Method for producing in vitro metabolic or modified products in the small intestine

Abstract

To produce a product equal to a product that is created when a substance is metabolized and / or modified in a small intestine of an organism in vitro.SOLUTION: A method for producing a product that is created when a substance is metabolized and / or modified includes: arranging a bag-shaped cell structure which has a villus layer on an external surface and includes an intestinal epithelium cell and liquid containing the substance such that the liquid contacts the villus layer; and accumulating the product inside the cell structure.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a method for producing a product resulting from the metabolism and / or modification of a substance, a method for producing a pharmaceutical containing, as an active ingredient, a product resulting from the metabolism and / or modification of a substance, a method for evaluating the behavior of a test substance in the small intestine, and a kit for evaluating the behavior of a test substance in the small intestine. [Background technology]

[0002] Evaluating the efficacy and pharmacokinetics of drugs requires assessment of their metabolic pathways and their effects on enzyme function in vivo, particularly in human small intestinal tissue. To perform this assessment appropriately, it is desirable to use human biospecimens or biosamples similar to them. However, human small intestinal tissue biospecimens are difficult to maintain in culture and are prone to metabolic inactivation, making them difficult to handle. The colon cancer cell line Caco-2, widely used in drug absorption evaluation studies, is not derived from small intestinal tissue and lacks metabolic enzyme activity, making it unsuitable for evaluating pharmacokinetics in human small intestinal tissue.

[0003] In recent years, there have been reports that small intestinal epithelial cells can be induced to differentiate from pluripotent stem cells and used to evaluate pharmacokinetics. Patent Document 1 describes that small intestinal epithelial-like cells induced to differentiate from pluripotent stem cells express drug-metabolizing enzymes and drug transporters, and describes a method for evaluating pharmacokinetics using small intestinal epithelial-like cells. Patent Document 2 describes a method for evaluating the pharmacokinetics of a test substance using intestinal epithelial cells induced to differentiate from induced pluripotent stem cells. Reports on metabolism by the inventors of these patent documents include Non-Patent Documents 1 and 2. However, the tissues containing cells obtained by differentiation induction in these documents are not morphologically similar to small intestinal tissue, and therefore cannot be said to be suitable as an evaluation system for pharmacokinetics in human small intestinal tissue.

[0004] Patent Document 3, Patent Document 4, and Non-Patent Document 3 describe the preparation of tissues with a structure similar to that of the intestine by culturing pluripotent stem cells on a substrate with a pattern of cell adhesion sites and inducing their differentiation. In particular, the intestinal-like organoids with a pouch-like structure described in Patent Document 4 and Non-Patent Document 3 contain cells such as enterocytes, goblet cells, enteroendocrine cells, and Paneth cells, have a higher-order structure similar to that of the small intestine, and exhibit intestinal peristalsis-like movements (Non-Patent Document 4).

[0005] Meanwhile, in drug metabolism, which is one mode of metabolism, metabolic enzymes function to break down and excrete foreign substances such as drugs and poisons. Drug metabolism mainly depends on the liver and intestines as biological tissues, and metabolic enzymes such as CYP family enzymes and CES family enzymes are activated and function in these tissues. For example, Non-Patent Documents 5 and 6 provide detailed explanations of each metabolic enzyme. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2016 / 147975 [Patent Document 2] WO2017 / 154795 [Patent Document 3] Patent No. 6151097 [Patent Document 4] WO2018 / 230102 [Patent Document 5] Patent No. 5070565 [Non-patent literature]

[0007] [Non-Patent Document 1] Ozawa T et al., Scientific Report, 5, 16479 (2015) [Non-patent document 2] Onozato D et al., Drug Metab Dispos, 46, (2018) 1572-1580 [Non-patent document 3] Uchida et al., JCI Insight Vol. 2 e86492 2017 [Non-patent document 4] Toru Sugawara, Hidenori Akutsu, Medical Progress Vol. 264 No. 8 2017 [Non-patent document 5] Zhang QY et al., Drug Metab Dispo, 27, (1999) 804-9 [Non-patent document 6] S. Casey Laizure et al., Pharmacotherapy, 33, (2013) 210-222 [Non-Patent Document 7] Okochi et al., Langmuir, Vol. 25, pp. 6947-6953, 2009 Summary of the Invention [Problem to be solved by the invention]

[0008] If it is possible to produce in vitro the products that are produced by the metabolism and / or modification of substances in the small intestine of a living organism, it would be useful in studying the behavior of drugs in the small intestine.In addition, since the products that are produced by the metabolism and / or modification of physiologically active substances in the small intestine of a living organism may have stronger physiological activity than the substances themselves, if these products can be produced in vitro, it would also be useful in the development of medicines.However, as mentioned above, there has not been provided a system that can efficiently produce in vitro the products that are produced by the metabolism and / or modification of substances using intestinal-like organoids with a structure similar to the small intestine. [Means for solving the problem]

[0009] The present invention includes the following inventions. (1) A method for producing a product resulting from metabolism and / or modification of a substance, comprising: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; and Accumulating the product within the cell structure. A method comprising: (2) The method according to (1), further comprising recovering the product accumulated in the cell structure, or recovering the cell structure in which the product has accumulated. (3) A method according to (1) or (2), wherein the length of the cell structure in the longitudinal direction is 5 mm or more. (4) The method according to any one of (1) to (3), wherein the cell structure has the properties of a small intestine. (5) The method according to any one of (1) to (4), wherein the cell structure is a cell structure formed by inducing differentiation from pluripotent stem cells. (6) A method for producing a medicine containing, as an active ingredient, a product produced by metabolism and / or modification of a substance, disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing said product to accumulate within said cellular structure; recovering the product accumulated in the cell structure; and formulating the recovered product into a pharmaceutically acceptable preparation. A method comprising: (7) A method for producing a medicine containing, as an active ingredient, a product produced by metabolism and / or modification of a substance, disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing said product to accumulate within said cellular structure; recovering the cell structure that has accumulated the product; and formulating the recovered cell structures into a pharmaceutically acceptable preparation. A method comprising: (8) A method for evaluating the behavior of a test substance in the small intestine, comprising: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the test substance, so that the liquid comes into contact with the villi layer; Culturing the cell construct; and Analyzing the composition of components accumulated inside the cell structure after culturing. A method comprising: (9) A kit for evaluating the behavior of a test substance in the small intestine, comprising: A pouch-like cellular structure containing small intestinal epithelial cells having a villi layer on its outer surface; and A medium for culturing the cell structure Kit including: (10) The kit according to (9), further comprising an instrument for removing components accumulated inside the cell structure. [Effects of the Invention]

[0010] According to one embodiment of the present invention, a method for producing a product resulting from the metabolism and / or modification of a substance accumulates the target product inside a sac-shaped cell structure, allowing the target product to be efficiently and easily recovered and utilized. According to a method for producing a pharmaceutical product according to one embodiment of the present invention, a pharmaceutical product containing a product generated by metabolism and / or modification of a substance as an active ingredient can be efficiently produced.

[0011] According to a method for evaluating the behavior of a test substance in the small intestine according to one embodiment of the present invention, the behavior of the test substance in the small intestine can be efficiently evaluated by analyzing the composition of components accumulated inside the cell structure. Furthermore, since the pouch-shaped cell structure can be cultured for a long period of time, the behavior of the test substance in the small intestine can be evaluated over a long period of time. A kit according to one embodiment of the present invention can be used in this evaluation method. [Brief explanation of the drawings]

[0012] [Figure 1]Figure 1 shows the results of the analysis of gene expression patterns over time in Example 1. Pluripotent stem cells (Edom iPS) were sampled at 7 days (D7), 14 days (D14), and 21 days (D21) of patterned culture, and the expression of each gene was observed over time. Differentiation induction of gut-like organoids was observed using definitive endoderm markers (FOXA2, SOX17, CXCR4), stem cell marker (Oct), early endoderm / mesoderm markers (GATA4, GATA6, T), intestinal epithelial marker (CDX2), and ectoderm marker (SOX1). The expression trends of metabolic enzymes CYP3A4 and CES2 were also observed. [Figure 2] Figure 2 shows the results of immunostaining in Example 2. Human intestinal tissue, the colon cancer cell line Caco-2, and intestinal-like organoids (Mini-Gut) were used to observe the expression of the metabolic enzyme CYP3A4 and the multidrug efflux transporter p-gp by immunostaining. [Figure 3] Figure 3 shows the results of quantitative PCR in Example 3. Intestinal-like organoids (mini-gut) were treated with vitamin D3 (VD3) to observe mRNA expression of the metabolic enzyme CYP3A4 and the multidrug efflux transporter p-gp. Human intestinal tissue (Intestine) and cDNA from the colon cancer cell line Caco-2 were used as controls. DMSO was used as a blank. [Figure 4A] Figure 4A shows the results of immunostaining in Example 4. In Example 4, iPS cell-derived intestinal-like organoids were treated with vitamin D3 (VD3), and mRNA expression of the metabolic enzyme CYP3A4 and the multidrug efflux transporter p-gp and protein expression were observed by immunostaining. [Figure 4B] FIG. 4B shows the results of quantitative PCR in Example 4. [Figure 5A] Figure 5A is a schematic diagram showing the procedure for the P450-Glo™ CYP3A4 Assay in Example 5. The P450-Glo™ CYP3A4 Assay was performed using intestinal-like organoids. After drug addition and reaction, the medium supernatant and the internal fluid of the intestinal-like organoids were aspirated using instruments such as syringes and needles. The luminescence intensity of each medium supernatant and the aspirated internal fluid was measured as samples. [Figure 5B] Figure 5B shows the results of the P450-Glo™ CYP3A4 Assay of Example 5. The luminescence intensity correlates with the amount of substrate metabolized by CYP3A4. [Figure 6] Figure 6 shows the results of LC / MS / MS in Example 6. In Example 6, intestinal-like organoids were used to measure the metabolites of the CYP3A4 substrate midazolam and the CES2 substrate irinotecan. After the addition of each drug, samples were taken over time to measure the concentrations of the metabolites. [Figure 7A] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism in Example 7. The control sample, human small intestine tissue (left side of each graph), was set to 1, and the expression of metabolism-related genes in the intestinal-like organoids was comprehensively analyzed as a relative evaluation. [Figure 7B] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. [Figure 7C] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. [Figure 7D] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. [Figure 7E] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. [Figure 7F] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. [Figure 7G] 7A to 7G show the results of the RT2 Profiler PCR Array Human Drug Metabolism of Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0013] <1.Cellular structures> The cell structure that can be used in the present invention is a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on the outer surface.

[0014] Small intestinal epithelial cells can be identified by the expression of transcription factors CDX2 and HNF4 in the cell nuclei, villin in the villi, and the expression of endodermal markers such as E-cadherin. The presence of these markers can be detected by tissue immunostaining using antibodies or PCR evaluation of mRNA. The presence of the villi can be confirmed by the expression of villin, and the presence of the villi can also be confirmed by microscopic observation of the outer surface of the cellular structure. Preferably, the cell structure has further crypts developed on its outer surface.

[0015] The cell structure that can be used in the present invention contains small intestinal epithelial cells and is useful as an intestinal-like organoid with functions equivalent to those of the intestine. "Intestinal-like organoid" refers to a cell structure (tissue) that has functions similar to those of the intestine of the organism from which the cells originate, particularly the intestine of mammals such as humans, and in particular the human intestine (specifically, the function of peristalsis, the function of mucus secretion, the function of substance absorption, etc.).

[0016] A sac-shaped cell structure is a cell structure that contains a space (cavity) that can hold a liquid without any cells inside. The space may be entirely closed or partially open. The contour shape of the sac-shaped cell structure is not particularly limited, but it is usually granular. "Granular" also includes spherical shapes.

[0017] The cell structure that can be used in the present invention includes a villus layer on at least a portion of its outer surface. This configuration allows substances present outside the cell structure to be absorbed into the cavity via the small intestinal epithelial cells that make up the villus layer on the outer surface. If the substance is metabolized and / or modified during absorption, the metabolic and / or modified products are also absorbed and accumulated in the cavity. Furthermore, it is preferable that the small intestinal epithelial cells that make up the villus layer on the outer surface are transporter-positive and capable of transporter-mediated substance uptake. In other words, a cell structure containing transporter-positive small intestinal epithelial cells has a substance absorption ability similar to that of the intestine. Note that in mammalian intestines, small intestinal epithelial cells face the inside of the intestinal tract, which is a hollow cavity, and are therefore different from the cell structure of this embodiment.

[0018] The cell structure that can be used in the present invention has a longitudinal length of 5 mm or more, preferably 8 mm or more, more preferably 10 mm or more, more preferably 12 mm or more, and more preferably 15 mm or more. The use of such large cell structures is preferred because it makes it easier to recover products accumulated inside.

[0019] Here, "longitudinal length" refers to the longest distance between two points on the contour of the observed image of the cell structure in an appropriate buffer solution that can be connected by a single straight line passing only within the contour, when the cell structure is observed visually or using an optical microscope. The contour of each cell structure may deform due to peristaltic movement, but the maximum measured value should be taken as the longitudinal length. The cell structure that can be used in the present invention preferably includes endodermal cells, ectodermal cells, and mesodermal cells.

[0020] The endoderm forms the digestive tract as well as tissues of organs such as the lungs, thyroid gland, pancreas, and liver, cells of secretory glands that open into the digestive tract, the peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most of the urethra, and part of the ureter). Differentiation of pluripotent stem cells such as ES cells and iPS cells into endodermal cells can be confirmed by measuring the expression levels of endoderm-specific genes. In addition to those described below, endoderm-specific genes include, for example, AFP, SERPINA1, SST, ISL1, IPF1, IAPP, EOMES, HGF, ALBUMIN, PAX4, and TAT.

[0021] The small intestinal epithelial cells contained in the cell structure are a type of endodermal cell. The cell structure preferably contains one or more small intestinal epithelial cells selected from enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. It is particularly preferable that the intestinal epithelial cells contain all of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells. The presence of endodermal cells in the cell structure can be determined based on the positive expression of an endodermal cell marker. Examples of an enterocyte marker include CDX2, a goblet cell marker includes MUC2, an enteroendocrine cell marker includes CGA, and a Paneth cell marker includes DEFA6. Other markers for intestinal epithelial cells include ECAD, Na+ / K+-ATPase, and villin. Definitive endoderm markers FOXA2, SOX17, and CXCR4 can also be used as markers for identifying endodermal cells. Early endoderm and mesoderm markers GATA4, GATA6, and T (Brachyury) can also be used as markers for identifying endodermal cells.

[0022] The ectoderm forms the epidermis of the skin, the epithelium of the distal end of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the distal parts of the oral cavity, pharynx, nose, and rectum, and salivary glands), and the lens. Part of the ectoderm invaginates into a groove during development to form the neural tube, which also gives rise to neurons and melanocytes of the central nervous system, such as the brain and spinal cord. It also forms the peripheral nervous system. Differentiation of pluripotent stem cells, such as ES cells and iPS cells, into ectodermal cells can be confirmed by measuring the expression levels of ectoderm-specific genes. Examples of ectoderm-specific genes include β-tublin, nestin, galanin, GCM1, GFAP, neurod1, olig2, synapthysin, desmin, and th.

[0023] Ectoderm cells that can be contained in the cell structure include cells that make up the enteric plexus. The presence of ectodermal cells in the cell structure can be determined based on the positive expression of ectodermal cell markers. Markers that can be used to identify ectodermal cells include the enteric plexus marker PGP9.5 and the neural progenitor cell marker SOX1.

[0024] The mesoderm forms the body cavity and its lining, the mesothelium, muscles, skeleton, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, and gonads (testes, uterus, and gonadal epithelium). Differentiation of pluripotent stem cells such as ES cells and iPS cells into mesodermal cells can be confirmed by measuring the expression levels of mesoderm-specific genes. Examples of mesoderm-specific genes include FLK-1, COL2A1, FLT1, HBZ, MYF5, MYOD1, RUNX2, and PECAM1.

[0025] Mesodermal cells that can be contained in the cell structure include smooth muscle cells and interstitial cells of Cajal. The presence of mesodermal cells in the cell structure can be determined based on the positive expression of mesodermal cell markers. Examples of mesodermal cell markers that can be used include the smooth muscle cell marker α-smooth muscle actin (SMA) and the interstitial cell markers of Cajal, CD34 and CKIT (in double-positive cases). Additionally, GATA4, GATA6, or T (Brachyury), which are markers for early endoderm and mesoderm, can also be used as markers for distinguishing mesodermal cells. The cell structure more preferably further comprises intestinal stem cells. The presence of intestinal stem cells can be determined by an index of positivity for the intestinal stem cell marker LGR5. Preferably, the cell structure further comprises serotonin-positive enteroendocrine cells.

[0026] Preferably, the cell structure further comprises transporter-positive cells and is capable of uptake of substances via transporters, such as the intestinal oligopeptide transporter (PEPT1) and ATP-binding cassette (ABC) transporters ABCB1 and ABCG2.

[0027] Preferably, the cell structure further comprises cystic fibrosis transmembrane conductance regulator (CFTR)-positive intestinal epithelial cells. CFTR-positive intestinal epithelial cells are involved in mucus secretion. That is, a cell structure having CFTR-positive intestinal epithelial cells has mucus secretion ability similar to that of the intestine. Preferably, the cell structure further comprises histamine H1 receptor positive cells.

[0028] The cell structure that can be used in the present invention preferably has the ability to contractile movements similar to peristalsis. Such a function is generated by the development of neural networks and smooth muscles. In the following explanation, the ability to contractile movements similar to peristalsis may be referred to as "peristaltic activity." Cell structures with peristaltic activity particularly preferably exhibit drug responsiveness similar to that of the intestine, in that the frequency of contractions increases with histamine treatment and decreases with atropine treatment. As a cell structure having the above characteristics, the intestinal organoids described in Patent Document 4 and Non-Patent Document 3 are particularly preferred.

[0029] <2. Manufacturing method of cell structures> The cell structure that can be used in the present invention can be produced by culturing stem cells and inducing their differentiation. The stem cells used here may be stem cells capable of differentiating into small intestinal epithelial cells, but are preferably stem cells capable of differentiating into endodermal cells (such as small intestinal epithelial cells), ectodermal cells, and mesodermal cells, and are more preferably pluripotent stem cells. Embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) are particularly suitable as pluripotent stem cells.

[0030] The embryonic stem cells (ES cells) used in the present invention are preferably mammalian-derived ES cells, such as ES cells derived from rodents (e.g., mice) or primates (e.g., humans). Mouse or human-derived ES cells are particularly preferred. ES cells are stem cell lines derived from the inner cell mass of a blastocyst-stage embryo, an early stage of animal development. These cells can be proliferated in vitro almost indefinitely while maintaining the pluripotency to theoretically differentiate into all tissues. For example, ES cells can be used that incorporate a reporter gene near the Pdx1 gene to facilitate confirmation of their degree of differentiation. Examples include a 129 / Sv-derived ES cell line incorporating the LacZ gene at the Pdx1 locus, or the SK7 ES cell line carrying a GFP reporter transgene under the control of the Pdx1 promoter. Alternatively, the PH3 ES cell line carrying an mRFP1 reporter transgene under the control of an Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of the Pdx1 promoter can also be used. In addition, ES cell lines such as SEES1, SEES2, SEES3, SEES4, SEES5, SEES6, or SEES7, which were established at the Department of Reproductive and Cellular Medicine at the National Center for Child Health and Development and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, or cell lines into which additional genes have been introduced into these ES cell lines, can also be used.

[0031] The induced pluripotent stem cells (iPS cells) used in the present invention are pluripotent cells obtained by reprogramming somatic cells. Several groups have successfully produced induced pluripotent stem cells, including the group of Professor Shinya Yamanaka et al. at Kyoto University, the group of Rudolf Jaenisch et al. at Massachusetts Institute of Technology, the group of James Thomson et al. at the University of Wisconsin, and the group of Konrad Hochedlinger et al. at Harvard University. For example, International Publication WO 2007 / 069666 describes somatic nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes, as well as somatic nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes. The publication also describes a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes contacting somatic cells with the nuclear reprogramming factors.

[0032] The type of somatic cells used to generate iPS cells is not particularly limited, and any somatic cells can be used. In other words, the somatic cells referred to in the present invention encompass all cells other than germ cells that constitute a living organism, and may be differentiated somatic cells or undifferentiated stem cells. The somatic cells may be derived from any of mammals, birds, fish, reptiles, and amphibians, but are not particularly limited thereto. They are preferably mammals (e.g., rodents such as mice, or primates such as humans), and are particularly preferably mice or humans. Furthermore, when human somatic cells are used, they may be from a fetus, a newborn, or an adult. Specific examples of somatic cells include fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiac myocytes), hepatic parenchymal cells, non-hepatic parenchymal cells, adipocytes, osteoblasts, cells that constitute periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells that constitute the kidney, eye, and ear.

[0033] iPS cells are stem cells that have the ability to self-renew over a long period of time under specific culture conditions (e.g., conditions for culturing ES cells) and have the pluripotency to differentiate into ectoderm, mesoderm, and endoderm under specific differentiation-inducing conditions. Furthermore, iPS cells in the present invention may be stem cells that have the ability to form teratomas when transplanted into test animals such as mice.

[0034] To produce iPS cells from somatic cells, at least one reprogramming gene is first introduced into the somatic cells. Reprogramming genes encode reprogramming factors that reprogram somatic cells to iPS cells. Specific examples of combinations of reprogramming genes include, but are not limited to, the following: (i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

[0035] The cell structure that can be used in the present invention is: Step 1 of seeding the stem cells as described above onto a cell culture substrate as described below; Step 2: Cultivating the seeded stem cells and inducing their differentiation It can be produced by a method comprising:

[0036] The cell culture substrate preferably includes a support substrate having a surface including cell adhesive portions and non-cell adhesive portions surrounding the cell adhesive portions. In the cell culture substrate, at least one, and preferably multiple, cell adhesive portions are preferably arranged in an island-like manner within the non-cell adhesive portions. Particularly preferred examples of such cell culture substrates include the cell culture substrates described in Patent Document 4 and Non-Patent Document 3.

[0037] The support substrate is not particularly limited as long as it is made of a material capable of forming cell-nonadhesive and cell-adhesive regions on its surface. Specific examples include support substrates containing inorganic materials such as glass, metal, ceramic, and silicon, and organic materials such as elastomers and plastics (e.g., polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, and vinyl chloride resin). Glass substrates are particularly preferred as support substrates. The shape of the support substrate is also not limited, and examples include flat shapes such as plates, flat membranes, films, and porous membranes, as well as three-dimensional shapes such as cylinders, stamps, multiwell plates, and microchannels.

[0038] In the present invention, "cell adhesiveness" refers to the strength of cell adhesion, i.e., the ease with which cells adhere. A "cell adhesive portion" refers to a region on a surface with good cell adhesiveness, and a "cell non-adhesive portion" refers to a region on a surface with poor cell adhesiveness. Therefore, when cells are seeded on a surface on which cell adhesive portions and non-cell adhesive portions are arranged in a predetermined pattern, the cells adhere to the cell adhesive portions but do not adhere to the non-cell adhesive portions, resulting in a patterned arrangement of cells on the surface of the cell culture substrate.

[0039] The "cell adhesive portion" is defined as the portion to which cells to be actually cultured, preferably stem cells, adhere when seeded on the cell culture substrate, and the "cell non-adhesive portion" is defined as the portion to which cells to be actually cultured, preferably stem cells, adhere when seeded. When cells are seeded on the cell culture substrate, the surface of the cell culture substrate may be coated with a protein or the like to enhance cell adhesiveness. The cell non-adhesive portion may be covered by cells that have adhered to and proliferated on the cell adhesive portion.

[0040] The cell adhesion spreading rate during actual cell culture can be used as an index for determining whether a region is a cell adhesion region or a cell non-adhesive region. The surface of a cell adhesion region having cell adhesive properties is preferably a surface with a cell adhesion spreading rate of 60% or more, more preferably a surface with a cell adhesion spreading rate of 80% or more. A high cell adhesion spreading rate allows efficient cell culture. In the present invention, the cell adhesion spreading rate is determined when the seeding density is 4000 cells / cm. 2 More than 30000 cells / cm 2 Cells to be cultured within this range are seeded on the surface to be measured, and stored in an incubator at 37°C and 5% CO2 concentration. After 14.5 hours of culture, the percentage of cells that have adhered and spread is defined as {(number of adhered cells) / (number of seeded cells)} x 100(%).

[0041] In the above measurement, cells are seeded onto the surface to be measured by suspending them in DMEM medium containing 10% FBS, and then the surface to be measured is slowly shaken to distribute the cells as uniformly as possible. Furthermore, the cell adhesion spreading rate is measured after changing the medium immediately before the measurement to remove unadhered cells. When measuring the cell adhesion spreading rate, the measurement points are areas excluding areas where the cell density tends to be specific (e.g., the center of a specified area where the cell density tends to be high, or the periphery of a specified area where the cell density tends to be low).

[0042] On the other hand, the non-cell-adhesive portion is a surface region that has the property of being difficult for cells to adhere (cell non-adhesiveness). Cell non-adhesiveness is determined by whether or not cell adhesion and spreading are difficult to occur depending on the chemical and physical properties of the surface. The surface of the non-cell-adhesive portion is preferably a surface with a cell adhesion spreading rate as defined above of less than 60%, more preferably less than 40%, even more preferably 5% or less, and most preferably 2% or less.

[0043] The cell adhesive portion may be a region on the surface of the support substrate where a cell adhesive layer is formed, or, if the surface of the support substrate is cell adhesive (e.g., the surface of a glass substrate), it may be a region where the surface of the support substrate is exposed. However, it is preferably a region where the cell adhesive surface of the support substrate is exposed. The cell non-adhesive portion may be a region on the surface of the support substrate where a cell non-adhesive layer is formed. The cell adhesive portion and the cell non-adhesive portion can be formed using various materials and methods. Preferably, the cell non-adhesive portion is a portion of the surface of the support substrate covered with a cell non-adhesive layer, such as a layer containing a hydrophilic organic compound such as a hydrophilic polymer. As described in Patent Document 5, the average thickness of the cell non-adhesive layer constituting the cell non-adhesive portion is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. An average thickness of 0.8 nm or more is preferable because protein adsorption and cell adhesion are less affected by regions of the support substrate not covered with the cell non-adhesive layer. Furthermore, coating is relatively easy if the average thickness is 500 μm or less. In particular, when the non-cell adhesive layer is formed of a polyethylene glycol layer, the thickness can be 5 nm to 10 nm, for example.

[0044] As a method for producing a cell culture substrate containing polyethylene glycol (PEG) as a hydrophilic polymer as a non-cell adhesive layer, the methods described in Patent Document 5 and Non-Patent Document 7 can be used.

[0045] Examples of hydrophilic organic compounds include hydrophilic polymers (including hydrophilic oligomers), water-soluble organic compounds, surface-active substances, and amphiphilic substances, with hydrophilic polymers being particularly preferred.

[0046] Specific examples of hydrophilic polymers include polyalkylene glycols, zwitterionic polymers having phospholipid polar groups, polyacrylamides, polyacrylic acids, polymethacrylic acids, polyvinyl alcohols, polysaccharides, etc. These specific examples of hydrophilic polymers also include derivatives thereof. The molecular shape of the hydrophilic polymers may be linear, branched, or dendrimer.

[0047] Specific examples of polyalkylene glycols include polyethylene glycol, polypropylene glycol, and copolymers of polyethylene glycol and polypropylene glycol, such as Pluronic F108 and Pluronic F127.

[0048] Specific examples of amphoteric polymers having a phospholipid polar group include poly(methacryloyloxyethyl phosphorylcholine) (=MPC polymer), copolymers of methacryloyloxyethyl phosphorylcholine and acrylic monomers, and the like. A specific example of polyacrylamide is poly(N-isopropylacrylamide). A specific example of polymethacrylic acid is poly(2-hydroxyethyl methacrylate). Specific examples of polysaccharides include dextran and heparin.

[0049] A cell culture substrate can be produced by forming a layer of a hydrophilic organic compound on the surface of a support substrate to form a cell non-adhesive portion, and then partially removing the layer to form a cell adhesive portion where the surface of the support substrate is exposed. Methods for partially removing the hydrophilic organic compound layer include ultraviolet irradiation treatment, photocatalytic treatment, and treatment with an oxidizing agent. For partial treatment, a mask such as a photomask or stencil mask or a stamp can be used. Alternatively, the layer can be removed by a direct imaging method, such as a method using a laser, such as an ultraviolet laser.

[0050] In the cell culture substrate used in the present invention, the area of ​​each cell adhesion portion is not particularly limited. 2 The above are examples, and preferably 0.5 mm 2 More than 0.785mm, preferably 0.785mm 2 More than 1.0mm, preferably 1.0mm 2 More than 1.2mm, preferably 1.2mm 2 More preferably 1.5 mm 2 Above 1.7mm, most preferably 1.7mm 2 or more, preferably 25 mm 2 Less than 15mm, preferably 2 Less than 10 mm, more preferably 2 Less than 5mm, most preferably 2 The pattern is formed so as to fall within the following range: When the area of ​​the cell adhesion portion falls within this range, it is easy to culture large cell structures with a length in the major axis direction exceeding 5 mm.

[0051] The shape of each cell adhesion portion is not particularly limited, but may be polygonal, including square, circular, elliptical, etc. Circular is preferred. In the case of a circular portion, the diameter may preferably be a diameter that satisfies the above-mentioned area range; specifically, the diameter of the circle may be 0.35 mm or more, for example, preferably 0.8 mm or more, preferably 1.0 mm or more, preferably 1.2 mm or more, more preferably 1.5 mm or more, and preferably 6 mm or less, more preferably 4 mm or less, even more preferably 3 mm or less, and even more preferably 2 mm or less. Multiple cell adhesion portions present in one cell culture substrate preferably have the same area, and more preferably have the same area and shape, although different areas and shapes may be present.

[0052] Another example of the shape of each cell adhesive portion is a ring shape. A ring-shaped cell adhesive portion has one non-cell adhesive portion disposed in the center, and other cell adhesive portions are formed to surround the periphery of the central non-cell adhesive portion. The outer periphery of the ring-shaped cell adhesive portion can preferably be a polygon such as a rectangle, a circle, an ellipse, or the like, and is more preferably a circle. The area of ​​the region surrounded by the periphery of the ring-shaped cell adhesive portion (the total area of ​​the ring-shaped cell culture portion and the central non-cell adhesive portion) is as described above for the area of ​​the cell adhesive portion. When the outer periphery of the ring-shaped cell adhesive portion is circular, the diameter of the circle is as described above for the area of ​​the circular cell adhesive portion. The width of the ring-shaped cell adhesive portion (the width in the direction along the line passing through the center of gravity of the central non-cell adhesive portion) is not particularly limited, but is preferably more than 30 μm and not more than 400 μm, and more preferably 60 μm or more and not more than 300 μm.

[0053] Next, steps 1 and 2 of the method for producing a cell structure will be described.

[0054] Step 1 is a step of seeding stem cells onto the cell culture substrate described below.

[0055] In step 1, the stem cells are maintained in an undifferentiated state using a non-differentiation-inducing medium before being seeded onto the cell culture substrate. Before and after seeding onto the surface of the cell culture substrate, the medium is switched to a differentiation-inducing medium, and the stem cells are seeded onto the substrate surface.

[0056] The non-differentiation-inducing medium is not particularly limited as long as it is a medium that does not induce differentiation of stem cells, and examples include media containing leukemia inhibitory factor, which is known to have the property of maintaining the undifferentiated state of mouse embryonic stem cells and mouse induced pluripotent stem cells.

[0057] In step 1, the seeding density of stem cells on the cell culture substrate may be any conventional method and is not particularly limited. In one embodiment of the present invention, stem cells are seeded on the cell culture substrate at a density of 3×10 4 cells / cm 2 It is preferable to sow at a density of 3 × 10 or more. 4~5×10 5 cells / cm 2 It is more preferable to sow at a density of 3 × 10 4 ~2.5×10 5 cells / cm 2 It is more preferable to sow the seeds at a density of 1000 to 15000.

[0058] Step 2 is a step of culturing the stem cells seeded in step 1 and inducing their differentiation. The culture temperature in step 2 is usually 37° C. It is preferable to culture in an atmosphere with a CO 2 concentration of about 5% using a CO 2 cell culture device or the like.

[0059] Step 2 is carried out in a differentiation-inducing medium. The differentiation-inducing medium is not particularly limited as long as it induces differentiation of stem cells such as embryonic stem cells and induced pluripotent stem cells. Examples include serum-containing media and serum-free media containing known components with serum-substitute properties. Depending on the type of cells used, MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, and RPMI1640 medium can be used. Various growth factors, antibiotics, amino acids, and the like may also be added to the medium. Examples include 0.05 mM to 1.0 mM non-essential amino acids, 1 mM to 5 mM GlutaMAX-I, 0.01 mM to 0.1 mM β-mercaptoethanol, 0.1 mM to 2 mM pyruvic acid, 10 U / ml to 200 U / ml penicillin, 10 μg / ml to 200 μg / ml streptomycin, 10 μg / ml to 200 μg / ml L-ascorbic acid 2-phosphate, and 1 μM to 20 μM ROCK inhibitor (e.g., Y-27632).

[0060] In step 2, cells are cultured in a differentiation-inducing medium. Approximately three days after seeding, the cells become confluent within the cell adhesion zone and form a cell pattern. Further culture then transforms the cell pattern into a hemispherical, dome-shaped cell mass above the cell adhesion zone, and differentiation progresses within the cell mass. Approximately 30 days after seeding, the cell mass detaches from the cell adhesion zone and floats in the medium. Further culture in the floating state, as needed, results in intestinal-like organoids. The culture period is not particularly limited; culture may be terminated when the cells detach from the cell adhesion zone, but typically, culture is carried out for 30 to 130 days after seeding. The medium is replaced as needed during this period. This method is preferred because autonomous differentiation progresses within the cell mass, resulting in intestinal-like organoids. Furthermore, the resulting intestinal-like organoids are believed to have functions more similar to those of the natural intestine. More specific examples of the method for producing a cell structure including steps 1 and 2 are described in Patent Document 4 and Non-Patent Document 3.

[0061] <3. Methods for producing products resulting from metabolism and / or modification of substances> A first embodiment of the present invention comprises: A method for producing a product obtained by metabolizing and / or modifying a substance, comprising: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; and Accumulating the product within the cell structure. The present invention relates to a method comprising: This method makes it possible to produce in vitro products that are produced when substances are metabolized and / or modified in the small intestine.

[0062] Examples of raw material substances include precursor compounds that are metabolized and / or modified in the small intestine to produce products with useful properties such as pharmaceutical activity, and test substances such as physiologically active compounds that are the subject of evaluation of their behavior in the small intestine, i.e., what products are produced when metabolized and / or modified in the small intestine.

[0063] The present inventors have surprisingly found that a sac-like cellular structure containing small intestinal epithelial cells and having a villi layer on its outer surface expresses one or more genes selected from the following metabolic enzyme genes and has the enzyme activity corresponding to said genes: Drug transporter genes: MT2A, MT3, ABCB1, ABCC1, GPI

[0064] Phase I metabolic enzyme genes: CYP11B2, CYP17A1, CYP19A1, CYP1A1, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP3A4, CYP3A5

[0065] Phase II metabolic enzyme genes: CES1, CES2, CES3, GAD1, GAD2, ADH1B, ADH1C, ADH4, ADH5, ADH6, ALAD, ALADH1A1, HSD17B1, HSD17B2, HSD17B3, GPX1, GPX2, GPX3, GPX4, GPX5, GSTA3, GSTA4, GSTM2, GSTM3, GSTM5, GSTP1, GSTT1, GSTZ1, LP O, MPO, ALOX12, ALOX15, ALOX5, APOE, ASNA1, EPHX1, FAAH, FPB1, HK2, PKLR, PKM, AOC1, BLVRA, BLVRB, CYB5R 3, GSR, MTHFR, NOS3, NQO1, SRD5A1, SRD5A2, PON1, PON2, PON3, MGST1, MGST2, MGST3, CHST1, NAT1, NAT2, COMT Other drug metabolism genes: AHR, ARNT, GCKR, SNN

[0066] The cell constructs used in the present invention have expression levels of these metabolic enzyme genes, particularly CYP family enzyme genes (CYP11B2, CYP17A1, CYP19A1, CYP1A1, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP3A4, CYP3A5) and CES family enzyme genes (CES1, CES2, CES3), comparable to those in biological samples from the human small intestine. Therefore, the products accumulated inside the cell construct after a substance is absorbed through the villi on the outer surface of the cell construct and metabolized and / or modified are equivalent to those produced by metabolism and / or modification in the small intestine. Therefore, the first method of the present invention allows for the production of metabolic and / or modified products of raw materials using one or more selected from these metabolic enzymes.

[0067] In the first method of the present invention, first, a sac-shaped cell structure and a liquid containing a raw material substance are placed so that the liquid comes into contact with the villus layer of the cell structure. Here, an aqueous solution in which the substance is dissolved in water can be used as the liquid containing the substance. The aqueous solution of the substance is preferably a buffer solution or culture medium in which the cell structure can be cultured. By placing such a liquid containing the substance so that it comes into contact with the villus layer of the sac-shaped cell structure, the substance in the liquid is taken up into the interior of the cell structure, metabolized and / or modified by the metabolic enzymes contained in the cell structure as described above, and accumulated inside the sac-shaped cell structure.

[0068] In the first method of the present invention, the pouch-shaped cell structure is preferably suspended in a liquid containing the substance. In this case, the liquid containing the substance is preferably a buffer solution or culture medium containing the substance. The buffer solution or culture medium is not particularly limited as long as it is a medium or buffer solution capable of culturing the pouch-shaped cell structure, but preferably, DMEM medium containing 15% Xeno-Free KSR, phosphate-buffered saline (PBS), etc. can be used. The liquid containing the substance preferably further contains a component that promotes the expression of metabolic enzymes. An example of a component that promotes the expression of metabolic enzymes is 1α,25-dihydroxyvitamin D3. The pouch-shaped cell structure used in the present invention has a function similar to that of the small intestine, and the expression of metabolic enzymes is promoted by adding a component that promotes the expression of metabolic enzymes in the small intestine.

[0069] A further feature of the first method of the present invention is that a product is accumulated inside the sac-shaped cell structure. The sac-shaped cell structure has the surprising ability to absorb substances in a liquid through the outward-facing villi, take them into the cell structure, and accumulate products (metabolic modification products) that have been metabolized and / or modified by metabolic enzymes inside. Due to this function, the raw material is present outside the cell structure, and the metabolic modification products are accumulated inside the cell structure, preventing the two from being mixed together, allowing the desired metabolic modification products to be easily recovered.

[0070] The method for recovering metabolic modification products accumulated inside the pouch-shaped cell structure is not particularly limited. For example, an instrument such as a syringe can be used to extract and recover the internal fluid containing the metabolic modification products from the pouch-shaped cell structure in which the metabolic modification products have accumulated. The recovered internal fluid may be further purified to obtain the metabolic modification products. The pouch-shaped cell structure from which the internal fluid has been extracted may be further used to produce the next metabolic modification product. Alternatively, the metabolic modification products can be obtained by recovering the pouch-shaped cell structure itself in which the metabolic modification products have accumulated (and further disrupting or purifying it as necessary).

[0071] <4. Pharmaceutical manufacturing methods> The present invention secondly provides a method for producing a medicament containing, as an active ingredient, a product generated by metabolism and / or modification of a substance, comprising the steps of: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing said product to accumulate within said cellular structure; recovering the product accumulated in the cell structure; and formulating the recovered product into a pharmaceutically acceptable preparation. The present invention relates to a method comprising: A third aspect of the present invention provides a method for producing a pharmaceutical comprising, as an active ingredient, a product generated by metabolism and / or modification of a substance, the method comprising the steps of: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing said product to accumulate within said cellular structure; recovering the cell structure that has accumulated the product; and formulating the recovered cell structures into a pharmaceutically acceptable preparation. The present invention relates to a method comprising:

[0072] The substance used as the raw material in the second or third method of the present invention can be, for example, a precursor compound whose product obtained by metabolism and / or modification in the small intestine has useful properties such as pharmaceutical activity. In patients with impaired small intestinal function, normal metabolic modification products are not produced in the small intestine when the precursor compound is administered, and the desired pharmaceutical effect may not be achieved. Therefore, the second or third method of the present invention makes it possible to produce normal metabolic modification products in the small intestine ex vivo and to manufacture a pharmaceutical containing the normal metabolic modification products as active ingredients.

[0073] In the second or third method of the present invention, the step of placing the sac-shaped cell structure and a liquid containing a substance and the step of accumulating metabolic modification products are as described for the first method of the present invention.

[0074] The metabolic modification product, which is the active ingredient of the pharmaceutical produced by the second or third method of the present invention, is typically a metabolic modification product of the raw material by one or more of the above-mentioned metabolic enzymes. For example, when the raw material is midazolam, a metabolic modification product by CYP3A4 can be obtained, and when the raw material is irinotecan, a metabolic modification product by CES2 can be obtained.

[0075] In the second method of the present invention, the method for recovering metabolic modification products accumulated in the cell structure is not particularly limited. For example, an instrument such as a syringe can be used to remove and recover the internal fluid containing the metabolic modification products from the sac-shaped cell structure in which the metabolic modification products have accumulated. The recovered internal fluid may be further purified to obtain the metabolic modification products. The sac-shaped cell structure from which the internal fluid has been removed may be further used to produce the next metabolic modification product. Alternatively, the metabolic modification products can be obtained by recovering the sac-shaped cell structure itself in which the metabolic modification products have accumulated (and further disrupting or purifying it as necessary). In the second method of the present invention, the recovered metabolic modification products can be combined with a pharmaceutically acceptable carrier or the like to be formulated as a pharmaceutical.

[0076] In the third method of the present invention, the sac-like cell structures in which metabolic modification products have accumulated are collected and combined with a pharmaceutically acceptable carrier, etc. to form a pharmaceutical formulation. The sac-like cell structures in which metabolic modification products have accumulated can be used as active ingredients because they themselves encapsulate the metabolic modification products.

[0077] 5. Methods for evaluating the behavior of test substances in the small intestine A fourth aspect of the present invention is a method for evaluating the behavior of a test substance in the small intestine, comprising: disposing a pouch-shaped cell structure containing small intestinal epithelial cells and having a villi layer on its outer surface, and a liquid containing the test substance, so that the liquid comes into contact with the villi layer; Culturing the cell construct; and Analyzing the composition of components accumulated inside the cell structure after culturing. The present invention relates to a method comprising:

[0078] Test substances used in the fourth method of the present invention include pharmaceutical compounds whose behavior in the small intestine needs to be known and compounds whose safety in living organisms needs to be confirmed. The pouch-shaped cell structure used in the present invention has metabolic enzyme activity and function similar to that of the small intestine, making it possible to produce products equivalent to metabolic modification products of the test substance in a small intestine sample in vitro. Therefore, the fourth method of the present invention can evaluate the behavior of the test substance in the small intestine with high accuracy. Furthermore, unlike small intestine samples from living organisms, the pouch-shaped cell structure used in the present invention can be cultured for long periods of time, making it possible to evaluate the behavior of the test substance in the small intestine over long periods of time.

[0079] The metabolic modification product of the test substance produced by the fourth method of the present invention is typically a metabolic modification product of the starting substance by one or more of the above-mentioned metabolic enzymes. For example, when the test substance is midazolam, a metabolic modification product by CYP3A4 can be obtained, and when the test substance is irinotecan, a metabolic modification product by CES2 can be obtained.

[0080] The step of placing the sac-shaped cell structure and the substance-containing solution in the fourth method of the present invention is as described for the first method of the present invention. Furthermore, the step of culturing the cell structure in the fourth method of the present invention can be carried out in the same manner as the culturing of the cell structure for the step of accumulating metabolic modification products inside the cell structure in the first method of the present invention.

[0081] In the fourth method of the present invention, the step of analyzing the composition of components accumulated inside the cell structure after culture is a step of recovering the internal fluid of the cell structure after culture using an instrument such as a syringe, further purifying it as necessary, and analyzing the composition of the recovered material by conventional analytical means such as liquid chromatography or mass spectrometry. The bag-shaped cell structure from which the internal fluid has been removed may be further used to carry out the next evaluation method. Alternatively, the step may be a step of recovering the cell structure itself after culture, further disrupting or purifying it as necessary, and analyzing the composition of the recovered material by conventional analytical means.

[0082] <6. Kit for evaluating the behavior of test substances in the small intestine> Fifthly, the present invention provides A kit for evaluating the behavior of a test substance in the small intestine, comprising: A pouch-like cellular structure containing small intestinal epithelial cells having a villi layer on its outer surface; and A medium for culturing the cell structure This kit can be used to carry out the fourth method of the present invention. The pouch-like cell structure and the medium for culturing the cell structure are as described above.

[0083] The kit of the present invention preferably further comprises a tool for removing components accumulated inside the cell structure, such as a syringe for removing the internal fluid of the cell structure. The present invention will be described below with reference to specific experimental results, but the scope of the present invention is not limited to the scope of the experimental results. [Example]

[0084] Example 1 To evaluate the expression patterns of metabolic enzymes during the intestinal-like organoid formation process, we performed time-course sampling and quantitative PCR analysis. The methods are described below.

[0085] Human iPS cells were prepared from Edom iPS cells derived from endometrial cells or human ES cell lines. The cell lines were grown and maintained in StemFit medium (Ajinomoto Co.) on vitronectin (Life Technologies)-coated cell culture dishes.

[0086] Vitronectin coating was performed by dropping a vitronectin solution diluted 1 / 100 with phosphate-buffered saline (PBS) in an amount sufficient to coat the substrate or culture dish, leaving it at room temperature for 30 minutes, and then washing with PBS.

[0087] A glass substrate (substrate size 5 cm × 5 cm) on whose surface were formed cell non-adhesive areas covered with a polyethylene glycol film and circular patterns of cell adhesive areas with a diameter of 1500 μm, which were scattered like islands in the cell non-adhesive areas and from which the film had been decomposed and removed, was produced by the method described in Patent Document 5 and Non-Patent Document 7. This glass substrate was placed in a circular culture dish (Falcon) with a diameter of 10 cm, and then coated with vitronectin. The cultured cells were then detached by EDTA treatment and then cultured in a 10 cm diameter dish. 7 cells were seeded.

[0088] The seeded cells were cultured to obtain intestinal-like organoids according to the methods described in the examples of Non-Patent Document 3 and Patent Document 4. The day after seeding, the cells were transferred to a 10 cm diameter circular culture dish (BD Falcon) and the medium was maintained at approximately 15 ml, with the medium being replaced every two days. Undifferentiated cells and cells cultured on the substrate for 7, 14, and 21 days were sampled. To the collected sample, 900 μl of QiaZol solution (Qiagen) was added, the cells were disrupted by homogenization, and the mixture was allowed to stand for 5 minutes.

[0089] mRNA extraction from disrupted cells was performed according to the protocol for the RNeasy Plus Universal Mini Kit (Qiagen). 100 μl of gDNA Eliminator was added to the cell lysate and mixed. Then, 180 μl of chloroform (Nacalai Tesque) was added, mixed, and allowed to stand for 2 minutes. The mixture was then centrifuged at 12 krpm, 4°C, for 15 minutes, and the supernatant was collected. The supernatant was mixed with 600 μl of 70% ethanol and placed in an RNeasy Mini spin column, which was centrifuged at 10 krpm, 4°C, for 15 seconds. 700 μl of RWT buffer was added and centrifuged once at 10 krpm, 4°C, for 15 seconds. 500 μl of RPE buffer was added and centrifuged twice at 10 krpm, 4°C, for 15 seconds. Finally, 30 μl of RNase-free water was added and the column was centrifuged at 10 krpm, 4°C, for 1 minute. The OD value of the contained mRNA was quantified using a Nanodrop device (Thermo Fisher).

[0090] Reverse transcription to cDNA was performed using the Superscript IV VILO Master Mix Kit (Thermo Fisher). 0.5–1.0 μg of mRNA sample, 4 μl of Superscript IV VILO Master Mixture, and RNase-free water were added to a total volume of 20 μl, and the PCR reaction was carried out under the following PCR conditions: 10 minutes at 25°C, 10 minutes at 50°C, and 5 minutes at 80°C.

[0091] For quantitative PCR, each well of a 96-well plate was charged with 5-10 ng of cDNA, 1 μl each of 10 μM forward primer and 10 μM reverse primer for the target gene (final concentration 0.2 μM), 12.5 μl of 2× SYBR Green (final concentration 1×), 0.5 μl of ROX Reference dye (final concentration 0.5 μM), and MilliQ to adjust the total volume to 25 μl. The reaction was performed under the following PCR conditions: 50°C for 2 minutes, 95°C for 10 minutes (1 cycle), 95°C for 15 seconds, 60°C for 1 minute (40 cycles), 95°C for 15 seconds, 60°C for 1 minute, 95°C for 1 minute (1 cycle). The numerical value of the expression level of each target gene was a relative evaluation, and the Ct of each gene was corrected with the Ct of the internal control GAPDH and calculated by the ΔΔCT method.

[0092] The results of evaluating the expression levels of each gene when using intestinal-like organoids derived from Edom iPS cells as samples are shown in Figure 1. Expression of metabolic enzymes CYP3A4 and CES2 was observed in the later stages, correlating with an increase in the intestinal marker CDX2, which indicates intestinal-like organoid formation. These results suggest that intestinal-like organoids may have metabolic functions. In the following experiments, intestinal-like organoids derived from Edom iPS cells or ES cells produced by the above-mentioned method were used.

[0093] <Example 2> To confirm whether the intestinal-like organoids express CYP3A4 and the multidrug efflux transporter p-gp, immunostaining was performed. The methods are described below.

[0094] After 120 days of culture, ES cell-derived intestinal-like organoids were sampled, and a hole was made in the pouch structure using a 27G syringe needle (Terumo Corporation). The pouch structure was transferred to a 1.5 ml tube and 50 μl of culture medium was added. Next, for gel embedding, 10 μl of solution A from the iPGell Kit (GenoStaff) was added and pipetted three times, followed by 50 μl of solution B, which was pipetted three times and left to stand at room temperature for 1 minute. After gel embedding, the organoids were fixed overnight at 4°C with 1 ml of 4% paraformaldehyde (Wako Pure Chemical Industries). Paraffin sections were prepared by slicing paraffin-embedded specimen blocks with a microtome and attaching them to glass slides.

[0095] Paraffin sections were deparaffinized by immersing them in xylene solution (Muto Chemical Co., Ltd.) five times and then in alcohol solution (Muto Chemical Co., Ltd.) five times. Next, to inactivate the antigen, they were placed in boiling pH 8.0 TE buffer and microwaved for 10 minutes. They were then blocked with Protein Block (Dako) for 30 minutes at room temperature. Primary antibodies, rabbit IgG-labeled anti-p-gp antibody (Abcam; dilution 1 / 1000) and mouse IgG-labeled anti-CYP3A4 antibody (Santa Cruz; dilution 1 / 200) diluted in 0.1% BSA / PBS, were applied dropwise to the section area and incubated overnight at 4°C. After the primary antibody reaction, the sections were washed three times with PBS, and then Alexa488-labeled anti-rabbit IgG antibody (Molecular Probes, dilution 1 / 1000), Alexa546-labeled anti-mouse IgG antibody (Molecular Probes, dilution 1 / 1000), and DAPI (Sigma, dilution 1 / 1000) diluted in 0.1% BSA / PBS were added dropwise to the sections. The reaction was allowed to proceed at room temperature for 30 minutes, after which the sections were mounted and observed under a confocal microscope. As controls, the small intestine of a living organism and the colon cancer cell line Caco-2 were also subjected to the same treatment and observed.

[0096] The results are shown in Figure 2. While CYP3A4 and p-gp proteins were expressed in the intestinal-like organoids, similar to the small intestine in vivo, CYP3A4 was not expressed in Caco-2, which is derived from colonic tissue. These results suggest that metabolic enzymes similar to those in human small intestinal tissue may be expressed and functioning in the intestinal-like organoids.

[0097] Example 3 Quantitative PCR analysis was performed to confirm whether the intestinal-like organoids have CYP3A4 expression and its induction pathway similar to that in small intestinal tissue. The quantitative PCR analysis procedure was the same as in Example 1. The expression induction treatment is shown below.

[0098] After 78 days of culture, ES cell-derived intestinal-like organoids were induced by adding 0.1% DMSO (blank) or 1α,25-dihydroxyvitamin D3 (SIGMA-ADLRICH) at a final concentration of 100 nM and reacting for 24 hours at 37°C. Samples were then collected, reverse-transcribed, and analyzed by quantitative PCR under the same conditions as in Example 1. As a control, colon cancer cells Caco-2 were treated in the same manner and quantitative PCR analysis was performed.

[0099] The results are shown in Figure 3. The expression of both CYP3A4 and p-gp genes in intestinal-like organoids (abbreviated as "mini-gut" in Figure 3) ("mini-gut+DMSO" in Figure 3) was comparable to that in human small intestine samples ("Intestine" in Figure 3), at 30-70% of the level. CYP3A4 was expressed poorly in Caco-2 cells derived from colonic tissue. Furthermore, treatment with 1α,25-Dihydroxyvitamin D3 (abbreviated as "VD3" in Figure 3), a small intestine-specific metabolic enzyme derivative, induced the expression of CYP3A4 and p-gp in intestinal-like organoids ("mini-gut+DV3" in Figure 3). These results suggest that intestinal-like organoids have metabolic enzyme induction pathways similar to those in human small intestinal tissue.

[0100] Example 4 Quantitative PCR analysis and immunostaining were performed to confirm that CYP3A4 expression and its induction pathway in iPS cell-derived intestinal-like organoids are similar to those in ES cell-derived intestinal-like organoids and are not affected by the origin of the cell line. Quantitative PCR analysis was performed using the same method as in Example 1, and immunostaining was performed using the same method as in Example 2. The expression induction treatment is described below.

[0101] After 116 days of culture, iPS cell-derived intestinal-like organoids were induced by adding 0.1% DMSO or 1α,25-Dihydroxyvitamin D3 (SIGMA-ADLRICH) at a final concentration of 100 nM and reacting for 24 hours at 37°C. Samples were then collected, reverse-transcribed, and analyzed by quantitative PCR under the same conditions as in Example 1. Paraffin sections of the iPS cell-derived intestinal-like organoids after 62 days of culture were prepared and immunostained under the same conditions as in Example 2.

[0102] The results are shown in Figures 4A and 4B. Expression of both CYP3A4 and p-gp genes in iPS cell-derived intestinal-like organoids was observed by immunohistochemistry (Figure 4A). Furthermore, treatment with 1α,25-Dihydroxyvitamin D3 (abbreviated as "VD3" in Figure 4B), a small intestine-specific metabolic enzyme derivative, induced expression of CYP3A4 and p-gp in iPS cell-derived intestinal-like organoids ("mini-gut+DV3" in Figure 4B). These results suggest that iPS cell-derived intestinal-like organoids possess metabolic enzyme induction pathways similar to those in human small intestinal tissue, and that the metabolic function of the generated intestinal-like organoids is cell line-independent.

[0103] <Example 5> In order to evaluate metabolic enzyme activity (CYP3A4 activity) using intestinal-like organoids with a pouch structure, we investigated whether it is effective to extract and analyze the internal fluid of intestinal-like organoids. TM CYP3A4 Assay (Promega) and sampling of the internal solution used for this assay were performed. The methods are described below. The procedure is outlined schematically in Figure 5A.

[0104] Two to three intestinal-like organoids derived from iPS cells after 160 days of culture were placed in each well of a 24-well culture plate (Corning) containing 500 μl of liquid medium. 0.5 μl of Luciferin-IPA, an artificial substrate for CYP3A4, was added and incubated at 37°C for 2 hours. After incubation, in some test groups, the intestinal-like organoids were separated from the medium, and 25 μl of the medium supernatant (supernatant A) without the intestinal-like organoids was collected. In other test groups, 25 μl of the medium supernatant (supernatant C) containing the intestinal-like organoids was collected.

[0105] The isolated intestinal-like organoids were placed in fresh medium, and the internal fluid was aspirated using a needle and syringe (Ito Seisakusho). Medium was added to the aspirated internal fluid to adjust the volume to 25 μl (contents). Additionally, 25 μl of the supernatant (supernatant B) of the medium used to aspirate the internal fluid from the intestinal-like organoids was collected.

[0106] The collected supernatants A, B, C, and the contents (internal solution) were transferred to a 96-well assay plate (Corning), and 25 μl of luciferin detection reagent was added. The mixture was incubated at room temperature for 20 minutes. The luminescence intensity was then measured using a luminometer (BioTek). The luminescence intensity of the contents (internal solution) was calculated based on the dilution ratio.

[0107] The results are shown in Figure 5B. The luminescence intensity, which correlates with the metabolic activity of CYP3A4, was approximately 2 to 4 times higher in the contents (internal fluid) than in supernatants A, B, and C. These results suggest that it is possible to evaluate the metabolic activity of intestinal-like organoids, and that evaluation based on the internal fluid extracted from intestinal-like organoids is particularly effective.

[0108] Example 6 To evaluate the metabolic enzyme activity (CYP3A4 activity and CES activity) of intestinal organoids with a pouch structure, we evaluated the metabolic activity of the CYP3A4 substrate midazolam and the CES substrate irinotecan. The metabolites of midazolam and irinotecan were analyzed by LC / MS / MS (Kamakura Techno Science Co., Ltd.). The methods are described below.

[0109] One intestinal-like organoid derived from ES cells after 90 days of culture was added to 300 μl of 100 mmol / L phosphate buffer in a tube, and the organoid was crushed with a spatula or other tool on ice, followed by sonication. Two stainless steel beads were then added to the tube, and a homogenate was prepared using a bead crusher.

[0110] A metabolic reaction mixture was prepared on ice using 200 μl of intestinal organoid-derived homogenate, 156 μl of 100 mmol / L potassium phosphate buffer, pH 7.4, and 4 μl of 0.2 mM midazolam (50% MeCN). For irinotecan, 4 μl of 2 mM irinotecan (50% MeCN) was added.

[0111] 180 μl of metabolic reaction mixture prepared for each intestinal organoid-derived homogenate was transferred to a 1.5 ml microtube and subjected to metabolic reactions at 37°C. After a 5-minute preincubation, 20 μl of 10 mmol / L NADPH was added and mixed. After a set reaction time of 5, 15, 30, and 60 minutes, 20 μl samples were taken. Samples were also taken immediately after the homogenate was added, marking the reaction time as 0 minutes. After the sample was taken, the mixture was immediately added to 20 μl of MeCN containing 100 nmol / L phenacetin, mixed, cooled on ice, and then 20 μl of 25% MeCN was added, mixed, and centrifuged at 1900 × g for 5 minutes. 100 μl of 20% MeCN was added to the 20 μl supernatant.

[0112] To prepare the calibration curve standard samples, 20 μL of 100 mmol / L phosphate buffer was placed in eight PP tubes (Blank + 7 concentrations). 20 μL of MeCN containing 100 nmol / L phenacetin (MeCN for Blank) was added, stirred, and then cooled on ice. 20 μL of standard solution (25% MeCN for Blank) was added, stirred, and centrifuged at 1900 × g for 5 minutes. 100 μL of 20% MeCN was added to the resulting 20 μL supernatant. The calibration curve range was: Blank, 0.0003 μM, 0.001 μM, 0.003 μM, 0.01 μM, 0.03 μM, 0.1 μM, and 0.3 μM. To calculate metabolic activity, the protein in each prepared suspension was quantified using DC Protein Assay Kit II (Bio-Rad).

[0113] The results are shown in Figure 6. We observed that the metabolites of midazolam and irinotecan, which are indicators of CYP3A4 activation and CES activation in intestinal-like organoids, increased over time. These results suggest that intestinal-like organoids have metabolic enzyme activity and are capable of metabolizing drugs.

[0114] Example 7 To evaluate the expression of other metabolic genes in the intestinal-like organoids, we performed a comprehensive analysis using the RT2 Profiler PCR Array (Qiagen). The methods are described below.

[0115] To recover mRNA samples from ES cell-derived intestinal-like organoids after 90 days of culture, one intestinal-like organoid was placed in a 1.5 ml tube and disrupted by homogenization. 1 ml of TRIzol solution (Life Technologies) was added and incubated at 37°C for 15 minutes. After incubation, the solution was transferred to a 15 ml centrifuge tube, and 4 ml of TRIzol solution was added, mixed, and incubated at 37°C for 5 minutes. 1 ml of chloroform (Nacalai Tesque) was then added, mixed, and allowed to stand for 2 minutes. The mixture was centrifuged at 3 krpm, 4°C, and 15 minutes, and the supernatant was collected. 2.5 ml of isopropanol (Nacalai Tesque) was added to the supernatant, mixed, and allowed to stand for 10 minutes. The mixture was centrifuged at 3 krpm, 4°C, and 10 minutes, and the supernatant was removed. After removing the supernatant, 5 ml of cold 75% ethanol was added, and the mixture was centrifuged at 3 krpm at 4°C for 10 minutes. After removing the supernatant, the mixture was suspended in 30 μl of UltraPure Distilled Water (Invitrogin), transferred to a 1.5 ml tube, and eluted by heat treatment at 60°C for 10 minutes. The OD value of the contained mRNA was quantified using a Nanodrop device (Thermo Fisher).

[0116] Reverse transcription to cDNA was performed using the RT2 First Strand Kit (Qiagen). The experiment was performed according to the protocol provided with the kit. A total volume of 10 μl was prepared using 1.0 μg of mRNA sample, 2 μl of Buffer GE, and RNase-free water. The mixture was incubated at 42°C for 5 minutes and then placed on ice for 1 minute. Next, 4 μl of 5xBuffer BC3, 1 μl of Control P2, 2 μl of RE3 Reverse Transcriptase Mix, and 3 μl of RNase-free water were added to the 10 μl reaction mixture to adjust the total volume to 20 μl. The mixture was incubated at 42°C for 15 minutes and then at 95°C for 5 minutes. 91 μl of RNase-free water was added to the reaction mixture and mixed.

[0117] Global quantitative PCR was performed using the RT2 Profiler PCR Array Human Drug Metabolism (Qiagen). Experiments were performed according to the protocol provided with the kit. 102 μl of cDNA sample, 1350 μl of 2x RT2 SYBR Green Mastermix, and 1248 μl of RNase-free water were mixed in a 5 ml tube. 25 μl of the mixture was added to each well of a 96-well plate. After adding all wells, the plate was tightly sealed with Optical Thin-Wall 8-Cap Strips. The mixture was centrifuged at 1 krpm for 1 minute at room temperature to remove air bubbles. The PCR reaction was then performed under the following PCR conditions: 1 cycle of 95°C for 1 minute, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute, 95°C for 1 minute, 60°C for 2 minutes, and 95°C at 2°C / min. Human small intestine tissue cDNA was used as a control sample and the same conditions were used.

[0118] The values ​​are relative evaluations, and the Ct of each gene in human small intestinal tissue (INTESTIN) and intestinal-like organoids (MGUT) was corrected by the average Ct of multiple internal controls included in the kit and calculated using the ΔΔCT method. The human small intestinal tissue (INTESTIN) was set to 1, and the relative evaluation was shown.

[0119] The results are shown in Figures 7A to 7G. The left column of each graph shows the relative expression level of each gene in human small intestinal tissue (INTESTIN), and the right column shows the relative expression level of each gene in intestinal-like organoids (MGUT). Compared to human small intestinal tissue, intestinal-like organoids it can be observed that multiple drug-metabolizing enzymes, including the metabolic enzyme CYP, are expressed at a similar level. These results suggest that intestinal-like organoids possess multiple metabolic pathways and metabolic functions, and are comparable to intestinal tissue in vivo.

Claims

1. A method for producing a product resulting from metabolism and / or modification of a substance, comprising: disposing a cell structure having a villi layer on its outer surface and containing a sac-shaped cavity containing small intestinal epithelial cells, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; and Accumulating the product in the cavity within the cell structure. A method comprising:

2. The method of claim 1 , further comprising recovering the product accumulated in the cell structure or recovering the cell structure that has accumulated the product.

3. The method according to claim 1 or 2, wherein the length of the cell structure in the longitudinal direction is 5 mm or more.

4. The method according to any one of claims 1 to 3, wherein the cellular structure has the properties of a small intestine.

5. The method according to any one of claims 1 to 4, wherein the cell structure is a cell structure formed by inducing differentiation from a pluripotent stem cell.

6. A method for producing a medicament containing, as an active ingredient, a product generated by metabolism and / or modification of a substance, comprising: disposing a cell structure having a villi layer on its outer surface and containing a sac-like cavity containing small intestinal epithelial cells, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing the product to accumulate in the cavity within the cell structure; recovering the product accumulated in the cell structure; and formulating the recovered product into a pharmaceutically acceptable preparation. A method comprising:

7. A method for producing a medicament containing, as an active ingredient, a product generated by metabolism and / or modification of a substance, comprising: disposing a cell structure having a villi layer on its outer surface and containing a sac-like cavity containing small intestinal epithelial cells, and a liquid containing the substance, so that the liquid comes into contact with the villi layer; allowing the product to accumulate in the cavity within the cell structure; recovering the cell structure that has accumulated the product; and formulating the recovered cell structures into a pharmaceutically acceptable preparation. A method comprising:

8. A method for evaluating what products are produced when a test substance is metabolized and / or modified in the small intestine, comprising: disposing a cell structure having a villi layer on its outer surface and containing a sac-like cavity containing small intestinal epithelial cells, and a liquid containing the test substance, so that the liquid comes into contact with the villi layer; Culturing the cell construct; and Analyzing the composition of components accumulated in the cavity inside the cell structure after culturing. A method comprising:

9. The method according to any one of claims 1 to 8, wherein the metabolism is drug metabolism.

10. The method according to any one of claims 1 to 9, wherein the cell structure expresses a phase II metabolic enzyme gene, and the product is a product produced by metabolism and / or modification of the substance by an enzymatic activity corresponding to the metabolic enzyme gene.

11. A kit for evaluating what kind of product is produced when a test substance is metabolized and / or modified in the small intestine, wherein the component to be evaluated is a component accumulated in an internal cavity of a cellular structure; A cellular structure containing a sac-like cavity containing small intestinal epithelial cells, the sac-like cavity having a villi layer on its outer surface; and A medium for culturing the cell structure Kit including:

12. The kit of claim 11 , further comprising an instrument for removing components accumulated in the cavity within the cell structure.

13. The kit according to claim 11 or 12, wherein the metabolism is drug metabolism.

14. The kit according to any one of claims 11 to 13, wherein the cell structure expresses a phase II metabolic enzyme gene, and the product is a product produced by metabolism and / or modification of the substance by an enzymatic activity corresponding to the metabolic enzyme gene.

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