Intestinal organoids and method for producing the same

Abstract

To provide an intestinal organoid equipped with the same function as an intestine, and a method for manufacturing the organoid.SOLUTION: An intestinal organoid of the present invention is derived from an embryonic stem cell and / or an artificial pluripotent stem cell, has a structure including a cavity, and a length in a long axis direction is 5 mm or more. A method for manufacturing an intestinal organoid of the present invention includes: a process 1 of inoculating a cell selected from an embryonic stem cell and an artificial pluripotent stem cell onto a substrate and a cell culture substrate including a cell adhesion region and a cell non-adhesion region surrounding the cell adhesion region which are formed on the surface of the substrate; and a process 2 of culturing a cell inoculated in the process 1. The process 2 includes differentiating a part of a cell into an entodermal cell and differentiating a part of a cell into an ectodermal cell.SELECTED DRAWING: Figure 1D

Description

Technical Field

[0001] The present invention relates to an intestinal organoid having a function similar to that of the intestine and a method for producing the same.

Background Art

[0002] The intestine is a complex organ containing cells derived from the three germ layers (endoderm, ectoderm, mesoderm). The intestine , intestinal epithelial cells (enterocytes, goblet cells, endocrine cells, brush cells, Paneth cells , M cells, etc.) derived from the endoderm, lymphoid tissue, smooth muscle cells, Cajal interstitial cells derived from the mesoderm, and the intestinal nerve plexus derived from the outer lung lobe are complexly combined to perform functions such as secretion, absorption, and peristaltic movement. .

[0003] On the other hand, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can be induced to differentiate into target cells and are expected to be applied in the field of regenerative medicine.

[0004] In recent years, techniques for producing "intestinal organoids" having functions similar to those of the intestine from pluripotent stem cells and isolated tissue progenitor cells have been reported (Non-Patent Document 1, etc.).

[0005] In Patent Document 1, (a) a step of collecting undifferentiated embryonic stem cells and culturing the collected undifferentiated embryonic stem cells in a hanging drop culture in a medium containing BDNF to induce embryoid bodies, and (b) a step of attaching the embryoid bodies induced in step (a) onto a culture dish and further culturing them are included to disclose a method for constructing an intestinal tube-like cell mass having an enteric nervous system.

[0006] In Patent Document 2, embryoid bodies are formed and cultured from purified iPS cells using a three-dimensional solid culture system, and then the embryoid bodies are cultured using a two-dimensional adherent culture system to induce differentiation of the intestinal tube. A method for producing an artificial intestinal tract, including the following, is disclosed.

[0007] In Patent Document 3, a method for inducing differentiation of induced pluripotent stem cells into intestinal epithelial cells, including the following steps (1) to (3): (1) a step of differentiating induced pluripotent stem cells into endoderm-like cells; (2 ) a step of differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (3 ) a step of differentiating the intestinal stem cell-like cells obtained in step (2) into intestinal epithelial cell-like cells, which includes culturing in the presence of at least one compound selected from the group consisting of a MEK1 inhibitor, a DNA methylation inhibitor, and a TGFβ receptor inhibitor and EGF, is disclosed.

[0008] In Patent Document 4, a method for producing an intestinal structure from embryonic stem cells and / or induced pluripotent stem cells, which includes performing cell culture on a substrate on which a pattern of a cell adhesion region having a predetermined area is formed, is disclosed.

[0009] In Patent Document 5, a method for inducing differentiation of induced pluripotent stem cells into endoderm-derived cells, which includes performing cell culture on a substrate on which a pattern of a cell adhesion region having a predetermined area is formed, is disclosed.

[0010] On the other hand, as a method for testing the effect of a drug acting on the intestine, a bidirectional transcellular transport ability test (Caco-2 cell membrane permeability test) using Caco-2 cells or cells overexpressing a specific transporter is known.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

[0012] [Non-Patent Document 1] Spence JR, et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature. 2011;470(7332):105-109 Summary of the Invention [Problem to be solved by the invention]

[0013] Caco-2 is a colon adenocarcinoma cell line that accurately reflects the absorption of small molecules by small intestinal epithelial cells. In addition, the Caco-2 cell membrane permeability test is a semi-permeable membrane (trans A monolayer is formed on the swell (no leaks) and transepithelial electrical resistance is measured before the assay. The procedure was complicated because it required the measurement of the TEER (tetanic oxygen saturation) and other parameters. -2 There was a problem that it easily damaged the cell membrane.

[0014] If we could create intestinal organoids with intestinal functions similar to those of the intestine, we could potentially predict and treat intestinal-related diseases. This is expected to be useful in the development of drugs to prevent or treat intestinal diseases and in pathological research into intestinal-related diseases. However, the intestinal organoids provided to date have not necessarily been satisfactory.

[0015] The method described in Non-Patent Document 1 involves culturing human pluripotent stem cells with activin treatment or the like to differentiate them into endoderm while suppressing differentiation into mesoderm and ectoderm, thereby producing an intestinal epithelial structure, and on the other hand, producing neural crest cells and combining the two to produce intestinal organoids. This method is complex in operation and does not mimic the natural development of the three germ layers in vivo.

[0016] In addition, the cell structures produced by the methods described in Patent Documents 1 to 5 do not have functions equivalent to those of the intestine and still have room for improvement.

Means for Solving the Problems

[0017] Therefore, the present invention provides an intestinal organoid having functions equivalent to those of the intestine, a method for producing the same, a medium suitable for producing the intestinal organoid, and a kit suitable for producing the intestinal organoid. Specifically, the present invention includes the following inventions.

[0018] (1) An intestinal organoid derived from embryonic stem cells and / or induced pluripotent stem cells, having a structure enclosing a cavity, characterized in that the length in the long axis direction is 5 mm or more. (2) The intestinal organoid according to (1), comprising endoderm-derived cells, ectoderm-derived cells, and mesoderm-derived cells. (3) The intestinal organoid according to (1) or (2), wherein the outer surface contains intestinal epithelial cells. (4) Step 1 of seeding cells selected from embryonic stem cells and induced pluripotent stem cells on a cell culture substrate comprising a substrate, a cell adhesion region formed on the surface of the substrate, and a cell non-adhesion region surrounding the cell adhesion region, and Step 2 of culturing the cells seeded in Step 1 ​​​​​​ comprising step 2 is a part of the cells seeded in step 1 differentiating into endodermal cells, and a part of the cells seeded in step 1 differentiating into ectodermal cells A method for producing an intestinal organoid comprising. (5) In step 2, the differentiation timing of a part of the cells seeded in step 1 into endodermal cells is earlier than the differentiation timing of a part of the cells seeded in step 1 into ectodermal cells, the method according to (4). (6) Step 2 is such that a part of the cells seeded in step 1 differentiates into endodermal cells within 14 days after seeding, the method according to (4) or (5). (7) Step 2 is such that a part of the cells seeded in step 1 differentiates into ectodermal cells after 15 days of seeding, the method according to any one of (4) to (6). (8) Step 2 includes culturing the cells in a medium containing insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF), the method according to any one of (4) to (7). (9) The method according to (8), wherein the medium further contains heregulin. (10) The method according to any one of (4) to (9), wherein step 2 is performed under conditions without the presence of a heterologous component. (11) A cell culture substrate comprising a substrate, a cell adhesion region formed on the surface of the substrate, and a cell non-adhesion region surrounding the cell adhesion region, seeding step 1 of a cell selected from embryonic stem cells and induced pluripotent stem cells on the cell culture substrate, and step 2 of culturing the cells seeded in step 1 comprising step 2 includes culturing the cells in a medium containing insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF) A method for producing an intestinal organoid. (12) The method according to (11), wherein the medium further contains heregulin. (13) The method according to (11) or (12), wherein step 2 is carried out in the absence of heterogeneous components. (14) A differentiation induction medium for producing intestinal organoids, comprising insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF). (15) The differentiation induction medium for producing intestinal organoids according to (14), further comprising heregulin. (16) The differentiation induction medium for producing intestinal organoids according to (14) or (15), which does not contain heterogeneous components. (17) A differentiation induction medium for producing intestinal organoids according to any one of (14) to (16), and a cell culture substrate comprising a substrate, a cell adhesion region formed on the surface of the substrate, and a cell non-adhesion region surrounding the cell adhesion region. A kit for producing intestinal organoids. [Advantages of the Invention]

[0019] The intestinal organoids of the present invention have a sufficient size, have functions equivalent to those of the intestine, and can take in substances from the outside into the enclosed lumen. Therefore, they are useful for the development of drugs for preventing or treating intestinal-related diseases and for the pathological study of intestinal-related diseases. According to the method for producing intestinal organoids of the present invention, intestinal organoids can be easily produced from cells selected from embryonic stem cells and induced pluripotent stem cells. The differentiation induction medium and kit of the present invention are useful for the production of intestinal organoids. [Brief Description of the Drawings]

[0020]

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Mode for Carrying Out the Invention

[0021] 1. Intestinal organoid In the present invention, the "intestinal organoid" refers to a tissue structure having functions similar to those of the intestine of the origin organism of the cells, particularly the intestine of mammals such as humans intestine, particularly functions similar to those of the human intestine (specifically, functions of peristaltic movement, mucus secretion function, substance absorption function, etc.).

[0022] The intestinal organoids of the present invention are intestinal organoids derived from embryonic stem cells (ES cells) and / or induced pluripotent stem cells (iPS cells ).

[0023] The embryonic stem cells (ES cells) used in the present invention are preferably ES cells derived from mammals and can use, for example, ES cells derived from rodents such as mice or primates such as humans. Particularly preferably, ES cells derived from mice or humans are used. ES cells are stem cell lines created from the inner cell mass belonging to a part of the embryo at the blastocyst stage, which is the early stage of animal development, and can theoretically proliferate almost infinitely while maintaining pluripotency to differentiate into all tissues in vitro. As ES cells, for example, in order to facilitate confirmation of the degree of their differentiation, cells into which a reporter gene has been introduced near the Pdx1 gene can be used. For example, an ES cell line derived from 129 / Sv in which the LacZ gene has been integrated into the Pdx1 locus or an ES cell line SK7 having a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, an ES cell line PH3 having an mRFP1 reporter transgene under the control of the 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 SEES1, SEES2, SEES 3, SEES4, SEES5, SEES6 or SEES7, which were established by the Reproductive and Cellular Medicine Research Department of the National Center for Child Health and Development and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, and cell lines in which additional genes have been introduced into these ES cell lines can also be used.

[0024] Induced pluripotent stem cells (iPS cells) used in the present invention are cells having pluripotency obtained by reprogramming somatic cells. The production of induced pluripotent stem cells was achieved by the group of Prof. Shinya Yamanaka of Kyoto University and Rudolf Jaenisch of the Massachusetts Institute of Technology (Rudolf Ja Groups such as the group of Yamanaka et al., the group of James Thomson of the University of Wisconsin, the group of Konrad Hochedlinger of Harvard University, etc. have been successful. For example, International Publication WO2007 / 06966 Publication No. 6 describes somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes, and somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes. Further described is a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes the step of contacting the somatic cells with the above nuclear reprogramming factors. The type of somatic cells used for the production of iPS cells is not particularly limited, and any somatic cells can be used. That is, the somatic cells referred to in the present invention include all cells other than germ cells among the cells constituting a living body, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the somatic cells may be any of mammals, birds, fish, reptiles, and amphibians, and is not particularly limited, but preferably is a mammal (for example, rodents such as mice, or primates such as humans), and particularly preferably is a mouse or a human. Also, when using human somatic cells, any somatic cells of a fetus, a newborn, or an adult may be used. Specific examples of somatic cells include, for example, fibroblasts (for example, skin fibroblasts), epithelial cells (for example, gastric epithelial cells, liver epithelial cells, alveolar epithelial cells), endothelial cells (for example, blood vessels, lymphatic vessels), nerve cells (for example, neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (for example, skeletal muscle cells, smooth muscle cells, cardiac muscle cells),

[0025] The type of somatic cells used for the production of iPS cells is not particularly limited, and any somatic cells can be used. That is, the somatic cells referred to in the present invention include all cells other than germ cells among the cells constituting a living body, and may be differentiated somatic cells or undifferentiated stem cells. The origin of the somatic cells may be any of mammals, birds, fish, reptiles, and amphibians, and is not particularly limited, but preferably is a mammal (for example, rodents such as mice, or primates such as humans), and particularly preferably is a mouse or a human. Also, when using human somatic cells, any somatic cells of a fetus, a newborn, or an adult may be used. Specific examples of somatic cells include, for example, fibroblasts (for example, skin fibroblasts), epithelial cells (for example, gastric epithelial cells, liver epithelial cells, alveolar epithelial cells), endothelial cells (for example, blood vessels, lymphatic vessels), nerve cells (for example, neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (for example, skeletal muscle cells, smooth muscle cells, cardiac muscle cells), cells, etc. Hepatocytes, non-parenchymal liver cells, adipocytes, osteoblasts, cells constituting periodontal tissues (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), cells constituting the kidneys, eyes, ears, etc. can be mentioned.

[0026] iPS cells have the ability of self-renewal over a long period under predetermined culture conditions (e.g., conditions for culturing ES cells), and also have the pluripotency to differentiate into ectoderm, mesoderm, and endoderm under predetermined differentiation induction conditions. In addition, the iPS cells in the present invention may also be stem cells having the ability to form teratomas

[0027] when transplanted into test animals such as mice. To produce iPS cells from somatic cells, first, at least one or more reprogramming genes are introduced into somatic cells. A reprogramming gene is a gene encoding a reprogramming factor having the action of reprogramming somatic cells into iPS cells. Specific examples of combinations of reprogramming genes include, but are not limited to, the following combinations. (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 large T gene

[0028] In one embodiment, the intestinal organoid has a structure enclosing a cavity. The cavity is preferably a closed cavity capable of retaining liquid inside. The intestinal organoid enclosing the cavity can take in substances present outside into the cavity, and thus can evaluate It is useful for the intended use.

[0029] The intestinal organoid according to one embodiment has a length in the major axis direction of 5 mm or more, preferably 8 mm or more, more preferably 10 mm or more, still more preferably 12 mm or more, and even more preferably 15 mm or more. Including the reports mentioned in the background art section, conventionally, there has been no example of producing an intestinal organoid with such a large size in the major axis direction. A large-sized intestinal organoid is preferable because it enables a higher-precision test compared to a small-sized intestinal organoid when used in a test for examining the effect of a drug, and is easy to handle. For example, since a large amount of liquid can be retained in the cavity enclosed by a large-sized intestinal organoid, it is possible to include a test drug in the buffer solution with the large-sized intestinal organoid floating in the buffer solution, and after a certain period of time, it is easy to perform an analysis that was impossible with conventional intestinal organoids, such as lifting the intestinal organoid and taking out and analyzing the liquid in the cavity. Also, because the volume of the liquid contained in the cavity is large, simple evaluation methods such as fluorescence observation can be used without using mass spectrometry. Here, the "length in the major axis direction" refers to the longest distance among the distances between two points that can be connected by a straight line passing through the inside of the observation image of the intestinal organoid on the contour of the observation image of the intestinal organoid when observing the intestinal organoid in an appropriate buffer solution visually or using an optical microscope. Although the contour of an individual intestinal organoid can be deformed by peristaltic movement, the maximum value measured may be taken as the length in the major axis direction. The overall shape of the intestinal organoid is not particularly limited, but it is usually granular. "Granular" includes spherical. The intestinal organoid according to one embodiment has a length in the major axis direction of 5 mm or more, preferably 8 mm or more, more preferably 10 mm or more, still more preferably 12 mm or more, and even more preferably 15 mm or more. Including the reports mentioned in the background art section, conventionally, there has been no example of producing an intestinal organoid with such a large size in the major axis direction. A large-sized intestinal organoid is preferable because it enables a higher-precision test compared to a small-sized intestinal organoid when used in a test for examining the effect of a drug, and is easy to handle. For example, since a large amount of liquid can be retained in the cavity enclosed by a large-sized intestinal organoid, it is possible to include a test drug in the buffer solution with the large-sized intestinal organoid floating in the buffer solution, and after a certain period of time, it is easy to perform an analysis that was impossible with conventional intestinal organoids, such as lifting the intestinal organoid and taking out and analyzing the liquid in the cavity. Also, because the volume of the liquid contained in the cavity is large, simple evaluation methods such as fluorescence observation can be used without using mass spectrometry. Here, the "length in the major axis direction" refers to the longest distance among the distances between two points that can be connected by a straight line passing through the inside of the observation image of the intestinal organoid on the contour of the observation image of the intestinal organoid when observing the intestinal organoid in an appropriate buffer solution visually or using an optical microscope. Although the contour of an individual intestinal organoid can be deformed by peristaltic movement, the maximum value measured may be taken as the length in the major axis direction. The overall shape of the intestinal organoid is not particularly limited, but it is usually granular. "Granular" includes spherical.

[0030] Here, the "length in the major axis direction" refers to the longest distance among the distances between two points that can be connected by a straight line passing through the inside of the observation image of the intestinal organoid on the contour of the observation image of the intestinal organoid when observing the intestinal organoid in an appropriate buffer solution visually or using an optical microscope. Although the contour of an individual intestinal organoid can be deformed by peristaltic movement, the maximum value measured may be taken as the length in the major axis direction. In the observation image, it is the longest distance between two points on the contour of the observation image of the intestinal organoid that can be connected by a straight line passing through the inside of the contour. The contour of each intestinal organoid can be deformed by peristaltic movement, but the maximum measured value can be used as the length in the major axis direction. The overall shape of the intestinal organoid is not particularly limited, but it is usually granular. "Granular" includes spherical. That's all.

[0031] The overall shape of the intestinal organoid is not particularly limited, but it is usually granular. "Granular" includes spherical. "Granular" includes spherical. The intestinal organoids preferably contain endodermal cells, ectodermal cells, and mesodermal cells.

[0032] In addition to the digestive tract, the endoderm forms tissues of organs such as the lungs, thyroid gland, pancreas, and liver, cells of exocrine glands that open into the digestive tract, the peritoneum, pleura, larynx, eustachian tube, trachea, bronchi, urinary tract (most of the bladder, urethra, and part of the ureter). Differentiation of ES cells or iPS cells into endodermal cells can be confirmed by measuring the expression levels of genes specific to the endoderm. Examples of genes specific to the endoderm include, in addition to those described below, AFP, SERPINA1, SST, ISL1, IPF1, IAPP, EOMES, HGF, ALBUMIN, PAX4, TAT, etc.

[0033] Endodermal cells that can be included in the intestinal organoids include, in particular, intestinal epithelial cells. The intestinal organoids preferably contain, as intestinal epithelial cells, one or more selected from intestinal cells, goblet cells, enteroendocrine cells, and Paneth cells, and particularly preferably contain all of intestinal cells, goblet cells, enteroendocrine cells, and Paneth cells as intestinal epithelial cells. The presence of endodermal cells in the intestinal organoids can be determined based on the positive expression of markers for endodermal cells. Examples of intestinal cell markers include CDX2, goblet cell markers include MUC2, enteroendocrine cell markers include CGA, and Paneth cell markers include DEFA6. In addition, ECAD, Na+ / K+-ATPase, and villin are markers for intestinal epithelial cells. Also, the embryonic endoderm markers FOXA2, SOX17, or CXCR4 can be used as markers for discriminating endodermal cells. In addition, GATA4, GATA6, or T (Brachyury), which are markers for early endoderm and mesoderm, can also be used to discriminate endodermal cells. ​​​​​​​​​​​​​​ It can be used as a marker for doing so.

[0034] The ectoderm forms the epidermis of the skin, the epithelium at the terminal part of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands ), sense organs (including the epithelium at the terminal parts of the oral cavity, pharynx, nose, and rectum, salivary glands), the lens, etc. A part of the ectoderm invaginates in a groove-like manner during development to form the neural tube, which also gives rise to neurons of the central nervous system such as the brain and spinal cord and melanocytes, etc. It also forms the peripheral nervous system. The differentiation of ES cells or iPS cells into ectodermal cells can be confirmed by measuring the expression level of genes specific to the ectoderm . Examples of genes specific to the ectoderm include, in addition to those described later, for example, β-TUBLIN, NESTIN, GALANIN, GCM1, GFAP, NEUROD1, OLIG2, SYNAPTPHYSIN , DESMIN, TH, etc.

[0035] Examples of ectodermal cells that can be included in intestinal organoids include, in particular, cells that make up the enteric nerve plexus . The presence of ectodermal cells in intestinal organoids can be determined based on the positive expression of markers for ectodermal cells . As markers for discriminating ectodermal cells, the enteric nerve plexus marker PGP9.5 and the neural progenitor cell marker SOX1 can be used.

[0036] The mesoderm forms the body cavity and the mesothelium lining it, muscles, skeleton, dermis of the skin, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, gonads ( testes, uterus, gonadal epithelium). The differentiation of ES cells or iPS cells into mesodermal cells can be confirmed by measuring the expression level of genes specific to the mesoderm . As specific genes, in addition to those described later, for example, FLK-1, COL2A1, FLT1, HBZ, MYF5, MYOD1, RUNX2, PECAM1 and the like can be mentioned.

[0037] Mesenchymal cells that can be included in intestinal organoids include, in particular, smooth muscle cells and Cajal interstitial cells can be mentioned. The presence of mesenchymal cells in intestinal organoids can be determined based on the positive expression of mesenchymal cell markers. As mesenchymal cell markers, smooth muscle cell marker α-smooth muscle actin (SMA), Cajal interstitial cell markers CD34 and CKIT (in the case of double positivity) can be used. Also, GATA4, GATA6 or T (Brachyury), which are markers of early endoderm and mesoderm, can also be used as markers for discriminating mesenchymal cells.

[0038] Intestinal organoids more preferably further contain intestinal stem cells. The presence of intestinal stem cells can be determined using the positivity of the intestinal stem cell marker LGR5 as an indicator.

[0039] Intestinal organoids preferably further contain serotonin-positive enteroendocrine cells. Intestinal organoids further contain transporter-positive cells, and it is preferable that substances can be taken up through transporters. Examples of transporters include intestinal oligopeptide transporter (PEPT1), ABCB1 and ABCG2 which are ATP-binding cassette (ABC) transporters. etc. can be exemplified.

[0040] Intestinal organoids preferably further contain cystic fibrosis transmembrane conductance regulator (CFTR)-positive intestinal epithelial cells CFTR-positive intestinal epithelial cells are involved in mucus secretion. That is, Intestinal organoids with CFTR-positive intestinal epithelial cells have mucus-secreting ability similar to that of the intestine. The intestinal organoids preferably further contain histamine H1 receptor-positive cells.

[0041] The intestinal organoids of the present invention preferably contain intestinal epithelial cells on at least a part of the outer surface. . According to this embodiment, it is preferable because a substance outside the intestinal organoid can be absorbed into the cavity through the intestinal epithelial cells on the outer surface. Also, in this embodiment, when the intestinal epithelial cells on the outer surface are further positive for the transporter, it is more preferable because the substance can be taken up through the transporter. That is, intestinal organoids containing transporter-positive intestinal epithelial cells have a substance absorption ability similar to that of the intestine. In the intestine of a mammal, the intestinal epithelial cells face the inside of the intestinal tract which is a cavity, which is different from the intestinal organoids according to this embodiment.

[0042] The intestinal organoids of the present invention more preferably have development of microvilli and crypts on the outer surface.

[0043] The intestinal organoids of the present invention preferably have the ability to perform a contraction movement similar to peristaltic movement. Such a function is caused by the development of the neural network and smooth muscle. In the following description, the ability to perform a contraction movement similar to peristaltic movement may be referred to as "peristaltic ability". Intestinal organoids with peristaltic ability particularly preferably show the same drug responsiveness as the intestine, that is, the frequency of contraction increases by histamine treatment and decreases by atropine treatment. Intestinal organoids with peristaltic ability showing the same drug responsiveness as the intestine can be suitably used for the purpose of evaluating the effect of a drug on the peristaltic movement of the intestine. ​​​​​​​​​​​

[0044] As described above, the intestinal organoids of the present invention have a sufficient size and functions equivalent to those of the intestine, and can take in substances from the outside into the enclosed cavity, so they are useful for the development of drugs for preventing or treating intestinal-related diseases and for pathological research on intestinal-related diseases. By using the intestinal organoids of the present invention, there is no need to perform complicated steps such as forming a fragile monolayer membrane on a semi-permeable membrane, like the conventional drug test method using Caco-2, or measuring the transepithelial electrical resistance (TEER) to indicate the integrity of the membrane. Moreover, if the intestinal organoids of the present invention are prepared from disease-specific iPS cells, it becomes easier to study intestinal diseases and evaluate drugs involving genetic factors. The intestinal organoids of the present invention can be produced by a method including the following steps: Step 1 of seeding cells selected from embryonic stem cells and induced pluripotent stem cells on a cell culture substrate comprising a substrate, a cell adhesion region formed on the surface of the substrate, and a cell non-adhesion region surrounding the cell adhesion region, and

[0045] Step 2 of culturing the cells seeded in Step 1. The cells selected from embryonic stem cells and induced pluripotent stem cells used in this method are as described above.

[0046] 2. Method for producing intestinal organoid A preferred embodiment of the cell culture substrate used in the present invention is as follows. In the cell culture substrate used in the present invention, preferably, a plurality of cell adhesion regions exist in an island shape in the cell non-adhesion region.

[0047]

[0048]

[0049] ​​​​​​​​​The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate.

[0050] In the present invention, "cell adhesiveness" means the strength of cell adhesion, that is, the ease of cell adhesion. The cell adhesive region means a region with good cell adhesiveness, and the cell non-adhesive region means a region with poor cell adhesiveness. Therefore, when cells are seeded on a substrate in which the cell adhesive region and the cell non-adhesive region are patterned, the cells adhere to the cell adhesive region, but do not adhere to the cell non-adhesive region, so that the cells are arranged in a pattern on the surface of the cell culture substrate.

[0051] As an index for judging cell adhesiveness, the cell adhesion and spreading rate during actual cell culture can be used. The surface with cell adhesiveness is preferably a surface with a cell adhesion and spreading rate of 60% or more, and more preferably a surface with a cell adhesion and spreading rate of 80% or more. A higher cell adhesion and spreading rate enables efficient cell culture. The cell adhesion and spreading rate in the present invention is defined as the ratio of the number of adhered and spread cells ({(number of adhered cells) / (number of seeded cells)}×100 (%)) when the cells to be cultured within the range of a seeding density of 4000 cells / cm 2 2

[0052] In the above measurement, the cells are seeded by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low). On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized. In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low). On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized. In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low).

[0053] On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized. In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low). On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized.

[0054] In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low). On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized. In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low). On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether cell adhesion and spreading are difficult to occur due to chemical properties, physical properties, etc. of the surface. The surface of cell non-adhesiveness is preferably a surface where the cell adhesion and spreading rate defined above is less than 60%, more preferably less than 40%, still more preferably 5% or less, and most preferably 2% or less. The cell culture substrate used in the present invention preferably has a cell non-adhesive region formed by immobilizing polyethylene glycol on the substrate, and a cell adhesive region formed by oxidizing and / or decomposing at least a part of the polyethylene glycol immobilized on the substrate. Such a cell culture substrate can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the substrate, and then subjecting the region where cell adhesion is desired to oxidation treatment and / or decomposition treatment to impart cell adhesiveness. The portion not subjected to the treatment is a cell non-adhesive region where PEG is immobilized. In the above measurement, the seeding of cells is carried out by suspending them in DMEM medium containing 10% FBS and seeding them on the object to be measured, and then gently shaking the object to be measured on which the cells are seeded so that the cells are distributed as uniformly as possible. Further, the measurement of the cell adhesion and spreading rate is carried out after replacing the medium immediately before the measurement to remove non-adherent cells. In the measurement of the cell adhesion and spreading rate, the measurement site is a site excluding the sites where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, and the periphery of a predetermined region where the density is likely to be low).

[0055] Polyethylene glycol (PEG) consists of one or more ethylene glycol units ((CH2)2-O) and contains at least an ethylene glycol chain (EG chain), which may be linear or branched. The ethylene glycol chain has, for example, the following formula: -((CH2)2-O)m- (where m is an integer representing the degree of polymerization) -((CH2)2-O)m- (m is an integer representing the degree of polymerization) m is preferably an integer from 1 to 13, more preferably an integer from 1 to 10.

[0056] PEG also includes ethylene glycol oligomers. PEG also includes those with functional groups introduced. Examples of functional groups include epoxy groups, carboxyl groups, N-hydroxysuccinimide groups, carbodiimide groups, amino groups, glutaraldehyde groups, (meth)acryloyl groups, etc. The functional groups are preferably introduced at the terminal, optionally via a linker. Examples of PEG with functional groups introduced include PEG (meth)acrylate and PEG di(meth)acrylate. The substrate used for the cell culture substrate is not particularly limited as long as it is made of a material capable of forming a PEG thin film on its surface. Specifically, inorganic materials such as metals, glasses, ceramics, and silicon, elastomers, 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, vinyl chloride resin) are representative. -((CH2)2-O)m- (where m is an integer representing the degree of polymerization) m is preferably an integer from 1 to 13, more preferably an integer from 1 to 10. PEG also includes ethylene glycol oligomers. PEG also includes those with functional groups introduced. Examples of functional groups include epoxy groups, carboxyl groups, N-hydroxysuccinimide groups, carbodiimide groups, amino groups, glutaraldehyde groups, (meth)acryloyl groups, etc. The functional groups are preferably introduced at the terminal, optionally via a linker. Examples of PEG with functional groups introduced include PEG (meth)acrylate and PEG di(meth)acrylate.

[0057] The substrate used for the cell culture substrate is not particularly limited as long as it is made of a material capable of forming a PEG thin film on its surface. Specifically, inorganic materials such as metals, glasses, ceramics, and silicon, elastomers, 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, vinyl chloride resin) are representative. Metals, glasses, ceramics, silicon, etc. Inorganic materials such as metals, glasses, ceramics, and silicon, elastomers, 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, vinyl chloride resin) are representative. -((CH2)2-O)m- (where m is an integer representing the degree of polymerization) m is preferably an integer from 1 to 13, more preferably an integer from 1 to 10. ​Examples of the organic material to be used can be given. Its shape is not limited either. For example, flat shapes such as flat plates, flat films, films, porous membranes, etc., and three-dimensional shapes such as cylinders, stamps, multi-well plates, microfluidic channels, etc. can be mentioned. When using a film, its thickness is not particularly limited, but it is usually 0.1 to 1000 μm, preferably 1 to 500 μm, more preferably 10 to 200 μm. When using a film, its thickness is not particularly limited, but it is usually 0.1 to 1000 μm, preferably 1 to 500 μm, more preferably 10 to 200 μm. When using a film, its thickness is not particularly limited, but it is usually 0.1 to 1000 μm, preferably 1 to 500 μm, more preferably 10 to 200 μm. When using a film, its thickness is not particularly limited, but it is usually 0.1 to 1000 μm, preferably 1 to 500 μm, more preferably 10 to 200 μm. There is.

[0058] The average thickness of the PEG thin film formed on the substrate is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, even more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. The average thickness of the PEG thin film formed on the substrate is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, even more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region. If the average thickness is 0.8 nm or more, it is preferable because it is less likely to be affected by the regions on the substrate surface not covered with the PEG thin film in protein adsorption and cell adhesion. Also, if the average thickness is 500 μm or less, coating is relatively easy. Furthermore, by making the thickness of the PEG thin film equal to or more than a certain value, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film equal to or less than a certain value, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region.

[0059] As methods for forming the PEG thin film on the substrate surface, methods such as directly adsorbing PEG to the substrate, directly coating PEG on the substrate, performing a crosslinking treatment after coating PEG on the substrate, forming an underlayer on the substrate to enhance adhesion and then coating PEG, forming a polymerization initiation point on the substrate surface and then polymerizing PEG, etc. can be mentioned. The method of forming an underlayer on the substrate and then coating PEG is preferable. As methods for forming the PEG thin film on the substrate surface, methods such as directly adsorbing PEG to the substrate, directly coating PEG on the substrate, performing a crosslinking treatment after coating PEG on the substrate, forming an underlayer on the substrate to enhance adhesion and then coating PEG, forming a polymerization initiation point on the substrate surface and then polymerizing PEG, etc. can be mentioned. The method of forming an underlayer on the substrate and then coating PEG is preferable. As methods for forming the PEG thin film on the substrate surface, methods such as directly adsorbing PEG to the substrate, directly coating PEG on the substrate, performing a crosslinking treatment after coating PEG on the substrate, forming an underlayer on the substrate to enhance adhesion and then coating PEG, forming a polymerization initiation point on the substrate surface and then polymerizing PEG, etc. can be mentioned. The method of forming an underlayer on the substrate and then coating PEG is preferable. As methods for forming the PEG thin film on the substrate surface, methods such as directly adsorbing PEG to the substrate, directly coating PEG on the substrate, performing a crosslinking treatment after coating PEG on the substrate, forming an underlayer on the substrate to enhance adhesion and then coating PEG, forming a polymerization initiation point on the substrate surface and then polymerizing PEG, etc. can be mentioned. The method of forming an underlayer on the substrate and then coating PEG is preferable. As methods for forming the PEG thin film on the substrate surface, methods such as directly adsorbing PEG to the substrate, directly coating PEG on the substrate, performing a crosslinking treatment after coating PEG on the substrate, forming an underlayer on the substrate to enhance adhesion and then coating PEG, forming a polymerization initiation point on the substrate surface and then polymerizing PEG, etc. can be mentioned. The method of forming an underlayer on the substrate and then coating PEG is preferable.

[0060] The underlying layer can be formed, for example, by the method described in JP-A-2012-175983, preferably using a silane coupling agent having a functional group that can react with a hydroxyl group at the end of PEG or an introduced functional group to form a covalent bond, or a functional group that can be converted into such a functional group. Examples of such functional groups include (meth)acryloyl groups, (1H-imidazol-1-yl)carbonyl groups, succinimidyl oxycarbonyl groups, glycidyl groups, epoxy groups, aldehyde groups, amino groups, thiol groups, carboxyl groups, azide groups, cyano groups, active ester groups (1H-benzotriazol-1-yloxycarbonyl groups, pentafluorophenyloxycarbonyl groups, paranitrophenyloxycarbonyl groups, etc. ), halogenated carbonyl groups, isocyanate groups, maleimide groups, etc. Among them, the (meth)acryloyl group, glycidyl group or epoxy group is preferable, and the glycidyl group or epoxy group is most preferable.

[0061] For example, taking a silane coupling agent having a methacryloyl group at the end (methacryloyl silane) as an example, the water contact angle of the surface of the substrate to which methacryloyl silane is added is typically 45° or more, desirably 47° or more, more desirably 48° or more, and even more desirably 50° or more. Thereby, a sufficient cell non-adhesive region can be formed by immobilizing PEG next.

[0062] The density of PEG immobilized on the substrate and the cell non-adhesiveness can be easily evaluated using the contact angle of water on the surface as an index. For example, the water contact angle of the surface after PEG immobilization is typically If it is 48° or less, preferably 40° or less, more preferably 30° or less, PEG has a sufficient density and is considered to be cell non - adhesive. In the present invention, the water contact angle refers to the water contact angle measured at 23°C .

[0063] In the present invention, "oxidation" has a narrow meaning and refers to a reaction in which an organic compound, that is, PEG, reacts with oxygen and the oxygen content becomes higher than before the reaction. In the present invention, "decomposition" refers to a reaction in which the bond of an organic compound, that is, PEG, is broken. As "decomposition", typically there are decomposition due to oxidation, decomposition due to ultraviolet irradiation, etc., but it is not limited to these . When "decomposition" is decomposition accompanied by oxidation (that is, oxidative decomposition), "decomposition" and "oxidation" refer to the same treatment .

[0064] Decomposition by ultraviolet irradiation means that PEG absorbs ultraviolet light and decomposes through an excited state . When PEG is irradiated with ultraviolet light in a system where it exists together with molecular species containing oxygen (oxygen, water, etc.), in addition to the decomposition caused by the absorption of ultraviolet light by PEG, the molecular species may be activated and react with PEG . The latter reaction can be classified as "oxidation". And the reaction in which PEG decomposes due to oxidation by the activated molecular species can be classified as "decomposition by oxidation" rather than "decomposition by ultraviolet irradiation". As described above, "oxidation" and "decomposition" may overlap as operations and it is impossible to clearly distinguish between the two. Therefore, in this specification, the term "oxidation and / or decomposition" is used .

[0065] As methods of oxidation and / or decomposition, there are methods such as ultraviolet irradiation treatment of a PEG thin film, photocatalytic treatment treatment, treatment with an oxidizing agent, etc. The PEG thin film is partially oxidized and / or​ When decomposing, masks such as photomasks and stencil masks may be used, or stamps may be used. Also, it may be oxidized and / or decomposed by a direct drawing method such as a method using a laser such as an ultraviolet laser. / or decomposed.

[0066] In the case of ultraviolet irradiation treatment, lamps that emit ultraviolet light in the VUV region to the UV-C region, such as mercury lamps that emit ultraviolet light with wavelengths of 185 nm or 254 nm or excimer lamps that emit ultraviolet light with a wavelength of 172 nm, are preferably used as the light source. When performing photocatalytic treatment, it is preferable to use a light source that emits ultraviolet light with a wavelength of 365 nm or less, and it is more preferable to use a light source that emits ultraviolet light with a wavelength of 254 nm or less. As the photocatalyst, it is preferable to use a titanium oxide photocatalyst or a titanium oxide photocatalyst activated by metal ions or metal colloids. As the oxidizing agent, organic acids and inorganic acids can be used without particular limitation, but since high-concentration acids are difficult to handle, it is advisable to dilute them to a concentration of 10% or less before use. The optimum ultraviolet treatment time, photocatalytic treatment time, and oxidizing agent treatment time can be appropriately determined according to various conditions such as the ultraviolet intensity of the light source used, the activity of the photocatalyst, and the oxidizing power and concentration of the oxidizing agent.

[0067] The carbon content in the cell adhesion region (including the underlying layer if present) is preferably lower than the carbon content in the cell non-adhesion region (including the underlying layer if present). Specifically, it is preferable that the carbon content in the cell adhesion region is 20 to 99% of the carbon content in the cell non-adhesion region. The percentage value of the carbon combined with oxygen among the carbon in the cell adhesion region (including the underlying layer if present) is When including the underlying layer, the ratio (%) of carbon bonded to oxygen among the carbon in is preferably a small value with respect to the value. Specifically, the ratio (%) of carbon bonded to oxygen among the carbon in the cell adhesion region is preferably 35 to 99% with respect to the ratio (%) of carbon bonded to oxygen among the carbon in the cell non - adhesion region. As the ultraviolet exposure amount increases during patterning, the cell adhesion increases, but when recovering the cells, high adhesiveness makes it difficult for the cells to peel off and difficult to recover. In the present invention, "carbon amount" is defined as "the carbon amount obtained from the analysis value of the C1s peak obtained using an X - ray photoelectron spectrometer", and "the ratio of carbon bonded to oxygen" is defined as "the ratio of carbon bonded to oxygen obtained from the analysis value of the C1s peak obtained using an X - ray photoelectron spectrometer".

[0068] In the cell culture substrate used in the present invention, the area of each cell adhesion region is not particularly limited. Specific examples of the area of each cell adhesion region include 0.1 mm or more, preferably 0.5 mm or more

[0069] Preferably 0.785 mm or more, more preferably 1.0 mm 2 or more, more preferably 1.2 mm 2 or more Even more preferably 1.5 mm 2 or more, most preferably 1.7 mm 2 or more, and preferably 25 mm 2 or less, more preferably 15 mm or less, even more preferably 10 mm 2 or less, most preferably 5 mm 2 or less. 2 In the cell culture substrate used in the present invention, the area of each cell adhesion region is not particularly limited. 2 Specific examples of the area of each cell adhesion region include 0.1 mm 2 or more, preferably 0.5 mm 2 It is formed in a pattern within the following range. When the area of the cell adhesion region is within this range, it is easy to culture large-sized intestinal organoids with a major axis length exceeding 5 mm.

[0070] The shape of each cell adhesion region is not particularly limited, and it can be a polygon such as a quadrilateral, a circle, an ellipse or the like. A circular one is preferred. In the case of a circle, the diameter is preferably a diameter that satisfies the above described area range. Specifically, the diameter of the circle can be 0.35 mm or more as an example and is 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, preferably 6 mm or less, more preferably 4 mm or less, still more preferably 3 mm or less, and still more preferably 2 mm or less. In one cell culture substrate, a plurality of existing cell adhesion regions preferably all have the same area, and more preferably have the same area and shape but different areas and shapes may be mixed.

[0071] Also, in the cell culture substrate, each cell adhesion region is surrounded by a cell non-adhesion region, that is, they are isolated from each other, preferably separated from each other by 0.75 mm or more, more preferably 1.5 mm or more and are arranged. That is, the shortest distance between cell adhesion regions (in the case of a circle, the distance between the centers of two circles is the value obtained by adding the above value to the sum of each radius) is preferably 0.75 mm or more, more preferably 1.5 mm or more. By isolating each cell adhesion region by a certain distance or more the cells within each cell adhesion region can be cultured uniformly at a constant interval without forming cell-cell junctions with the cells of other cell adhesion regions, and an experimental system with high reproducibility can be constructed.

[0072] The ratio of the cell adhesion area in the cell culture substrate is usually 5 to 80%, preferably 20 to 70%, more preferably 40 to 60%. In addition, this ratio is calculated as the ratio of the cell adhesion area to the entire substrate excluding the bottom surface of the dish even when the substrate is placed in a dish or the like. By setting the amount of cells in the culture medium to a certain level or higher, cell death can be prevented, and by setting it to a certain level or lower, depletion of factors necessary for survival and damage to cells caused thereby can be prevented.

[0073] In addition, each cell adhesion area is preferably arranged regularly at a constant interval, for example, in a grid pattern, with the same pitch in both the vertical and horizontal directions. By making the paracrine effect of the products from the cells in each cell adhesion area constant, the influence on differentiation can be made constant.

[0074] For example, a pattern having a plurality of circular cell adhesion areas can be formed by using a photomask having a plurality of circular openings, arranging a glass substrate on which a PEG thin film is formed so as to face the photomask, irradiating ultraviolet rays from the side of the photomask, and subjecting the region corresponding to the openings of the photomask in the PEG thin film to an oxidation treatment.

[0075] The cell culture substrate used in the present invention is preferably pre-coated for the purpose of promoting the adhesion of embryonic stem cells (ES cells) and / or induced pluripotent stem cells ( iPS cells) to the cell adhesion area. The pre-coating treatment can be carried out by coating the cell culture substrate with an extracellular matrix (collagen, fibronectin, proteoglycan, laminin, vitronectin), gelatin, lysine, peptide, a gel-like matrix containing them, serum, etc. By carrying out the pre-coating treatment, the cell adhesion area of ES cells and iPS cells with low adhesiveness can be improved. It can promote adhesion to the domain and effectively perform cell adhesion culture and induction of differentiation.

[0076] Similarly, for the purpose of promoting adhesion to the cell adhesion region of ES cells and iPS cells, ES cells and iPS cells are seeded with feeder cells before seeding and cultured for about 24 hours, and ES cells and iPS cells are preferably cultured on the feeder cells. As the feeder cells, those commonly used in the art can be used and are not particularly limited. For example, fibroblasts and the like can be mentioned. The f eeder cells are seeded at a density of less than 1.26×10 5 cells / cm 2 , preferably at a density of 6.3× 10 4 cells / cm 2 or less, and preferably at a density of 3.15×10 4 cells / cm 2 or more.

[0077] In the present invention, both precoat treatment and seeding of feeder cells may be performed, or only pre coat treatment may be performed, or seeding of feeder cells may be performed without performing precoat treatment.

[0078] Subsequently, Step 1 and Step 2 of the method for producing intestinal organoids of the present invention will be described. In Step 1, embryonic stem cells and / or induced pluripotent stem cells before seeding on the cell culture substrate are maintained undifferentiated using an undifferentiation induction medium. Switch to a differentiation induction medium before and after seeding on the cell culture substrate surface, and seed on the substrate surface.

[0079] The undifferentiation induction medium is not particularly limited as long as it is a medium that does not induce differentiation of embryonic stem cells and / or induced pluripotent stem cells. For example, mouse embryonic stem cells and mouse induced pluripotent stem cells ​ containing leukemia inhibitory factor, which is known to have the property of maintaining differentiation The medium to be used can be mentioned.

[0080] In step 1, the seeding density of embryonic stem cells and / or induced pluripotent stem cells on the cell culture substrate may be according to a conventional method and is not particularly limited. In one embodiment of the present invention, embryonic stem cells and / or induced pluripotent stem cells are seeded on the cell culture substrate at a density of 3×10 4 cells / cm 2 or more, preferably seeded at a density of 3×10 4 ~5×10 5 cells / cm 2 , and more preferably seeded at a density of 3×10 ~2.5×10 4 ~2.5×10 5 cells / cm 2 of density.

[0081] Step 2 is a step of culturing the cells seeded in step 1. The culture temperature in step 2 is usually 37°C. It is preferably cultured in an atmosphere of about 5% CO2 concentration using a CO2 cell culture apparatus or the like.

[0082] Step 2 is carried out in a differentiation induction medium. The differentiation induction medium is not particularly limited as long as it is a medium for inducing differentiation of embryonic stem cells and / or induced pluripotent stem cells. For example, serum -containing medium, serum-free medium containing known components having properties to replace serum, etc. can be mentioned. According to the type of cells to be 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, RPMI1640 medium, etc. Various growth factors, antibiotics, amino acids, etc. may be added to the medium. For example, 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 of β-mercaptoethanol, 0.1 mM to 2 mM pyruvate, 10 U / ml to 200 U / ml penicillin, 10μg / ml~200μg / ml streptomycin, 10μg / ml~200μg / ml L-ascorbic acid 2-lysine and 1 μM to 20 μM of a ROCK inhibitor (for example, Y-27632).

[0083] In step 2, when the cells are cultured in a differentiation-inducing medium, the cell adhesion area increases about three days after seeding. The cells become confluent within the plate and form cell patterns. Then, the culture is continued. When the cell pattern is formed, it becomes a hemispherical dome-shaped cell mass on the cell adhesion area, and the cell pattern is then spun out. Differentiation proceeds within the cell clusters. Approximately 30 days after seeding, the cell clusters detach from the cell adhesion areas and float in the medium. If necessary, further culture in a floating state will yield the intestinal organoids of the present invention. The culture period is not particularly limited, and the culture may be terminated when the cells are detached from the cell adhesion region. Typically, the culture is carried out for 30 to 130 days after seeding. During this period, the medium is replaced as necessary. In the method of the present invention, unlike the method described in Non-Patent Document 1, the cells autonomously grow in the cell aggregate. The intestinal organoids are differentiated into intestinal organoids by the endothelial cells. Luganoids are preferred as they are believed to have functions closer to the natural intestine.

[0084] In a preferred embodiment of step 2, A part of the cells seeded in step 1 is differentiated into endodermal cells; and Some of the cells seeded in step 1 will differentiate into ectodermal cells. comprises

[0085] In this embodiment, more preferably, in step 2, the differentiation timing of some of the cells seeded in step 1 into endodermal cells is earlier than the differentiation timing of some of the cells seeded in step 1 into ectodermal cells. In the development of an embryo in a natural state, the intestinal tube structure is formed first, and then nerve cells invade and the enteric nervous system develops. Therefore, as in this embodiment, the differentiation into endodermal cells proceeds first and epithelial structures such as intestinal epithelium derived from the endoderm are formed, and then the differentiation into ectodermal cells proceeds and the nervous system derived from the ectoderm develops. This can be said to mimic the developmental mechanism. Therefore, the method for producing an intestinal organoid according to this embodiment can be expected to be used for research on elucidating the developmental mechanism of the nervous system in the intestine. Also the intestinal organoids obtained by this method are intestinal organoids that have undergone a process approximating the natural development process, so it is considered that their response to drugs is closer to that of a living body, and it can be expected to be suitable as a model for drug discovery research. In step 2, the fact that the differentiation timing of some of the cells seeded in step 1 into endodermal cells is earlier than the differentiation timing of some of the cells seeded in step 1 into ectodermal cells means that the period when the increase rate per unit time of the expression level of the endoderm marker in the cell culture is the maximum is earlier in time than the period when the increase rate per unit time of the expression level of the ectoderm marker is the maximum. This can be judged as an index. The endoderm marker in this case may be a gene specific to the endoderm. As a specific example, FOXA2, SOX17 or CXCR4 of the embryonic endoderm marker can be exemplified

[0086] In step 2, the fact that the differentiation timing of some of the cells seeded in step 1 into endodermal cells is earlier than the differentiation timing of some of the cells seeded in step 1 into ectodermal cells can be judged as an index by the fact that the period when the increase rate per unit time of the expression level of the endoderm marker in the cell culture is the maximum is earlier in time than the period when the increase rate per unit time of the expression level of the ectoderm marker in the cell culture. The endoderm marker in this case may be any gene specific to the endoderm. As a specific example, FOXA2, SOX17 or CXCR4 of the embryonic endoderm marker can be exemplified The ectodermal marker may be any gene specific to the ectoderm. As a specific example, the neural progenitor cell marker SOX1 can be exemplified. The expression level of each marker in the cell culture can be expressed as the relative amount to the mRNA expression level of GAPDH at the same time. As the increase width per unit time, the increase width per week can be adopted. Specifically, by measuring the expression level of each marker every week after seeding, the increase width of the expression level per week can be known.

[0087] In this embodiment, more preferably, step 2 includes that a part of the cells seeded in step 1 differentiates into endodermal cells from the time of seeding to 14 days after seeding. Specifically, the period when the increase width per unit time of the expression level of the endodermal marker in the cell culture is the largest is within 14 days after seeding. By the differentiation into endodermal cells at this time, it becomes easy to obtain intestinal organoids having functions similar to those of the intestine. Even in this case, there may be cells that differentiate into endodermal cells after 15 days after seeding, but it is preferable that most of the differentiation into endodermal cells occurs by 14 days after seeding.

[0088] In this embodiment, more preferably, step 2 includes that a part of the cells seeded in step 1 differentiates into ectodermal cells after 15 days after seeding. Specifically, the period when the increase width per unit time of the expression level of the ectodermal marker in the cell culture is the largest is after 15 days after seeding. By the differentiation into ectodermal cells at this time, it becomes easy to obtain intestinal organoids having functions similar to those of the intestine. Even in this case, there may be cells that differentiate into ectodermal cells by 14 days after seeding. Differentiating cells may be present, but most of the differentiation into ectodermal cells preferably occurs after 15 days from seeding. This is preferable.

[0089] In another preferred embodiment of Step 2, the differentiation-inducing medium does not contain components (heterologous components) derived from a mammal of a species different from the mammal of the cells to be cultured. Examples of the medium that does not contain heterologous components include a serum-free medium containing known components having properties to replace serum. Intestinal organoids differentiated in a medium that does not contain heterologous components have high safety when transplanted into a living body and are suitable for use in regenerative medicine. This is preferable. Examples of the medium that does not contain heterologous components include a serum-free medium containing known components having properties to replace serum. Intestinal organoids differentiated in a medium that does not contain heterologous components have high safety when transplanted into a living body and are suitable for use in regenerative medicine. This is preferable. This is preferable.

[0090] In another preferred embodiment of Step 2, the differentiation-inducing medium further contains insulin-like growth factor (IGF) and basic fibroblast growth factor (bFGF). The inventors have surprisingly found that when embryonic stem cells and / or induced pluripotent stem cells are cultured in the differentiation-inducing medium containing these two components, intestinal organoids having a structure containing a cavity and having a major axis length of 5 mm or more can be produced, preferably further having the above-described preferred characteristics. IGF-1 is particularly preferable as IGF. The concentration of IGF in the differentiation-inducing medium is preferably 20 ng / ml to 2 μg / ml. The concentration of bFGF in the differentiation-inducing medium is preferably 2 ng / ml to 200 ng / ml. IGF and bFGF are preferably derived from the same species as the cells to be cultured. Further, IGF and bFGF are not limited to those having a natural amino acid sequence, and may be functionally equivalent mutants or fragments. For example, as IGF-1, LONG R-IGF-1 (Sigma-Aldrich) can be used. The differentiation-inducing medium further contains heparin in addition to IGF and bFGF. This is preferable. This is preferable. This is preferable. This is preferable. This is preferable. This is preferable. This is preferable. This is preferable. This is preferable. 3 -IGF-1 (Sigma- Aldrich) can be used. The differentiation-inducing medium further contains heparin in addition to IGF and bFGF. It is particularly preferable to contain heregulin. As the heregulin, heregulin-1β is preferable. The here regulin-1β only needs to contain at least an EGF domain. As the concentration of heregulin in the differentiation-inducing medium described above, 1 ng / ml to 100 ng / ml is preferable. Further, when the differentiation-inducing medium contains heregulin, the heregulin is preferably derived from the same biological species as the cells to be cultured and is not limited to those consisting of the natural amino acid sequence, and may be a functionally equivalent mutant or fragment.

[0091] In the embodiment of performing differentiation into intestinal organoids using a differentiation-inducing medium containing IGF and bFGF in Step 2 the differentiation-inducing medium does not need to contain IGF and bFGF at the time of seeding in Step 1. For example, cells are seeded on a cell culture substrate in Step 1, and until the cells become confluent within the cell adhesion region (for example, about 3 days after seeding), they are cultured in a differentiation-inducing medium that does not contain IGF and bFGF and after becoming confluent, the medium is changed to a differentiation-inducing medium containing IGF and bFGF and further culture can be continued.

[0092] 3. Differentiation induction medium for producing intestinal organoid, kit for producing intestinal organoid The present invention also provides a differentiation-inducing medium for producing intestinal organoids, which contains IGF and bFGF. Details of this differentiation-inducing medium for producing intestinal organoids are as described above.

[0093] When embryonic stem cells and / or induced pluripotent stem cells are cultured in this differentiation-inducing medium for producing intestinal organoids intestinal organoids can be produced, which have a structure containing a cavity and have a length in the major axis direction of 5 mm or more, and preferably further have the above-described preferable characteristics. ​​​​​

[0094] The present invention also relates to a differentiation-inducing medium for producing intestinal organoids, a substrate, and a cell culture substrate comprising a cell adhesion region formed on the surface of the substrate and a cell non-adhesion region surrounding the cell adhesion region, which is used for producing intestinal organoids. The cell culture substrate in this kit is as described above with respect to the method for producing intestinal organoids. Hereinafter, the present invention will be described with reference to specific experimental results, but the scope of the present invention is not limited to the scope of the experimental results.

Example

[0095] 1. Preparation of cell culture substrate (First-stage reaction) Mix 58.5 g of toluene and 20.25 g of epoxy silane TSL8350 (manufactured by Momentive Performance Materials), add a catalytic amount of triethylamine while stirring this mixture, and further stir at room temperature for several minutes. Immerse a 5-inch square glass substrate that has been UV-cleaned in the above epoxy silane solution and leave it at room temperature for 20 hours. Then, wash the pretreated glass substrate with ethanol, then with water, and dry it. The average value of the water contact angle on the surface of the dried substrate was 56°. Thus, an epoxy silane-treated substrate was obtained.

[0096] (Second-stage reaction) While stirring 45 g of polyethylene glycol (molecular weight 400), slowly add a catalytic amount of concentrated sulfuric acid, and further stir at room temperature for several minutes. Immerse the above epoxy silane-treated substrate in the above polyethylene glycol and react it at 120°C for 30 minutes. After the reaction, wash the substrate thoroughly with water

[0097] ​​​​​​​​​​​Then, it was dried. As a result, a glass substrate with a hydrophilic thin film formed thereon could be produced. 。

[0098] (Patterning process) A photomask having a pattern with a plurality of circular openings of the same size was a 5-inch size. The photomask had circular openings of 1.5 mm formed, and the space between the openings, i.e., the shortest distance between the openings, all had a pattern of 0.35 mm. was.

[0099] The mask was gently placed on the film-forming surface of the glass substrate with the hydrophilic thin film formed thereon, and from the back side of the mask a xenon excimer lamp (172 nm, 10 mW / cm 2 ) was used as a light source to irradiate with vacuum ultraviolet rays for 1 minute. As a result, the region corresponding to the opening of the photomask on the surface of the hydrophilic thin film was oxidized. The substrate was cut into 5 cm squares and used for cell culture.

[0100] The shape of the cell adhesion region in the obtained cell culture substrate was circular, and its diameter was 1.5 mm. The space between the cell adhesion regions, i.e., the shortest distance between the cell adhesion regions, was all 0.35 mm. The ratio of the cell adhesion region in the cell culture substrate was 51.8%.

[0101] 2. Culture of intestinal organoids 2.1. Cell lines SEES1, SEES2, and SEES3 of human ES cell (hESC) lines were those established by the Reproductive ·Cell Medical Research Department of the National Center for Child Health and Development (Akutsu H, et al. Regen. Ther. 2015;1:18-2 9). These ES cells were cultured in commercially available serum-free DMEM trade name: KnockOut TMD-MEM (Thermo Fisher Sc ientific) was supplemented with 20% KnockOut TM Serum Replacement (Life Technol ogies), 0.1 mM non-essential amino acids (NEAA), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.055 mM β-mercaptoethanol, 50 U / ml penicillin / 50 μg / ml streptomycin (Pen-St rep), and 8 ng / ml recombinant human bFGF (all purchased from Life Technologies), and maintained on a γ-irradiated mouse embryonic fibroblast (MEF) feeder layer in the resulting medium. The medium was changed every two days. Cells were passaged approximately once a week using either an enzymatic method (dispase; Wako Pure C hemical Industries) or a mechanical method (EZPassage; Life Technologies).

[0102] Human induced pluripotent stem cells (hiPSC) were established at the Division of Reproductive and Cellular Medicine, National Center for Child Health and Development, by expressing the four Yamanaka factors (Oct3 / 4, Sox2, Klf4, c-Myc) in the human fetal lung-derived fibroblast MRC5 cell line using a retroviral vector (Makino H, et al. Exp Ce ll Res. 2009;315(16):2727-2740; Nishino K, et al. PLoS ONE. 2010;5(9):e13017; To yoda M, et al. Genes Cells. 2011;16(1):1-11). These human iPS cells were cultured in the presence of 10 ng / ml bFGF. ​​​​In the added iPSellon medium (Cardio Incorporated), on the γ-irradiated MEF feeder layer It was maintained.

[0103] 2.2. Formation of human intestinal organoids In previous studies, the present inventors have established xeno-free (XF), component-free conditions for establishing and growing human ES cells (Akutsu H, et al. Regen. Ther. 2015;1:18-29). Human ES cells were stably maintained in XF hESC medium containing 85% KnockOut D-MEM, 15% KnockOut TM serum replacement (Knockout Serum R TM eplacement) XF CTS (XF-KSR; Life Technologies), 1 mM pyruvate, 2 mM GlutaMAX-I , 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 ng / ml heregulin-1β (recombinant human NRG-β1 / HRG-β1 EGF domain; R&D System s), 200 ng / ml recombinant human IGF-1 (LONG R -IGF-1; Sigma-Aldrich), and 20 n 3 g / ml human bFGF (Life Technologies). Three types of media were prepared as differentiation induction media.

[0104] Regarding the medium composition, “%” refers to volume % unless otherwise specified. Differentiation induction medium 1 (sometimes referred to as “XF-KSR(-) medium” in this specification): 80% KnockOut D TM -MEM, 20% KnockOut serum replacement TMKnockout Serum Replacement XF CTS (XF-KSR; Life Technologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 0.055 m M β-mercaptoethanol, and 10 μM Y-27632-containing medium Differentiation induction medium 2 (sometimes referred to as "XF hESC medium" herein): 85% KnockOut TM D-M EM, 15% KnockOut TM Serum Replacement (Knockout Serum Replacement) XF CTS (XF-KSR; Life T echnologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich), 10 ng / ml heregulin-1β (recombinant human NRG-β1 / HRG-β1 EGF domain; R&D Systems), 200 ng / ml recombinant human IGF -1 (LONG R3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Life Technologies) in the medium Differentiation induction medium 3 (sometimes referred to as "XF-KSR medium" herein): 80% KnockOut TM D-ME M, 20% KnockOut TM Serum Replacement (Knockout Serum Replacement) XF CTS (XF-KSR; Life Te chnologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 0.055 mM β-mercaptoethanol-containing medium

[0105] To prepare intestinal organoids, undifferentiated hESCs or hiPSCs were dispersed using dispase and the cell culture substrate prepared by the above procedure was placed in a 90 mm culture dish and seeded onto a dish coated with 0.1% human recombinant type I collagen peptide (RCP) (Fujifilm). Other materials such as vitronectin (Life Technologies) may be used in this protocol. The culture dish coated with the matrix was pre-incubated at 37°C for 1 hour, the coating solution was removed, and the substrate was washed 3 times with PBS. 4 × 10 cells in 3 ml of medium were seeded onto the substrate and held for 10 minutes. Then, the medium was aspirated off and 10 ml of fresh differentiation induction medium 1 was gently added. Differentiation induction medium 1 contains the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) as described above and does not contain growth factors. hESCs or hiPSCs were cultured in differentiation induction medium 1 containing Y-27632 and no growth factors for 1 day, and then cultured in differentiation induction medium 3 containing no growth factors and Y-27632. Three days later, differentiation induction medium 3 was replaced with differentiation induction medium 2 containing growth factors. Then, the cells were cultured while appropriately changing the medium in differentiation induction medium 2. The replacement of differentiation induction medium 2 was gently performed every 3 - 4 days. After 40 days, multiple organoids showing peristaltic movements similar to those of the intestine were collected and cultured together in the above differentiation induction medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). Also, in another experiment, undifferentiated hESCs or hiPSCs were seeded onto the substrate by the same procedure and cultured until the 4th day ×10 6 and held for 10 minutes. Then, the medium was aspirated off and 10 ml of fresh differentiation induction medium 1 was gently added. Differentiation induction medium 1 contains the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) as described above and does not contain growth factors. hESCs or hiPSCs were cultured in differentiation induction medium 1 containing Y-27632 and no growth factors for 1 day, and then cultured in differentiation induction medium 3 containing no growth factors and Y-27632. Three days later, differentiation induction medium 3 was replaced with differentiation induction medium 2 containing growth factors. Then, the cells were cultured while appropriately changing the medium in differentiation induction medium 2. The replacement of differentiation induction medium 2 was gently performed every 3 - 4 days. After 40 days, multiple organoids showing peristaltic movements similar to those of the intestine were collected and cultured together in the above differentiation induction medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). Also, in another experiment, undifferentiated hESCs or hiPSCs were seeded onto the substrate by the same procedure and cultured until the 4th day and held for 10 minutes. Then, the medium was aspirated off and 10 ml of fresh differentiation induction medium 1 was gently added. Differentiation induction medium 1 contains the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) as described above and does not contain growth factors. hESCs or hiPSCs were cultured in differentiation induction medium 1 containing Y-27632 and no growth factors for 1 day, and then cultured in differentiation induction medium 3 containing no growth factors and Y-27632. Three days later, differentiation induction medium 3 was replaced with differentiation induction medium 2 containing growth factors. Then, the cells were cultured while appropriately changing the medium in differentiation induction medium 2. The replacement of differentiation induction medium 2 was gently performed every 3 - 4 days. After 40 days, multiple organoids showing peristaltic movements similar to those of the intestine were collected and cultured together in the above differentiation induction medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). Also, in another experiment, undifferentiated hESCs or hiPSCs were seeded onto the substrate by the same procedure and cultured until the 4th day and held for 10 minutes. Then, the medium was aspirated off and 10 ml of fresh differentiation induction medium 1 was gently added. Differentiation induction medium 1 contains the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) as described above and does not contain growth factors. hESCs or hiPSCs were cultured in differentiation induction medium 1 containing Y-27632 and no growth factors for 1 day, and then cultured in differentiation induction medium 3 containing no growth factors and Y-27632. Three days later, differentiation induction medium 3 was replaced with differentiation induction medium 2 containing growth factors. Then, the cells were cultured while appropriately changing the medium in differentiation induction medium 2. The replacement of differentiation induction medium 2 was gently performed every 3 - 4 days. After 40 days, multiple organoids showing peristaltic movements similar to those of the intestine were collected and cultured together in the above differentiation induction medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). Also, in another experiment, undifferentiated hESCs or hiPSCs were seeded onto the substrate by the same procedure and cultured until the 4th day and held for 10 minutes. Then, the medium was aspirated off and held for 10 minutes. Then, the medium was aspirated off and 10 ml of fresh differentiation induction medium 1 was gently added. Differentiation induction medium 1 contains the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) as described above and does not contain growth factors. hESCs or hiPSCs were cultured in differentiation induction medium 1 containing Y-27632 and no growth factors for 1 day, and then cultured in differentiation induction medium 3 containing no growth factors and Y-27632. Three days later, differentiation induction medium 3 was replaced with differentiation induction medium 2 containing growth factors. Then, the cells were cultured while appropriately changing the medium in differentiation induction medium 2. The replacement of differentiation induction medium 2 was gently performed every 3 - 4 days. After 40 days, multiple organoids showing peristaltic movements similar to those of the intestine were collected and cultured together in the above differentiation induction medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). Also, in another experiment, undifferentiated hESCs or hiPSCs were seeded onto the substrate by the same procedure and cultured until the 4th day and cultured until the 4th day It was cultured in the differentiation induction medium 1 and the same operations were performed except that the medium was changed to the differentiation induction medium 2 on the 4th day. It was cultured.

[0106] Unless otherwise specified, the culture was carried out statically in an incubator at 37 °C with a CO2 concentration of 5%. It was carried out.

[0107] 2.3. Video recording of human intestinal organoids The floating organoids were transferred into a culture plate containing the differentiation induction medium 2, and video recording and analysis were performed using an inverted microscope equipped with a camera (Cohu 3600) of the ZILOS-tk system (Hamilton Thorne). In order to count the number of contracting intestinal organoids, all the human embryonic stem cell (hESC) floating organoids generated on one plate were transferred to a new dish and recorded for 10 minutes. The organoids showing peristalsis-like contraction movements were evaluated as positive. It was recorded and analyzed using an inverted microscope equipped with a camera (Cohu 3600) of the ZILOS-tk system (Hamilton Thorne). In order to count the number of contracting intestinal organoids, all the human embryonic stem cell (hESC) floating organoids generated on one plate were transferred to a new dish and recorded for 10 minutes. The organoids showing peristalsis-like contraction movements were evaluated as positive. It was recorded and analyzed using an inverted microscope equipped with a camera (Cohu 3600) of the ZILOS-tk system (Hamilton Thorne). In order to count the number of contracting intestinal organoids, all the human embryonic stem cell (hESC) floating organoids generated on one plate were transferred to a new dish and recorded for 10 minutes. The organoids showing peristalsis-like contraction movements were evaluated as positive. It was recorded and analyzed using an inverted microscope equipped with a camera (Cohu 3600) of the ZILOS-tk system (Hamilton Thorne). In order to count the number of contracting intestinal organoids, all the human embryonic stem cell (hESC) floating organoids generated on one plate were transferred to a new dish and recorded for 10 minutes. The organoids showing peristalsis-like contraction movements were evaluated as positive. It was recorded and analyzed using an inverted microscope equipped with a camera (Cohu 3600) of the ZILOS-tk system (Hamilton Thorne). In order to count the number of contracting intestinal organoids, all the human embryonic stem cell (hESC) floating organoids generated on one plate were transferred to a new dish and recorded for 10 minutes. The organoids showing peristalsis-like contraction movements were evaluated as positive.

[0108] 2.4. Quantitative RT-PCR analysis RNA was isolated from the organoids using the RNeasy Mini Kit (Qiagen), and the contaminating DNA was removed using DNase (Life Technologies). cDNA was synthesized using SuperScript III reverse transcriptase and oligo-dT primers (Life Technologies) according to the instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n = 3). The primer sequences are shown in the following table. RNA was isolated from the organoids using the RNeasy Mini Kit (Qiagen), and the contaminating DNA was removed using DNase (Life Technologies). cDNA was synthesized using SuperScript III reverse transcriptase and oligo-dT primers (Life Technologies) according to the instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n = 3). The primer sequences are shown in the following table. RNA was isolated from the organoids using the RNeasy Mini Kit (Qiagen), and the contaminating DNA was removed using DNase (Life Technologies). cDNA was synthesized using SuperScript III reverse transcriptase and oligo-dT primers (Life Technologies) according to the instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n = 3). The primer sequences are shown in the following table. RNA was isolated from the organoids using the RNeasy Mini Kit (Qiagen), and the contaminating DNA was removed using DNase (Life Technologies). cDNA was synthesized using SuperScript III reverse transcriptase and oligo-dT primers (Life Technologies) according to the instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n = 3). The primer sequences are shown in the following table. RNA was isolated from the organoids using the RNeasy Mini Kit (Qiagen), and the contaminating DNA was removed using DNase (Life Technologies). cDNA was synthesized using SuperScript III reverse transcriptase and oligo-dT primers (Life Technologies) according to the instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n = 3). The primer sequences are shown in the following table.

[0109] [Table 1]

[0110] After amplification, a dissociation curve was obtained to confirm that each PCR product was amplified. As a reference GAPDH was adopted as the housekeeping gene. QuantStudio 12K Flex software v1.0 was used to normalize the mRNA expression level of the target gene against the mRNA expression level of GAPDH, and the relative expression level of the mRNA of the target gene was quantified. Small intestinal tissues from healthy adults (R1234226-50, BioChain Institute) and cDNA of adult pancreas and liver (HA-188 and HA-149, Alpha Diagnostic International) were used as positive controls.

[0111] 2.5. Immunocytochemical analysis The organoids were fixed with PBS containing 4% paraformaldehyde (Wako Pure Chemical Industries) at 4°C for 5 minutes, permeabilized with 0.2% Triton X-100 at room temperature for 2 minutes, and further blocked with PBS containing 5% normal serum for each antibody as needed. The treated organoids were incubated overnight at 4°C with the primary antibody against the following antigens: villin (sc-7672, 1:50, Santa Cruz Biotechnology); E-cadherin (610181, 1:50, BD Pharmingen); CGA (ab16007, 1:100), CDX2 (ab76541, 1:100), and PGP9.5 (ab8189, 1:10) (from Abcam); DEFA6 (HPA019462, 1:500) and SMA (A25 ​​​​​​​​​47, 1:400) (from Sigma-Aldrich); MUC2 (sc-7314, 1:50, Santa Cruz Biotechnology); LGR5 (LMC-1235, 1:100, Medical & Biological Laboratories); CKIT (NB100-77477AF48 8, 1:10, Immuno-Biological Laboratories); Na+ / K+-ATPase (NB300-146, 1:100, Novus Biologicals); S-100 (422091, 1:100, Nichirei Biosciences); Neurofilament (M076229, 1:50, Dako); β-actin (A5316, 1:1,000, Sigma-Aldrich); Histamine H1 receptor (aa471-484, 1:200, LSBio); and CFTR (ab131553, 1:100, Abcam). Alexa 4 88- or Alexa 546-labeled anti-mouse, anti-rabbit or anti-goat secondary antibodies (BD Bioscienc es) were used. Cell nuclei were counterstained with DAPI. Fluorescence was analyzed using an LSM 510 Meta Laser Scanning Confoc al Microscope (Carl Zeiss Microscopy).

[0112] 2.6. Histochemical analysis Organoids were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm thick sections. Every other section was placed on a slide, stained with H&E, and analyzed. For Alcian Blue staining, incubation was performed in 1% Alcian Blue in 3% acetic acid solution at pH 2.5 for 20 minutes Beat and then incubate in 0.1% Nuclear Fast Red for 2 minutes and dehydrate in ethanol and clear with xylene.

[0113] Histochemical analysis of the organoids was performed using sections stained with antibodies against SMA and serotonin (ab16007, 1:20, A bcam). As the secondary antibody, polyclonal rabbit anti-mouse immunoglobulin-HRP (1:100; Dako) was used.

[0114] 2.7. Electron Microscopy Observation Electron microscopy observation of the intestinal organoid samples was performed according to the standard protocol (Tokai Electron Microscopy). That is, the organoids were fixed with 2% paraformaldehyde and 2% glutaraldehyde in PBS, dehydrated, and embedded in epoxy resin. After polymerization was completed the samples were cut into 70 nm sections, placed on copper grids, and observed with a JEM-1200EX TEM (Jeol Ltd.) equipped with a Veleta CCD camera (Olymp us).

[0115] 2.8. Cell Transfection To examine the differentiation during the formation process of the intestinal organs, the inventors constructed a reporter construct (pPB-hLgr5p-EGFP-neo) specific to the intestinal cell line. This pPB-hLgr5p-EGFP-neo contains a 5- kb LGR5 promoter and a phosphoglycerate kinase (PGK) promoter, which respectively promote the expression of EGFP and the neomycin resistance gene (neo). SEES1 cells were cultured with Y-27632 for 24 hours and then subjected to electroporation, washed with PBS, and treated with Accuutase solution (L They were collected using ife Technologies) and resuspended in iPSellon medium. Strong pipetting was used to dissociate the cells into a single-cell suspension, and 1 - 2×10 6 cells were pelleted, and in Opti-MEM ( Life Technologies), they were mixed with the pPB-hLgr5p-EGFP-neo reporter vector and the hyperactive PiggyBac transposase expression vector (pCMV-hyPBase) (provided by A. Bradley, Wellcome Trust Sanger In stitute, Hinxton, Cambge, United Kingdom). Thus, the resulting cell suspension was transferred to a cuvette and electroporation was performed using the NEPA21 Super Electroporator (Nepa Gene) to obtain transfected cells. The transfected cells were selected with G418 (Sigma-Aldrich).

[0116] To visualize the in vivo function of the transplanted intestinal organoids, human embryonic stem cells (hESC) were transfected with a vector (pmGENIE-EGFP) that constitutively expresses EGFP (provided by S. Moisyadi, University of Haw aii, Honolulu, Hawaii, USA), and after screening a stable GFP-positive human embryonic stem cell (hESC) line was established.

[0117] 2.9. Evaluation of the Uptake of β-Ala-Lys-AMCA In Vitro by Human Intestinal Organoids Intestinal organoids grown in differentiation induction medium 2 were washed with PBS and incubated with 25 μM of the fluorescently labeled dipeptide ​It was cultured in DMEM containing chid β-Ala-Lys-AMCA (Biotrend Chemicals) at 37 °C for 4 hours. For the inhibition experiment, it was cultured for 1 hour under conditions with or without the addition of 1 mM captopril, an angiotensin-converting enzyme inhibitor (Sigma-A ldrich), rinsed with PBS, placed in the same dish and observed with a fluorescence microscope (Olympus). Furthermore, to quantify the uptake amount of β-Ala-Lys-AMCA intestinal organoids were cultured under conditions with or without the addition of 10 μM, 100 μM or 1 mM captopril, and the AMCA-related signal was observed using a fluorescence microscope (BZ-X710; Keyence) equipped with a tops stage incubator ー(5% CO2, 37 °C), and the fluorescence signal intensity was quantified using Hybrid Cell Count / BZ-H3C (Keyence). All sample images were recorded under standard conditions. Three independent assays were performed for each concentration condition.

[0118] 2.10. In vitro contractility of human intestinal organoids One of the human embryonic stem cell (hESC)-derived intestinal organoids (80 - 90 days in culture) showing peristalsis-like movement was treated with histamine (0.2 μM) and atropine sulfate (0.2 μM), an anticholinergic drug, which stimulate peristalsis. The contractile response was recorded using an inverted microscope. The movement of the intestinal organoids was visualized using image analysis software CL-Qua nt version 3.10 (Nikon Corporation). First, in each frame of the time-lapse image, a specific region of the organoid was identified using the software. Second, an ellipse was fitted to the region by the software ​​​​​Measured the length of the wing, calculated the ratio of the longest diameter to the shortest diameter as the aspect ratio, and plotted the change in this aspect ratio over time on a chart. Video was taken at 30 frames per second. Performed video shooting at 30 frames per second.

[0119] 2.11. CFTR Transport Activity of Intestinal Organoids One organoid was placed into one well of a 12-well ART culture dish (NIPRO) containing 100 μl of medium. Organoids derived from EGFP-human embryonic stem cells (hESCs) that constitutively express EGFP under the control of the CMV promoter were used to visualize volume changes. 5 μM forskolin was added, and the morphology of the organoids was monitored using a time-lapse fluorescence laser confocal microscope (Keyence). To inhibit CFTR, the organoids were pre-incubated with 50 μM of the CFTR inhibitor CFT Rinh172 and 50 μM GlyH-101 (TOCRIS) for 3 hours. Images were acquired every minute for 20 minutes in a topside incubator (5% CO2, 37°C). Each experimental condition was evaluated in three wells. In all experimental conditions, the DMSO concentration was made the same within the range not exceeding 0.2% (w / v). The surface area of the organoids was measured using Hybrid Cell Count / BZ-H3C (Keyence). The normalized total surface area of the organoids was determined, and the average value of the measurements from three separate wells was obtained for each experimental condition. One organoid was placed into one well of a 12-well ART culture dish (NIPRO) containing 100 μl of medium. Organoids derived from EGFP-human embryonic stem cells (hESCs) that constitutively express EGFP under the control of the CMV promoter were used to visualize volume changes. 5 μM forskolin was added, and the morphology of the organoids was monitored using a time-lapse fluorescence laser confocal microscope (Keyence). To inhibit CFTR, the organoids were pre-incubated with 50 μM of the CFTR inhibitor CFT Rinh172 and 50 μM GlyH-101 (TOCRIS) for 3 hours. Images were acquired every minute for 20 minutes in a topside incubator (5% CO2, 37°C). Each experimental condition was evaluated in three wells. In all experimental conditions, the DMSO concentration was made the same within the range not exceeding 0.2% (w / v). The surface area of the organoids was measured using Hybrid Cell Count / BZ-H3C (Keyence). The normalized total surface area of the organoids was determined, and the average value of the measurements from three separate wells was obtained for each experimental condition. The normalized total surface area of the organoids was determined, and the average value of the measurements from three separate wells was obtained for each experimental condition. The normalized total surface area of the organoids was determined, and the average value of the measurements from three separate wells was obtained for each experimental condition.

[0120] 2.12. Transplantation of Human Intestinal Organoids To confirm the in vivo growth of intestinal organoids, they were transplanted into immunodeficient nude mice (BALB / cAJcl-nu / nu) purchased from CLEA Japan. One intestinal organoid on the 35th day of culture was transplanted into a mouse. One intestinal organoid on the 35th day of culture was transplanted into a mouse. It was transplanted under the renal capsule. The mice that had received the transplantation were euthanized by cervical dislocation 6 weeks later. Its kidney was observed with an MVX10 fluorescence microscope (Olympus). The site where the organoids were transplanted was further analyzed by H&E staining and immunocytochemistry.

[0121] 2.13. Statistics Quantitative data are shown as the mean ± SEM values obtained from at least 3 independent experiments. Statistical analysis was performed using unpaired, 2-tailed t-test or Mann-Whitney rank sum test. P < 0.05 was considered to indicate a statistically significant difference.

[0122] 3. Results Representative results are shown in Figures 1A to 9B. Figures 1A to 1D are diagrams explaining the formation of intestinal organoids with peristaltic ability from human pluripotent stem cells on a cell culture substrate with a pattern of cell adhesion areas. Figures 2A to 2D are diagrams for explaining the characteristics of the differentiation of intestinal organoids. Figures 3A to 3C are diagrams showing the characteristics of intestinal organoids and the results of detection of LGR5-E GFP-positive cells during the formation process of intestinal organoids. Figures 4A to 4F are diagrams for explaining the peristaltic ability of intestinal organoids. Figures 5A to 5C are diagrams explaining the absorption function of intestinal organoids. Figures 6A and 6B are diagrams explaining the CFTR transport activity of intestinal organoids. Figures 7A to 7D show the characteristics of intestinal organoids without peristaltic ability. Figures 8A to 8D show that when intestinal organoids (day 35 of culture) were transplanted, a highly organized structure of the intestinal tract was formed . Figures 9A and 9B show the changes during the culture process of intestinal organoids derived from human iPS cells.

[0123] The experimental results will be described below with reference to the drawings as appropriate. Specific descriptions of the drawings are provided in the section "Brief Description of the Drawings".

[0124] 3.1. Intestinal organoids are formed autonomously on a cell culture substrate with a pattern of cell adhesion regions formed Generally, tissue self-formation mainly consists of three steps: autonomous aggregation, self-patterning, and self morphogenesis (Sasai Y. Nature. 2013;493(7432):318-326). In this experiment, this concept was utilized to induce the morphological formation of the intestine through the stage where the cell pattern swells and the stage of self-morphogenesis (Figure 1A). In the first step, human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) cultured in XF medium (Akutsu H, et al. Regen. Ther. 2015;1:18-29) aggregated on the circular pattern of cell adhesion regions on the glass surface in the cell culture substrate (Okochi N, Okazaki T, Hattori H. Langmuir. 20 09;25(12):6947-6953) in differentiation induction medium 2. The cells aggregated in the cell adhesion regions formed a hemispherical dome structure after the 7th day of culture (Figure 1C ). Subsequently, a large structure with a cavity was formed. This structure was composed of self-formed cell masses, with the epithelium folded and presenting cyst-like protrusions, and was covered with cuboidal epithelial cells by the 20th day of culture. By the 30th day of culture, self-formed cyst-like spheroids detached from the cell culture substrate. The detached spheroids could be roughly classified into two types. One was a simple cyst-like spheroid with a thin wall composed of cells. The other had a solid part and a part ​​​​It was a two-component spheroid having cystic protrusions. It should be noted that later, those with motility were this two-component spheroid. Such spheroids were 4% of all dissociated spheroids (34 out of 791, n = 3) and were maintained over a long period (see Fig. 1D). This suggests that cell types derived from different germ layers formed a functional cell network and that the assembled organoids were formed. All of the dissociated spheroids were motile. Such spheroids were 4% of all dissociated spheroids (34 out of 791, n = 3) and were maintained over a long period (see Fig. 1D). This suggests that cell types derived from different germ layers formed a functional cell network and that the assembled organoids were formed. This was 4% of all dissociated spheroids (34 out of 791, n = 3) and was maintained over a long period (see Fig. 1D). This suggests that cell types derived from different germ layers formed a functional cell network and that the assembled organoids were formed. This suggests that cell types derived from different germ layers formed a functional cell network and that the assembled organoids were formed. This suggests that cell types derived from different germ layers formed a functional cell network and that the assembled organoids were formed.

[0125] Next, the inventors examined the expression of germ layer markers at different time points of differentiation. At the initial differentiation stage, the proliferating pattern cells expressed the definitive endoderm markers FOXA2, SOX17, and CXCR4, as well as the early endoderm and mesoderm markers GATA4, GATA6, and T (Brachyury) (Fig. 2A). The expression of these markers increased until day 14 of culture when the hemispherical dome-shaped structures were formed (Figs. 1A and 2A). The expression of CXCR4 decreased on day 21 of culture when differentiation further progressed, which was consistent with the reported downregulation of gene expression during hindgut formation in mouse embryo development (McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442 - 456). The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and At the initial differentiation stage, the proliferating pattern cells expressed the definitive endoderm markers FOXA2, SOX17, and CXCR4, as well as the early endoderm and mesoderm markers GATA4, GATA6, and T (Brachyury) (Fig. 2A). The expression of these markers increased until day 14 of culture when the hemispherical dome-shaped structures were formed (Figs. 1A and 2A). The expression of CXCR4 decreased on day 21 of culture when differentiation further progressed, which was consistent with the reported downregulation of gene expression during hindgut formation in mouse embryo development (McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442 - 456). The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and The expression of these markers increased until day 14 of culture when the hemispherical dome-shaped structures were formed (Figs. 1A and 2A). The expression of CXCR4 decreased on day 21 of culture when differentiation further progressed, which was consistent with the reported downregulation of gene expression during hindgut formation in mouse embryo development (McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442 - 456). The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and This was consistent with the reported downregulation of gene expression during hindgut formation in mouse embryo development (McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442 - 456). The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and This was consistent with the reported downregulation of gene expression during hindgut formation in mouse embryo development (McGrath KE, Koniski AD, Maltby KM, McGann JK, Pali s J. Dev Biol. 1999;213(2):442 - 456). The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and The expression level of the hindgut marker CDX2 increased at the initial proliferation stage and was maintained relatively high even as differentiation progressed (Fig. 2A). The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and The expression of the neural progenitor cell marker SOX1 increased at a relatively late stage (day 21) compared to other germ layer markers (Fig. 2A). In contrast, the expression level of the pluripotency marker OCT4 showed a significant decrease at the initial differentiation stage. Thus, the inventors' procedure led to the definitive endoderm of human ES cells by day 21 of culture and promoted differentiation into the hindgut and formed a hemispherical dome-shaped structure. Subsequent differentiation caused the cell aggregates to form a structure with a cavity and detach from the cell culture substrate (Figure 1C). The epithelium of this structure had a folded structure. When the peristaltic organoids were embedded in paraffin and stained with H&E, the intestinal structure consisting of the mucosa and submucosa was confirmed (Figure 2B). The intestinal mucosa is formed by the epithelium and the basement membrane. The intestinal epithelium consists of two types of cells, absorptive enterocytes and secretory cells, which include goblet cells that produce mucus, enteroendocrine cells, and Paneth cells (Gracz AD, Magness ST. Am J Physiol Gastrointest Liver Physiol. 2014;307(3): G260-G273.). Alcian Blue staining showed that goblet cells containing purple-stained mucopolysaccharides were present in the organoid epithelial layer (Figure 2C). Interestingly, in the mature organoids on the 50th day of culture, the relative expression levels of several late differentiation markers were equivalent to those in the human adult intestine (Figure 2D). Along with the maturation of intestinal epithelial cells, CDX2 (intestinal transcription factor), E-cadherin (epithelial cell-specific E-cadherin), and brush border-specific villin showed expression levels in intestinal organoids similar to those in the human adult intestine. As a result of immunostaining, it was confirmed that villin was localized on the apical side of the epithelium and CDX2 was present in the epithelial layer (Figure 3A). From the TEM (transmission electron microscope) image of the organoids, it was confirmed that a brush border was formed on the apical side of the microvilli (Figure 3B). Paneth cells were identified by staining for Paneth cell-specific defensin α-6 (DEFA6) (Figure 3A), and TEM showed that Paneth cells were present (Figure 34). were present (Figure 3B). By staining for Paneth cell-specific defensin α-6 (DEFA6), Paneth cells were identified (Figure 3A), and TEM showed that Paneth cells It was confirmed that they had secretory granules (Figure 3B). By H&E staining and Alcian Blue staining, it was identified that goblet cells were present in the epithelial layer. That they were goblet cells was identified by mucin-2 (MUC2) staining (Figure 3A) and the presence of mucin granules in TEM observation images (Figure 3B). The expression level of the epithelial enteroendocrine cell marker chromogranin A (CGA) was lower in intestinal organoids than in human adult intestine (Figure 2D), but from the immunostaining of ECAD and CGA, it was confirmed that both of these proteins were present in the epithelium (Figure 3A). In intestinal organoids, the intestinal stem cell marker LGR5 was co-expressed with CDX2 (Figure 3A). The expression of LGR5 was also confirmed during the formation of intestinal organoids from human embryonic stem cells (hESCs) transfected with an EGFP expression vector under the control of the LGR5 promoter (Figure 3C). From this, it was shown that the self-formed intestinal organoids according to the present invention contain various types of highly differentiated intestinal cell types. It was identified that goblet cells were present in the . That they were goblet cells was identified by mucin-2 (MUC2) staining (Figure 3A) and the presence of mucin granules in TEM observation images (Figure 3B). The expression level of the epithelial enteroendocrine cell marker chromogranin A (CGA) was lower in intestinal organoids than in human adult intestine (Figure 2D), but from the immunostaining of ECAD and CGA, it was confirmed that both of these proteins were present in the . The expression level of the epithelial enteroendocrine cell marker chromogranin A (CGA) was lower in intestinal organoids than in human adult intestine (Figure 2D), but from the immunostaining of ECAD and CGA, it was confirmed that both of these proteins were present in the epithelium . The expression level of the epithelial enteroendocrine cell marker chromogranin A (CGA) was lower in intestinal organoids than in human adult intestine (Figure 2D), but from the immunostaining of ECAD and CGA, it was confirmed that both of these proteins were present in the epithelium . In intestinal organoids, the intestinal stem cell marker LGR5 was co-expressed with CDX2 . The expression of LGR5 was also confirmed during the formation of intestinal organoids from human embryonic stem cells (hESCs) transfected with an EGFP expression vector under the control of the LGR5 promoter . From this, it was shown that the self-formed intestinal organoids according to the present invention contain various types of highly differentiated intestinal cell types . From this, it was shown that the self-formed intestinal organoids according to the present invention contain various types of highly differentiated intestinal cell types .

[0126] 3.2. Intestinal organoids have functions specific to mature intestine It was examined whether intestinal organoids have functions specific to the intestine, such as peristaltic ability, peptide absorptivity, and mucus secretion ability. Since intestinal organoids showed contractile movement, the presence of a functionally mature mesenchymal layer was suggested. Intestinal motility is controlled by pacemaker cells such as the enteric nervous system and interstitial cells of Cajal (ICC) (Sanders KM, Koh SD, Ward SM. Annu Rev Physiol. 2006;68:307-343; uizinga JD, Lammers WJ. Am J Physiol Gastrointest Liver Physiol. 2009;29 It was examined whether intestinal organoids have functions specific to the intestine, such as peristaltic ability, peptide absorptivity, and mucus secretion ability. Since intestinal organoids showed contractile movement, the presence of a functionally mature mesenchymal layer was suggested . It was examined whether intestinal organoids have functions specific to the intestine, such as peristaltic ability, peptide absorptivity, and mucus secretion ability. Since intestinal organoids showed contractile movement, the presence of a functionally mature mesenchymal layer was suggested . Intestinal motility is controlled by pacemaker cells such as the enteric nervous system and interstitial cells of Cajal (ICC) (Sanders KM, Koh SD, Ward SM. Annu Rev Physiol. 2006;68:307-343; uizinga JD, Lammers WJ. Am J Physiol Gastrointest Liver Physiol. 2009;29 . Intestinal motility is controlled by pacemaker cells such as the enteric nervous system and interstitial cells of Cajal (ICC) (Sanders KM, Koh SD, Ward SM. Annu Rev Physiol. 2006;68:307-343; uizinga JD, Lammers WJ. Am J Physiol Gastrointest Liver Physiol. 2009;29 . 6(1): G1 - G8). To identify which cell types are involved in the contractile movements of the intestine, immunochemical analysis was performed. Smooth muscle cells derived from the mesoderm were confirmed in the submucosal region by staining for α - smooth muscle actin (SMA) (Figure 4A). This indicates that the mesoderm is involved in the formation of organoids. Myofibroblasts under the intestinal epithelium support the in vivo and in vitro growth of human intestinal epithelium (Lahar N, et al. PLoS One. 2011;6(11):e26898). Using quantitative RT - PCR, cells expressing the enteric nervous system marker protein gene product 9.5 (PGP9.5) (Figure 3A), as well as the ICC markers CD34 and CKIT (Figure 2D) were identified. Also, using immunohistochemistry, it was identified that double - positive cells for the glial cell markers CKIT and S - 100 are localized in the submucosal region (Figure 4B). Serotonin is a major neurotransmitter that controls gastrointestinal functions (peristalsis, secretion, etc.) and is synthesized by enteroendocrine cells of the intestinal mucosa (Mawe GM, Hoffman JM. Nat Rev Gastroenterol Hepatol. 2013;10(8):473 - 486). As a result of immunohistochemical analysis, it became clear that serotonin - positive cells are present in the epithelial layer of intestinal organoids (Figure 4C). The contraction rate of intestinal organoids increased upon histamine treatment and decreased upon atropine treatment (Figure 4D). Thus, since the contractility changes after treatment with histamine or atropine, it became clear that intestinal organoids have motility similar to that of mature intestine (Mittal RK, Padda B, Bhalla V, Bhargava V, Liu J. Am J Physiol Gastrointes tinal Liver Physiol. 2013;304(1):G1 - G8). tinal Liver Physiol. 2013;304(1):G1 - G8). t Liver Physiol. 2006;290(3):G431-G438). Intestinal organoids with peristaltic ability respond to drugs in this way while intestinal organoids without peristaltic ability did not respond to histamine, which promotes contraction (n = 6, Figure 4E). On the other hand, the histamine H1 receptor was expressed in organoids with peristaltic ability and in the epithelial and mesenchymal regions of the human intestine (Figure 4F). Organoids without peristaltic ability also expressed the histamine H1 receptor, and quantitative RT-PCR and immunostaining analysis of intestinal tissue-specific genes confirmed that they were intestinal-like tissues (Figure 7A). Albumin and insulin, markers of mature endoderm-derived cells, were not expressed in intestinal organoids (Figure 7B). Organoids without peristaltic ability showed gene expression levels similar to those of the human adult intestine, but immunostaining revealed immaturity / defects in the mesenchymal layer of organoids without peristaltic ability. Staining for the smooth muscle marker SMA in organoids without peristaltic ability was at a lower level compared to organoids with peristaltic ability (Figure 7C). Neurofilament was distributed throughout the mesenchymal region in organoids with peristaltic ability, while it was sparse in organoids without peristaltic ability (Figure 7D). Insufficient development of the muscle cell layer and neurons in the mesenchymal region may be the cause of the lack of peristaltic ability.

[0127] To evaluate peptide uptake by intestinal organoids, the expression levels of the intestinal oligopeptide transporter (PEPT1) and the main ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 were examined. The expression levels of these genes were the same in intestinal organoids and adult intestine. It was as follows (Figure 5A). The uptake ability of the bioactive peptide was evaluated by an in vitro assay (Gronebe rg DA, Doring F, Eynott PR, Fischer A, Daniel H. Am J Physiol Gastrointest Liver Physiol. 2001;281(3):G697-G704). Intestinal organoids cultured with the fluorescently labeled dipeptide β-Ala-Lys-N(ε)-7-amino-4- methylcoumarin-3-acetic acid showed peptide absorbability, and peptide absorbability decreased with capt opril treatment (Figure 5B). Treatment with different concentrations of captopril was performed, but the captopril concentration-dependence of the inhibitory effect on peptide absorbability could not be confirmed (Figure 5C). (Figure 5C).

[0128] To examine the secretory activity of organoids, the expression level of cystic fibrosis transmembrane conductance regulator (CF TR) was confirmed, and an assay based on forskolin-induced swelling (FIS) was performed. CFTR is important for mucus secretion in intestinal epithelial cells. The inventors confirmed that CF TR is present in the epithelial layer of intestinal organoids, similar to the human intestine (Figure 6A). To confirm the function of CF TR, the FIS assay (Dekkers JF, et al. Nat Med . 2013;19(7):939-945) used in the art was performed, and it was confirmed that intestinal organoids swelled with forskolin . Also, the CFTR blocker completely inhibited this swelling (Figure 6B). From these results, the inventors concluded that intestinal organoids have absorption and secretion abilities similar to those of the mature intestine . .

[0129] From the experimental results, stem cell-derived intestinal organoids prepared under conditions free of heterologous components have the properties of the intestine It was confirmed that the main cell types related to the intestine have an organized structure and the functions of a mature intestine. In previous studies, it has been shown that when intestinal organoids are transplanted into the mouse kidney capsule, maturation and differentiation occur (Watson CL, et al. Nat Med. 2014;20(11):1310-1314). To confirm that intestinal organoids formed from stem cells in vitro are mature, one intestinal organoid on day 35 of culture, derived from human embryonic stem cells (hESC) and constantly expressing EGFP under the control of the CMV promoter, was transplanted under the kidney capsule of immunodeficient nude mice (Figure 8A). All intestinal organoids were transplanted without problems (n = 4) and had a highly organized structure of the intestinal tract, namely a lumen and a stratified structure (Figures 8B and 8C). In addition, it was confirmed that CDX2 (intestinal cells), MUC2 (goblet cells), CGA (enteroendocrine cells), DEFA6 (Paneth cells), as well as ECAD and Na+ / K+-ATPase (epithelium), which are gene markers of intestinal epithelium and mesenchymal tissue, and PGP9.5, which is an intestinal nerve marker, were expressed in the multi-layered SMA-positive mesenchyme (Figure 8D). That is, all intestinal cell types were present in the transplanted intestinal organoids. In the transplantation experiment, since the intestinal organoids did not grow much after transplantation, it was suggested that they were highly differentiated in vitro.

[0130] 4. Discussion The above experimental results indicate that human stem cell-derived intestinal organoids prepared under conditions without heterologous components have a highly organized structure of the main cell types related to the intestine and functions equivalent to those of a mature intestine. The intestinal organoids obtained in this experiment have at least three advantages . ​​​​​

[0131] First, intestinal organoids can be stably maintained for a long time under conditions without heterogeneous components and are suitable for application in medicine.

[0132] Second, intestinal organoids have a complex tissue structure and have functions equivalent to those of mature intestines. Different from epithelial organoids derived from epithelium, intestinal organoids contain both an intestinal epithelial layer and a mesenchymal layer. Intestinal organoids have epithelial functions such as peptide absorption and mucus secretion, and, like mature human intestines, perform peristalsis-like movements in response to histamine and atropine. Therefore, intestinal organoids can be used as "mini intestines" and have high utility value in the study of intestinal-related diseases.

[0133] Third, this experiment is the first example of creating functional organoids having cells derived from the three germ layers, the endoderm, mesoderm, and ectoderm, from stem cells in vitro.

Industrial Applicability

[0134] The intestinal organoids provided by the present invention are useful for studying the mechanisms of intestinal diseases and developing therapeutic drugs.

[0135] According to the method for producing intestinal organoids provided by the present invention, intestinal organoids can be easily produced in vitro for the study of diseases and drug development.

Claims

1. A method for evaluating a substance, comprising: derived from embryonic stem cells and / or induced pluripotent stem cells, having a structure enclosing a cavity, including an epithelial layer containing intestinal epithelial cells on at least a part of the outer surface, including muscle cells and / or nerve cells inside the epithelial layer, Villin being localized on the outer surface of the epithelial layer, and the intestinal epithelial cells being positive for CDX2 expression, characterized by arranging an intestinal organoid and a liquid containing the substance such that the liquid contacts the intestinal epithelial cells, wherein the method includes the above steps.

2. The method according to claim 1, wherein the intestinal organoid is arranged in a suspended state in the liquid.

3. The intestinal organoid has a structure enclosing a closed cavity, and the substance is retained in the closed cavity, wherein the method is according to claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the intestinal epithelial cells are positive for transporters.

5. The method according to any one of claims 1 to 4, wherein the intestinal organoid includes microvilli on the outer surface.

6. The method according to any one of claims 1 to 5, wherein the length of the intestinal organoid in the major axis direction is 5 mm or more.

7. When arranged so that the liquid contacts the intestinal epithelial cells, the area of contact between the intestinal epithelial cells and the liquid is 78 mm ２ or more. The method according to any one of claims 1 to 6.

8. A method for evaluating the absorption ability of a substance in the intestine, comprising: derived from embryonic stem cells and / or induced pluripotent stem cells, having a structure enclosing a cavity, including an epithelial layer containing intestinal epithelial cells on at least a part of the outer surface, including muscle cells and / or nerve cells inside the epithelial layer, Villin being localized on the outer surface of the epithelial layer, and the intestinal epithelial cells being positive for CDX2 expression, characterized by arranging an intestinal organoid and a liquid containing the substance such that the liquid contacts the intestinal epithelial cells, absorbing the liquid inside the intestinal organoid, and analyzing the liquid absorbed inside the intestinal organoid, wherein the method includes the above steps.

9. The intestinal organoid has a structure enclosing a closed cavity, and the liquid is retained in the closed cavity, wherein the method is according to claim 8.

10. The method according to claim 8 or 9, wherein the analysis is mass spectrometry or fluorescence observation.

11. A method for examining the effect of a drug in the intestine, comprising: derived from embryonic stem cells and / or induced pluripotent stem cells, having a structure enclosing a cavity, including an epithelial layer containing intestinal epithelial cells on at least a part of the outer surface, containing muscle cells and / or nerve cells inside the epithelial layer, Villin is localized on the outer surface of the epithelial layer, and the intestinal epithelial cells are positive for the expression of CDX2, characterized in that the intestinal organoid and the liquid containing the drug are arranged such that the liquid contacts the intestinal epithelial cells, the liquid being taken into the intestinal organoid, and analyzing the liquid taken into the intestinal organoid, the method comprising the above.

12. the intestinal organoid has a structure enclosing a closed cavity, and the liquid is retained in the closed cavity, The method according to claim 11.

13. the substance is a drug, and further comprising examining the contractile responsiveness of the intestinal organoid to the drug, the evaluation method being a method for evaluating the effect of the drug on intestinal peristaltic movement, The method according to claim 1.

14. The method according to claim 13, wherein the intestinal organoid contains an enteric nervous system and Cajal interstitial cells.

15. The method according to claim 13 or 14, wherein the contractile responsiveness is the change over time in the ratio of the longest diameter to the shortest diameter of an ellipse fitted to a specific region of the intestinal organoid.

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