intestinal epithelial progenitor cells were cultured for 20 to 25 days in medium 3 (hereinafter referred to as "medium 3-A") containing 3 nM LY2090314, 100 nM active vitamin D3, 10 μM SB431542, and 50 ng / mL human EGF to induce differentiation into small intestinal epithelial-like cells. The medium was changed every other day.

[0054] As a control, endoderm cells were induced to differentiate into small intestinal epithelial progenitor cells using medium 3 containing 5 μM BIO and 10 μM DAPT, and the small intestinal epithelial progenitor cells were induced to differentiate into small intestinal epithelial-like cells using medium 3 containing 1 μM BIO, 2 μM DAPT, 10 μM SB431542, and 50 ng / mL human EGF. The method for producing small intestinal epithelial-like cells of the present invention and the existing method are illustrated in FIG.

[0055] Example 2 Gene expression of drug metabolizing enzyme CYP3A4 On day 20 from the start of the culture of the small intestinal epithelial precursor cells in Example 1, each type of small intestinal epithelial-like cell was collected.

[0056] From these cells, total RNA samples for real-time PCR were obtained using Nucleo Spin RNA plus (Takara Bio). In addition, as another control, total RNA of human adult small intestine tissue (5 donors, Clontech) and total RNA of human colon cancer-derived Caco-2 cells were prepared. Gene expression of drug-metabolizing enzymes (CYP3A4) was evaluated by real-time PCR using the housekeeping gene GAPDH as an internal control. The relative values, with the gene expression in human adult small intestine tissue taken as 1, are shown in Figure 2. In Figure 2, the results of the existing method are shown as "(1)", the results of the method of the present invention are shown as "(2)", the results of the RNA of human adult small intestine tissue are shown as "Small intestine", and the results of Caco-2 cells are shown as "Caco2". As shown in Figure 2, the cells obtained by the production method of the present invention showed higher expression of drug-metabolizing enzymes than the cells obtained by the existing method and Caco-2 cells.

[0057] Example 3: Time course of gene expression in small intestinal epithelial-like cells Three types of human iPS cell lines, ChiPSC18 (C18), ChiPSC12 (C12), and ChiPSC22 (C22) (all from Takara Bio Inc.), were prepared, and differentiation of small intestinal epithelial-like cells was induced by the same method as in Example 1. The C18 cells were harvested on the 10th, 15th, and 21st days after the start of the culture of the small intestinal epithelial progenitor cells. The C12 and C22 cells were harvested on the 15th and 21st days after the start of the culture of the small intestinal epithelial progenitor cells. The gene expression of small intestinal markers (Villin, CDX-2, SI), drug metabolizing enzymes (CYP3A4), and transporters (P-gp) was measured by the same method as in Example 2. Total RNA derived from human adult small intestinal tissue was used as a control. The relative values, with the gene expression of human adult small intestinal tissue set to 1, are shown in Figure 3. As shown in Figure 3, the gene expression of Villin, CYP3A4, and P-gp tended to increase over time in all human iPS cell line-derived cells. In addition, in ChiPSC18, the expression of SI, CYP3A4, and P-gp was confirmed on the 10th day after the start of culture of small intestinal epithelial progenitor cells.

[0058] Example 4 Gene expression in small intestinal epithelial-like cells prepared by detachment and replated In order to confirm the effects of detachment and reseeding, gene expression was examined in small intestinal epithelial-like cells prepared by detaching and reseeding small intestinal epithelial progenitor cells.

[0059] Small intestinal epithelial progenitor cells were prepared in the same manner as in Example 1. Small intestinal epithelial progenitor cells were cultured in medium 3-A, and the medium was removed on the 10th day after the start of culture. The cells were washed once with calcium- and magnesium-free phosphate-buffered saline (PBS(-)), and then the cells were detached using a detachment agent, Accumax (Innovative Cell Technologies), and the dispersed cells were collected. The cells were washed and resuspended in medium 3-A, stained with 0.4% trypan blue solution (GIBCO), and the total number of live cells was calculated. The cells were diluted to 2.5 × 10 5 / cm 2 (1×10 6The cells were replated on a Matrigel-coated 12-well plate at a density of 1000 cells / well and cultured for 10 days using medium 3-A. As a control, small intestinal epithelial-like cells were prepared in the same manner as in Example 1, without detachment and reseeding.

[0060] On the 20th day after the start of culturing the small intestinal epithelial progenitor cells, each type of cell was collected. Using the same method as in Example 3, the expression of small intestinal markers (Villin, CDX-2, SI), drug metabolizing enzymes (CYP3A4), and transporters (P-gp, PEPT1, ANPEP, OSTα) genes was measured. In addition, total RNA (Clontech) derived from human adult small intestinal tissue was used as a control. As a result of the measurement, the detached and reseeded induced cells showed no significant difference in gene expression of SI, P-gp, CYP3A4, and ANPEP compared to cells that were not detached and reseeded.

[0061] Example 5 Cryopreservation of small intestinal epithelial progenitor cells Since it was confirmed in Example 4 that there was no significant effect when small intestinal epithelial progenitor cells were detached and reseeded, the detached cells were frozen, thawed, and then reseeded, and the cells were evaluated.

[0062] Small intestinal epithelial progenitor cells were prepared in the same manner as in Example 1. Small intestinal epithelial progenitor cells were cultured in medium 3-A, and the medium was removed on the 10th day after the start of culture. The cells were washed once with PBS(-), and then detached using a detachment agent, Accumax, and the dispersed cells were collected. The cells were washed and resuspended in medium 3-A, stained with 0.4% trypan blue, and the cells were counted to confirm a viability of 80-90%. The suspended cells were collected by centrifugation, and 2 x 10 cells were added to 1 mL of a cryoprotectant, CellBanker (registered trademark) 1plus (Takara Bio Inc.). 6 The cells were suspended at 100 cells / mL and frozen at -80° C. After freezing, storage in liquid nitrogen was started.

[0063] Example 6 Thawing and culturing of small intestinal epithelial progenitor cells The following medium 6 was prepared. "Medium 6" Four inducers were added to 10% KSR+DMEM (Dulbecco's modified Eagle medium containing 10% KSR, 100 unit / mL penicillin G, 100 μg / mL streptomycin sulfate, 2 mmol / mL alanyl-L-glutamine, and 1% MEM essential amino acid solution (×100)) at the following concentrations to prepare medium 6; 3 nM LY2090314, 100 nM active vitamin D3 (calcitriol), 10 μM SB431542, and 50 ng / mL human EGF.

[0064] Matrigel (registered trademark) was diluted 48-fold with DMEM to prepare a Matrigel solution, and while cooling on ice, 200 μL / cm was added to a culture vessel (12-well plate). 2 The culture vessel was kept in a cell culture incubator at 37°C for 30 minutes or more to coat the culture vessel with Matrigel, and the Matrigel solution was removed from the culture vessel. The frozen preserved cells prepared in Example 5 were thawed in a 37°C water bath and centrifuged at 200 x G for 5 minutes at room temperature to remove the supernatant containing the cryoprotectant. The cells were suspended in 1 mL of medium 6, and the total number of live cells was calculated using a 0.4% trypan blue solution, and the entire amount was poured into a Matrigel-coated 12-well plate at 4.5 x 10 5 cells / cm 2 After thawing, the cells were cultured for 10 days with medium 6 replaced every other day.

[0065] The cells were collected on the 10th day of culture after thawing. The expression of the small intestine type marker (Villin), drug metabolizing enzyme (CYP3A4), and transporter (P-gp) genes was measured in the same manner as in Example 3. As a control, the expression of marker genes was measured for each of the Total RNA (Clontech) derived from human adult small intestine tissue, Caco2 cells, the small intestine epithelial-like cells of Example 1 (cells cultured for 20 days from the start of culture of small intestine epithelial precursor cells), and the small intestine epithelial-like cells of Example 4 (cells detached and reseeded on the 10th day from the start of culture of small intestine epithelial precursor cells, and then cultured for 10 days). The results are shown in FIG. 4. In FIG. 4, the Total RNA derived from human adult small intestine tissue is shown as "small intestine", the small intestine epithelial-like cells of Example 1 as "continuous culture", the small intestine epithelial-like cells of Example 4 as "Day10 Seeding", and the frozen and thawed cells of this Example as "Day10 Freeze & Thaw". As shown in FIG. 4, frozen and thawed cells did not show any significant effect on gene expression compared to cells that were continuously cultured or cells that were detached and reseeded.

[0066] Example 7 Freezing and thawing of small intestinal epithelial progenitor cells and small intestinal epithelial-like cells A comparative study was performed on small intestinal epithelial-like cells prepared by freezing and thawing the small intestinal epithelial progenitor cells on the 11th and 20th days after the start of culture.

[0067] Small intestinal epithelial-like cells prepared by the method of Example 5, except that the small intestinal epithelial progenitor cells were cultured from the initiation of culture to the 11th day, were collected on the 11th day, were frozen by the method of Example 5, and were thawed by the method of Example 6. In addition, small intestinal epithelial-like cells prepared by the method of Example 1 were collected on the 20th day from the initiation of culture, were frozen by the method of Example 5, and were thawed by the method of Example 6.

[0068] It is known that small intestinal epithelial cells have strong intercellular adhesion, form tight junctions, and have a barrier function. To confirm the formation of tight junctions, all of the thawed cells were reseeded onto cell inserts of a 6-well plate coated with Matrigel. The cells were cultured for 8 days, with medium replacement for induction culture being performed every other day with Medium 6.

[0069] Microscopic images of the cells after culturing are shown in Figure 5. No morphological abnormalities were observed in the cells frozen on day 11 after the start of culturing the small intestinal epithelial progenitor cells (Day 11 cryopreservation), whereas morphological abnormalities were observed after thawing in the cells frozen on day 20 (Day 20 cryopreservation).

[0070] Furthermore, the TEER (transepithelial electrical resistance) of the cultured cells was measured using a Millicell ERS-2 resistance measurement system to confirm the function of tight junctions. 1.5 mL of 10% KSR+DMEM was added to the apical side (cell insert) and 2.7 mL to the basolateral side (companion plate), and measurements were taken of wells seeded with cells and blank wells not seeded with cells. Unit area resistance was calculated using the following formula: Resistance value (Ω) = sample (Ω) - blank (Ω) Unit area resistance = resistance value (Ω) × effective area of ​​membrane (cm 2 )(6 wells: 4.2cm 2 ) The results are shown in Table 1.

[0071] [Table 1]

[0072] The TEER measurement results showed that the TEER of cells frozen on the 20th day after the start of small intestinal epithelial progenitor cell culture (Day 20 cryopreservation) was no different from that of the blank, and no tight junction formation was confirmed. On the other hand, the formation of tight junctions was confirmed in cells frozen on the 11th day (Day 11 cryopreservation).

[0073] Furthermore, the permeability of Lucifer Yellow was measured for the cells after the same culture to confirm the tight junction function. Lucifer Yellow is a substance that permeates the intercellular space, but cannot permeate the intercellular space of cells that form tight junctions, such as the small intestinal epithelium. 10 μM Lucifer Yellow (1.5 mL) was added to the apical side, and buffer (2.7 mL) was added to the basolateral side, and the permeation to the basolateral side was measured and confirmed by the fluorescence wavelength. Standard solutions were prepared by serial dilution of known concentrations of Lucifer Yellow, and a standard curve was created to calculate the amount of Lucifer Yellow permeation and the permeability coefficient (Papp). The buffer used was HBSS_Ca(+), Mg(+) supplemented with 25 mM glucose and 10 mM HEPES pH 7.4. The results are shown in Table 2.

[0074] [Table 2]

[0075] Measurement of the permeability coefficient of Lucifer Yellow showed that the cells frozen on the 20th day after the start of the culture of small intestinal epithelial progenitor cells (frozen on Day 20) had a high permeability coefficient and no tight junction formation was confirmed. On the other hand, the formation of tight junctions was confirmed in the cells frozen on the 11th day (frozen on Day 11).

[0076] The results in Tables 1 and 2 show that cryopreservation of small intestinal epithelial-like cells at around day 20, when maturation has progressed, becomes difficult, whereas cryopreservation of small intestinal epithelial progenitor cells at around day 11 is possible.

[0077] Example 8 Addition of Vinca Alkaloid Antitumor Drugs The following medium 5 was prepared, containing vinblastine as a vinca alkaloid antitumor drug. "Medium 5" 10% KSR+DMEM containing 3nM LY2090314, 100nM active vitamin D3, 10μM SB431542, 50ng / mL human EGF, and 10nM vinblastine.

[0078] The frozen cells prepared in Example 5 were thawed in a 37°C water bath and centrifuged at 200×G for 5 minutes at room temperature to remove the supernatant containing the cryoprotectant. The cells were suspended in 1 mL of Medium 5, and the total number of live cells was calculated using a 0.4% trypan blue solution. The total amount was then plated onto a Matrigel-coated 12-well plate at 4.5×10 5 cells / cm 2 The cells were then reseeded with Medium 5. After thawing, the cells were cultured for 10 days with Medium 5 replaced every other day. Figure 6 shows a microscopic image taken on the 10th day of culture after reseeding using Medium 5. It was confirmed that the use of a medium containing 10 nM vinblastine resulted in more uniform growth of small intestinal epithelial-like cells than the use of a medium without vinblastine (0 nM).

[0079] Furthermore, the concentration of vinblastine contained in medium 5 was changed to 0 nM, 3 nM, and 10 nM to induce differentiation of small intestinal epithelial-like cells, and the expression of small intestinal markers (Villin, CDX-2), drug metabolizing enzymes (CYP3A4), and transporter (PEPT1, ANPEP, OSTα) genes was measured in the same manner as in Example 3. Total RNA (Clontech) derived from human adult small intestinal tissue was prepared as a control. The results are shown in Figure 7. In Figure 7, the total RNA derived from human adult small intestinal tissue is shown as "small intestine." As shown in Figure 7, it was shown that the addition of vinblastine increased the expression of marker genes in small intestinal epithelial cells.

[0080] Example 9 Addition of Rho kinase inhibitor A method was examined in which a medium containing Y27632 as a Rho kinase inhibitor was used for reseeding thawed small intestinal epithelial progenitor cells frozen by the procedure described in Example 5. The following medium 4 was prepared. "Medium 4" 10% KSR+DMEM basic medium containing 3nM LY2090314, 100nM active vitamin D3, 10μM SB431542, 50ng / mL human EGF, and 5μM Y27632.

[0081] In the same manner as in Example 6, the following four differentiation induction methods were compared: thawed cells were reseeded in medium 6 and cultured for 10 days (Y27632-, Vinblastine-), thawed cells were reseeded in medium 4 and cultured for 1 day, then cultured in medium 5 for 2 days, and then cultured in medium 6 for 7 days (Y27632+, Vinblastine+), thawed cells were reseeded in medium 4 and cultured for 1 day, then cultured in medium 6 for 9 days (Y27632+, Vinblastine-), thawed cells were reseeded in medium 6 and cultured for 1 day, then cultured in medium 5 for 2 days, and then cultured in medium 6 for 7 days (Y27632-, Vinblastine+). After reseeding of the cells, the adhesion and morphology of the cultured cells were confirmed by microscope 2 hours and 30 minutes later, on the 2nd day, and on the 5th day, and morphological images were obtained over time (Figure 8). It was shown that the addition of Y27632 increased adhesion efficiency, and the addition of 10 nM vinblastine resulted in a uniform morphology.

[0082] Example 10: Examination of reseeding density Small intestinal epithelial-like cells were prepared using the same medium and culture period, except that they were cultured for 8 days in medium 6, as compared with the Y27632+, Vinblastine+ cells in Example 9 (i.e., cells that were thawed and reseeded in medium 4 and cultured for 1 day, then cultured in medium 5 for 2 days, and then cultured in medium 6 for 7 days). Reseeding was performed by culturing small intestinal epithelial progenitor cells in a 24-well plate (1.9 cm 2 ) to 5×10 6 cells / well (2.5×10 5 cells / cm 2 ), and the cells were harvested on the 5th (Day 15), 8th (Day 18), and 11th (Day 21) days after reseeding. Furthermore, the reseeding density was changed to 3.75 × 10 5 cells / cm 2 , 5×10 5 cells / cm 2 The medium was changed to 1, and induction culture was performed, and the cells were harvested on day 21 after replated.

[0083] The expression of small intestinal markers (Villin, SI), drug metabolizing enzymes (CYP3A4), transporters (P-gp, PEPT1, ANPEP, OSTα), and tight junction (ZO-1) genes was measured using the same method as in Example 3. Total RNA (Clontech) derived from adult human small intestine tissue was prepared as a control. The results are shown in Figure 9. In Figure 9, total RNA derived from adult human small intestine tissue is shown as "small intestine", 3.75 x 10 5 cells / cm 2 The cells were reseeded at 375K, 5 × 10 5 cells / cm 2 In Figure 9, "Day 15," "Day 18," and "Day 21 (250K / cm2)" are the cells reseeded at 2.5 × 10 5 cells / cm 2 9. The figures show the cells on days 5, 8, and 11 after reseeding. As can be seen from FIG. 9, the culture after reseeding was for 5 to 8 days (15 to 18 days from the start of culture of the small intestinal epithelial progenitor cells), and the reseeding density was 3.75×10 5 cells / cm 2 ~5×10 5 cells / cm 2 was found to be preferred.

[0084] Example 11 Examination of added drugs 1 Small intestinal epithelial progenitor cells were frozen in the same manner as in Example 5. However, a solution of KSR:DMSO mixed at a ratio of 9:1 was used as a cryoprotectant. Various drugs were added to the medium 6 in which the thawed cells were reseeded. 10% fetal bovine serum (FBS, Bio West), GF [1% DEF-CS GF1 (Takara Bio), 50 ng / mL insulin-like growth factor (IGF), 100 ng / mL fibroblast growth factor (FGF)4], 1 μM PD0325901 (PD), 10 μM Br-cAMP, and 2% B-27 Supplement minus vitamin A (Thermo Fisher Scientific) were added to the medium under the conditions in Table 3. In the table, "〇" indicates the addition, and "×" indicates the absence of addition.

[0085] [Table 3]

[0086] In the same manner as in Example 6, the thawed cells were reseeded in medium 6 to which drugs were added according to conditions 1 to 7 in Table 3, and the cells were cultured for 8 days to recover. In the same manner as in Example 3, the expression of drug metabolizing enzyme (CYP3A4) and transporter (P-gp) genes was measured. As a control, total RNA (Clontech) derived from human adult small intestine tissue was prepared. The results are shown in FIG. 10. In FIG. 10, total RNA derived from human adult small intestine tissue is shown as "small intestine". From FIG. 10, it was confirmed that the addition of all drugs improved the expression of CYP3A4 compared to the case where none of the drugs were included. Furthermore, it was found that the expression of CYP3A4 was significantly reduced when PD0325901 was removed, and the expression of P-gp was reduced when PD0325901 and Br-cAMP were removed. From the above, it was shown that the addition of MEK inhibitors and cAMP or its derivatives was effective.

[0087] Example 12: Examination of added drugs 2 Endoderm cells prepared in the same manner as in Example 1 were diluted to 9.375 × 10 6 / Flask(1.25×10 5 cells / cm 2 ) in a Matrigel-coated T75 flask (75 cm 2 ) and cultured for 4 days in medium 3 containing 30 nM LY2090314 to induce differentiation into small intestinal epithelial progenitor cells.

[0088] Small intestinal epithelial progenitor cells were cultured using medium 3-A, and the medium was removed on day 8 after the start of culture. The cells were washed twice with PBS(-), then detached using Accumax detachment agent, and the dispersed cells were collected. The cells were washed and resuspended in medium 3-A, stained with 0.4% trypan blue, and counted to confirm a viability of 80-90%. The suspended cells were collected by centrifugation, and 7.5 × 10 cells were added to 1 mL of cryoprotectant (KSR:DMSO = 9:1 solution). 6The cells were suspended at 100 cells / mL and frozen at -80° C. After freezing, storage in liquid nitrogen was started.

[0089] The thawed cells were reseeded in medium 4 in the same manner as in Example 6 (Day 0). On Day 1, the medium was replaced with medium 5, and then on Days 3, 5, and 7, the medium was replaced with medium 6 containing the reagents in Table 4, and the cells were cultured for 7 days and then collected. The expression of drug metabolizing enzyme (CYP3A4) and transporter (P-gp) genes was measured in the same manner as in Example 3. Total RNA (Clontech) derived from human adult small intestine tissue was prepared as a control. The results are shown in Figure 11. In Figure 11, the total RNA derived from human adult small intestine tissue is shown as "small intestine". Figure 11 shows that the addition of PD0325901 and Br-cAMP is effective.

[0090] [Table 4]

[0091] Next, the following conditions were tested as shown in Table 5: the concentration of PD0325901 and Br-cAMP were fixed, the days of medium exchange for medium 6 were changed to days 2, 5, and 8, Br-cAMP was added from medium 4, in which cells were seeded on day 0, until the end of culture, and B-27 (trademark) Supplement minus vitamin A was added to medium 6. The expression of drug metabolizing enzymes (CYP3A4) and transporter (P-gp) genes was measured in the same manner as in Example 3. In addition, TEER (transepithelial electrical resistance) and permeability coefficient (Papp) were measured in the same manner as in Example 7.

[0092] [Table 5] [Industrial Applicability]

[0093] As described above in detail, the cells obtained by the production method of the present invention can be called "small intestinal epithelial-like cells" because the gene expression of each marker expressed by the small intestinal epithelium, drug-metabolizing enzymes and transporters has been confirmed, and the cells have tight junction function. In particular, the expression level of drug-metabolizing enzymes is superior to that of Caco-2 cells, which are used in existing technology. Furthermore, a method for producing these small intestinal type intestinal epithelial-like cells was established by freezing and storing small intestinal epithelial progenitor cells, and thawing and reseeding them with a simple operation of adding drugs, followed by induction culture. This makes it possible to produce and stably supply small intestinal type intestinal epithelial-like cells on a large scale, and also makes it possible to perform metabolic absorption tests of drugs and nutrients in a simple and short culture period. Therefore, the present invention provides small intestinal epithelial-like cells that are more versatile and convenient than primary cultured small intestinal epithelial cells, which are difficult to obtain and have variable performance. It is expected that the small intestinal epithelial-like cells obtained by the method of the present invention will greatly contribute to the development of drug metabolic absorption tests for pharmaceuticals, food analysis, and the like.

Claims

1. A method for producing small intestinal epithelial-like cells, comprising the steps of: (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D, a GSK-3 inhibitor, a TGFβ receptor inhibitor, and EGF, wherein the GSK-3 inhibitor is LY2090314 and the TGFβ receptor inhibitor is SB431542; and (ii) Obtaining small intestinal epithelial-like cells.

2. The method of claim 1 , further comprising the step of differentiating the endodermal cells into small intestinal epithelial progenitor cells.

3. The method of claim 2 , further comprising the step of differentiating the pluripotent stem cells into endoderm cells.

4. The method according to any one of claims 1 to 3, further comprising the steps of freezing the small intestinal epithelial progenitor cells and thawing the frozen small intestinal epithelial progenitor cells in the step (i), wherein the freezing step is performed 5 to 15 days after the start of the step (i).

5. The method according to claim 4, wherein the thawed small intestinal epithelial progenitor cells are cultured in a medium further containing one or more substances selected from a Rho kinase inhibitor, a vinca alkaloid antitumor drug, a MEK inhibitor, and cAMP and its derivatives.

6. (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D, a GSK-3 inhibitor, a TGFβ receptor inhibitor, and EGF, wherein the GSK-3 inhibitor is LY2090314 and the TGFβ receptor inhibitor is SB431542; and (ii) Obtaining small intestinal epithelial-like cells and producing small intestinal epithelial-like cells by a method comprising the steps of:

7. (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D, a GSK-3 inhibitor, a TGFβ receptor inhibitor, and EGF, wherein the GSK-3 inhibitor is LY2090314 and the TGFβ receptor inhibitor is SB431542; and (ii) Obtaining small intestinal epithelial-like cells 5. A method for testing drug interactions, comprising producing small intestinal epithelial-like cells by a method comprising the steps of:

8. (i) culturing small intestinal epithelial progenitor cells in a medium containing active vitamin D, a GSK-3 inhibitor, a TGFβ receptor inhibitor, and EGF, wherein the GSK-3 inhibitor is LY2090314 and the TGFβ receptor inhibitor is SB431542; and (ii) Obtaining small intestinal epithelial-like cells A method for testing induction of a drug-metabolizing enzyme, comprising producing small intestinal epithelial-like cells by a method comprising the steps of:

9. A medium comprising a TGFβ receptor inhibitor, a GSK-3 inhibitor, EGF and active vitamin D for use in the method according to any one of claims 1 to 5, wherein the TGFβ receptor inhibitor is SB431542 and the GSK-3 inhibitor is LY2090314.

10. The medium according to claim 9, further comprising one or more substances selected from the group consisting of a Rho kinase inhibitor, a vinca alkaloid antitumor drug, a MEK inhibitor, and cAMP and its derivatives.

11. The medium according to claim 10, wherein the Rho kinase inhibitor is Y27632, the vinca alkaloid antitumor drug is vinblastine, the MEK inhibitor is PD0325901, and the cAMP derivative is Br-cAMP.

Images