Cellular structure and its manufacturing method

Abstract

To provide a cell structure including a lumen, including an entodermal cell, an ectodermal cell, and a mesodermal cell, and including a macrophage. One or more embodiments of the disclosure relates to a cell structure including a lumen, including an entodermal cell, an ectodermal cell, and a mesodermal cell, and including a monocyte. Other one or more embodiments of the disclosure relates to a production method of a cell structure which includes a lumen, includes an entodermal cell, an ectodermal cell, and a mesodermal cell, and includes a macrophage, the method includes a step 11 for injecting monocyte to the cell structure including a lumen, including an entodermal cell, an ectodermal cell, and a mesodermal cell, a step 12 for growing the monocyte to the macrophage in the cell structure to which the monocyte is injected in the step 11.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] Disclosed herein are cellular structures comprising macrophages or monocytes and methods for their production. [Background technology]

[0002] Cell engineering has made it possible to form three-dimensional functional tissues consisting of a variety of cells. In addition, by using pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), it is now possible to form self-organizing structures similar to organs and tissues (organoids).

[0003] Patent Document 1 describes a method for producing intestinal organoids having functions equivalent to those of the intestine from embryonic stem cells or induced pluripotent stem cells.

[0004] Patent Documents 2 and 3 and Non-Patent Document 1 describe a method for obtaining a cell structure containing small intestinal epithelial cells, characterized in that the villus faces outward, useful components can be absorbed from the outside, and all three germ layers are contained, by culturing human ES cells or iPS cells on a cell culture substrate on which a pattern of cell culture regions is formed. Non-Patent Document 1 also describes that the obtained cell structure has peristaltic motility.

[0005] Patent Document 4 describes a bioprinted artificial three-dimensional living intestinal tissue model, which includes (i) a layer of intestinal stromal tissue containing myofibroblasts and (ii) a layer of intestinal epithelial cells on the layer of intestinal stromal tissue to form an artificial three-dimensional living intestinal tissue model, and describes that the layer of intestinal epithelial cells includes immune cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, and megakaryocytes.

[0006] Patent Document 5 describes a method for inducing the formation of human colon organoids (HCO) by differentiating definitive endoderm (DE) derived from embryonic stem cells, induced pluripotent stem cells, etc., and an intestinal colon organoid derived from HCO. Patent Document 5 also describes that expression of macrophage markers was confirmed in HCO. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2014-236716 A [Patent Document 2] Patent No. 6151097 [Patent Document 3] JP 2019-000014 A [Patent Document 4] Special Publication No. 2020-500018 [Patent Document 5] Special Publication No. 2020-501534 [Non-patent literature]

[0008] [Non-Patent Document 1] Uchida et al., JCI Insight Volume 2 e86492 2017 Summary of the Invention [Problem to be solved by the invention]

[0009] The small intestine is a complex organ that contains cells derived from three germ layers (endoderm, ectoderm, and mesoderm). The small intestine performs functions such as secretion, absorption, and peristalsis through a complex combination of small intestinal epithelial cells (enterocytes, goblet cells, endocrine cells, brush cells, Paneth cells, M cells, etc.) derived from the endoderm, lymphatic tissue, smooth muscle cells, and interstitial cells of Cajal derived from the mesoderm, and the enteric nerve plexus derived from the ectoderm.

[0010] Patent Document 3 and Non-Patent Document 1 disclose intestinal organoids which have a pouch-like structure containing an inner lumen, containing endodermal cells, ectodermal cells, and mesodermal cells, with a small intestinal epithelial cell layer including the villi arranged outward, and which have functions such as secretion, absorption, and peristalsis.

[0011] However, the intestinal organoids described in Patent Document 3 and Non-Patent Document 1 do not contain immune cells.

[0012] The bioprinted three-dimensional in vivo artificial intestinal tissue model described in Patent Document 4 includes immune cells such as macrophages. However, since it is not formed in a self-organizing manner by cell culture, the layer structure of the cells is different from the original layer structure of in vivo tissue.

[0013] The human colon organoid (HCO) expressing a macrophage marker described in Patent Document 5 does not have the function of the small intestine.

[0014] Thus, conventionally, no cell structure has been provided that contains endodermal cells, ectodermal cells, and mesodermal cells, as in the small intestine, and also contains immune cells. [Means for solving the problem]

[0015] The present disclosure includes the following inventions. (1) A cellular structure that contains an inner lumen, contains endodermal cells, ectodermal cells, and mesodermal cells, and contains macrophages. (2) A cell structure according to (1), in which macrophages are attached to the inner surface. (3) A cell structure described in (1) or (2), in which one or more selected from IBA1 and HAM56 are expressed. (4) A cell structure described in any one of (1) to (3), which has phagocytosis. (5) The cell structure described in any one of (1) to (4), further comprising a monocyte. (6) containing endodermal cells on the outer surface side; A cell structure according to any one of (1) to (5), comprising ectodermal cells and / or mesodermal cells on the inner surface side. (7) A cell structure described in any one of (1) to (6), comprising a small intestinal epithelial cell layer on the outer surface side. (8) The cell structure according to any one of (1) to (7), which is derived from an embryonic stem cell and / or an induced pluripotent cell. (9) A cellular structure containing an inner lumen, containing endodermal cells, ectodermal cells, and mesodermal cells, and containing monocytes. (10) containing endodermal cells on the outer surface side; The cell structure according to (9), comprising ectodermal cells and / or mesodermal cells on the inner surface side. (11) A cell structure according to (9) or (10), comprising a small intestinal epithelial cell layer on the outer surface side. (12) The cell structure according to any one of (9) to (11), which is derived from an embryonic stem cell and / or an induced pluripotent cell. (13) A method for producing a cell structure containing a lumen, containing endodermal cells, ectodermal cells, and mesodermal cells, and containing macrophages, comprising the steps of: Step 11 of injecting monocytes into a cell structure containing a lumen and including endodermal cells, ectodermal cells, and mesodermal cells; and Step 12: maturing the monocytes into macrophages in the cell structure into which the monocytes have been injected in step 11 The method includes: (14) seeding pluripotent stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface; Step 22: culturing the pluripotent stem cells to induce differentiation into a cell structure that contains a lumen and includes endodermal cells, ectodermal cells, and mesodermal cells; The method according to (13), further comprising producing the cell structure used in step 11 by a method comprising: Effect of the Invention

[0016] The cell structure according to one or more embodiments of the present disclosure has the functions of a macrophage and / or a monocyte. According to the method according to one or more embodiments of the present disclosure, it is easy to produce a cellular structure containing macrophages. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram for explaining the structural cross-section of a sac-like cell structure (intestinal organoid) before injection of monocytes. [Figure 2A] The "Minigut" in the upper part of Figure 2A shows the results of immunofluorescent staining for TGF-β and IL-8 and DAPI staining of cell nuclei of the intestinal organoids (sac-like cell structures) prepared in 2.2 before injection of monocytes. The "Small intestine" in the lower part of Figure 2A shows the results of immunofluorescent staining for TGF-β and IL-8 and DAPI staining of cell nuclei of human small intestine tissue. [Figure 2B] The left side of Figure 2B shows the results of quantitative RT-PCR analysis of TGF-β in human small intestine tissue (Small intestine), intestinal organoids (Minigut), and macrophage-resident intestinal organoids (MC-Minigut). The vertical axis shows the relative expression level, with the expression level of TGF-β in human small intestine tissue set to 1. The right side of Figure 2B shows the results of quantitative RT-PCR analysis of IL-8 in human small intestine tissue (Small intestine), intestinal organoids (Minigut), and macrophage-resident intestinal organoids (MC-Minigut). The vertical axis shows the relative expression level, with the expression level of IL-8 in human small intestine tissue set to 1. [Figure 3A]The upper left of Figure 3A shows the results of histochemical analysis of macrophage marker IBA1 in macrophage-resident intestinal organoids (MC-Minigut). The arrowhead indicates IBA1 expression. The scale bar indicates 100 μm. The lower left of Figure 3A shows the results of histochemical analysis of human small intestine tissue (Small intestine) for IBA1. The scale bar indicates 100 μm. The upper right of Figure 3A shows the results of histochemical analysis of another macrophage marker, HAM56, in macrophage-resident intestinal organoids (MC-Minigut). The arrowhead indicates HAM56 expression. The scale bar indicates 100 μm. The lower right of Figure 3A shows the results of histochemical analysis of human small intestine tissue (Small intestine) for HAM56. The scale bar indicates 100 μm. [Figure 3B] The upper part of Figure 3B shows the results of immunofluorescence staining for GFP (first from the left), IBA1 (second from the left), and DAPI (third from the left) at the same site of a GFP-expressing macrophage-resident intestinal organoid (MC(GFP+)-Minigut), and the right end shows the results of overlaying the two. The arrowheads indicate the area where GFP and DIBA1 were co-stained. The scale bar indicates 200 μm. The lower part of Figure 3B shows the results of immunofluorescence staining for GFP (first from the left), IBA1 (second from the left), and DAPI (third from the left) at another same site of a GFP-expressing macrophage-resident intestinal organoid (MC(GFP+)-Minigut), and the right end shows the results of overlaying the two. The scale bar indicates 50 μm. [Figure 3C] (C) Transmission electron microscopy of macrophages present on the surface surrounding the lumen of intestinal organoids containing GFP-expressing macrophages (scale bar 5 μm). [Figure 4] The upper part of Figure 4 shows the results of observation of intestinal organoids containing GFP-expressing macrophages using a fluorescent microscope. Green fluorescence indicates GFP-expressing macrophages. The lower part of Figure 4 shows an observation image of green fluorescence (left), an observation image of red fluorescence (center), and an overlapping image (right) of the two of intestinal organoids containing GFP-expressing macrophages incubated with pHrodoTMRed E. coli BioParticles (registered trademark) Conjugate. [Diagram 5] Figure 5 shows an overview of the procedure for dissociating GFP-expressing macrophage-resident intestinal organoids into single cells, performing fluorescence-activated cell sorting (FACS), and isolating GFP-positive macrophages. [Figure 6] The upper left of FIG. 6 is an image of macrophage-resident intestinal organoid (MC-Minigut) with immunofluorescence staining of CDX2 (intestinal cell marker) and villin (small intestinal epithelial cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. The lower left of FIG. 6 is an image of macrophage-resident intestinal organoid with immunofluorescence staining of ZO-1 (small intestinal epithelial cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. The upper right of FIG. 6 is an image of macrophage-resident intestinal organoid with immunofluorescence staining of CDX2 (intestinal cell marker) and MUC2 (goblet cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. The lower right image of Figure 6 shows a superposition of immunofluorescence staining of DEFA6 (Paneth cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei of the macrophage-resident intestinal organoid. The scale bar indicates 100 μm. [Figure 7]The upper left of FIG. 7 is an image of macrophage-resident intestinal organoid (MC-Minigut) with immunofluorescent staining of OLFM4 (intestinal stem cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. Green indicates OLFM4-positive cells. The upper right of FIG. 7 is an image of human small intestine tissue (Small intestine) with immunofluorescent staining of OLFM4 (intestinal stem cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. The arrowhead indicates OLFM4-positive cells. The lower left of FIG. 7 is an image of macrophage-resident intestinal organoid with immunofluorescent staining of GP2 (M cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei superimposed thereon. The scale bar indicates 100 μm. The arrowhead indicates OLFM4-positive cells. The lower right image of Figure 7 shows a superimposition of immunofluorescent staining of GP2 (M cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei in human small intestinal tissue. The scale bar indicates 100 μm. The arrowheads indicate OLFM4-positive cells. [Figure 8] Figure 8 shows the results of quantitative RT-PCR analysis of the expression levels of various intestinal-related markers, LGR5, OLFM4, ECAD, CDX2, MUC2, DEFA6, ChgA, and GP2, in macrophage-resident intestinal organoids (MC-Minigut), intestinal organoids (Minigut), and human small intestine tissue (Small intestine). [Figure 9] The upper left of Figure 9 shows macrophage-resident intestinal organoids (MC-Minigut), the upper center of Figure 9 shows intestinal organoids (Minigut), and the upper right of Figure 9 shows human small intestinal tissue (Small intestine), with immunofluorescence staining of PGP9.5 (enteric nerve plexus marker) and SMA (smooth muscle marker) superimposed with DAPI staining of cell nuclei. The scale bar indicates 100 μm. The lower part of Figure 9 shows the results of quantitative RT-PCR analysis of the expression levels of SMA, PGP9.5, and BMP2 in macrophage-resident intestinal organoids (MC-Minigut), intestinal organoids (Minigut), and human small intestinal tissue (Small intestine). [Figure 10A]FIG. 10A shows cytokine concentrations in the intestinal organoid lumen fluid (CF) and intestinal organoid culture supernatant (SF) of intestinal organoids (Minigut) that do not contain macrophages before the addition of LPS (lipopolysaccharide). [Figure 10B] FIG. 10B shows cytokine concentrations in the CF and SF of intestinal organoids (Minigut) that do not contain macrophages, 24 hours after LPS addition. [Figure 10C] FIG. 10C shows cytokine concentrations in the CF and SF of macrophage-resident intestinal organoids (MC-Minigut) before addition of LPS. [Figure 10D] FIG. 10D shows cytokine concentrations in the CF and SF of macrophage-resident intestinal organoids (MC-Minigut) 24 hours after LPS addition. [Figure 11A] Figure 11A shows the results of analyzing the concentration of the anti-inflammatory cytokine IL-10 in the lumen fluid (CF) of intestinal organoids before and 24 hours after addition of LPS for macrophage-resident intestinal organoids (MC-MGUT) (gray) and intestinal organoids (MGUT) (black). [Figure 11B] Figure 11B shows the results of quantitative RT-PCR analysis of IL-10 gene expression levels in macrophage-resident intestinal organoids (MC-Minigut) and intestinal organoids (Minigut) before and 24 hours after LPS addition, as well as IL-10 gene expression levels in human small intestine tissue (Small intestine). [Figure 12A] Figure 12A shows the results of histochemical analysis of CD14 (LPS receptor) in intestinal organoids co-cultured with monocytes (Mini-Gut(MCs+)), intestinal organoids (Mini-Gut(MCs-)), and human small intestine tissue (Small intestine). [Figure 12B] FIG. 12B shows the results of histochemical analysis of CD163 (scavenger receptor) in Mini-Gut (MCs+), Mini-Gut (MCs-), and small intestines. [Figure 12C]FIG. 12C shows the results of histochemical analysis of MHC II (antigen-presenting molecule) in Mini-Gut (MCs+), Mini-Gut (MCs-), and small intestine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] <1.Cellular structures> The cell structure according to one or more embodiments of the present disclosure is characterized in that it contains an internal lumen, contains endodermal cells, ectodermal cells, and mesodermal cells, and contains macrophages. The cell structure according to one or more embodiments of the present disclosure may also be characterized as containing an internal lumen, comprising endodermal, ectodermal and mesodermal cells, and comprising monocytes. The cell structure according to one or more embodiments of the present disclosure contains an internal lumen and has a bag-like structure overall, and therefore may be referred to as a "bag-like cell structure." The cell structure according to one or more embodiments of the present disclosure may be referred to as an "intestinal organoid" since it has a function similar to that of the small intestine.

[0019] The cellular constructs according to one or more embodiments of the present disclosure are preferably derived from pluripotent stem cells, such as embryonic stem cells or induced pluripotent stem cells.

[0020] Here, the embryonic stem cells (ES cells) are preferably mammalian ES cells, and for example, ES cells derived from rodents such as mice or primates such as humans can be used. Particularly preferably, ES cells derived from mice or humans are used. ES cells refer to stem cell lines made from inner cell masses belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development, and can be proliferated almost indefinitely in vitro while maintaining the pluripotency to theoretically differentiate into all tissues. As ES cells, for example, cells in which a reporter gene has been introduced near the Pdx1 gene can be used to facilitate confirmation of the degree of differentiation. For example, 129 / Sv-derived ES cell lines in which the LacZ gene has been incorporated into the Pdx1 locus or ES cell lines SK7 having a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, ES cell lines PH3 having an mRFP1 reporter transgene under the control of an Hnf3β endoderm-specific enhancer fragment and a GFP reporter transgene under the control of the Pdx1 promoter can also be used. In addition, ES cell lines SEES1, SEES2, SEES3, SEES4, SEES5, SEES6 or SEES7 established at the Department of Reproductive and Cellular Medicine at the National Center for Child Health and Development and disclosed in Akutsu H, et al. Regen Ther. 2015;1:18-29, or cell lines in which additional genes have been introduced into these ES cell lines can also be used.

[0021] Induced pluripotent stem cells (iPS cells) are cells with pluripotency obtained by reprogramming somatic cells. Several groups have succeeded in producing induced pluripotent stem cells, including the group of Professor Shinya Yamanaka at Kyoto University, the group of Rudolf Jaenisch at Massachusetts Institute of Technology, the group of James Thomson at the University of Wisconsin, and the group of Konrad Hochedlinger at Harvard University. For example, International Publication WO2007 / 069666 describes somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, and Myc family genes, as well as somatic cell nuclear reprogramming factors containing gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes, and further describes a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, including a step of contacting somatic cells with the above-mentioned nuclear reprogramming factors.

[0022] The type of somatic cells used to generate iPS cells is not particularly limited, and any somatic cells can be used. Somatic cells include all cells other than germ cells that constitute the living body, and may be differentiated somatic cells or undifferentiated stem cells. The origin of somatic cells is not particularly limited and may be any of mammals, birds, fish, reptiles, and amphibians, but is preferably mammals (e.g., rodents such as mice, or primates such as humans), and is particularly preferably mice or humans. When human somatic cells are used, any of fetal, neonatal, and adult somatic cells may be used. Specific examples of somatic cells include fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, hepatic epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), nerve cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiac myocytes), hepatic parenchymal cells, non-hepatic parenchymal cells, adipocytes, osteoblasts, cells that constitute periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), and cells that constitute the kidney, eye, and ear.

[0023] An iPS cell is a stem cell that has the ability to self-replicate over a long period of time under specific culture conditions (e.g., conditions for culturing ES cells) and has the ability to differentiate into ectoderm, mesoderm, and endoderm under specific differentiation-inducing conditions. An iPS cell may also be a stem cell that has the ability to form a teratoma when transplanted into a test animal such as a mouse.

[0024] To produce iPS cells from somatic cells, first, at least one type of reprogramming gene is introduced into the somatic cells. The reprogramming gene is a gene that encodes a reprogramming factor that has the effect of reprogramming somatic cells to 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 largeT gene (iv) Oct gene, Klf gene, Sox gene

[0025] Next, the characteristics of the cells that constitute the cell structure according to one or more embodiments of the present disclosure will be described. The endoderm forms the digestive tract, as well as the tissues of organs such as the lungs, thyroid gland, pancreas, and liver, cells of secretory glands that open into the digestive tract, the peritoneum, pleura, larynx, Eustachian tube, trachea, bronchi, and urinary tract (bladder, most part of the urethra, and part of the ureter). Differentiation of pluripotent stem cells into endodermal cells can be confirmed by measuring the expression level of endoderm-specific genes. Examples of endoderm-specific genes include AFP, SERPINA1, SST, ISL1, IPF1, IAPP, EOMES, HGF, ALBUMIN, PAX4, and TAT.

[0026] Endodermal cells that may be included in the cell structure according to one or more embodiments of the present disclosure include small intestinal epithelial cells. The cell structure according to one or more embodiments of the present disclosure preferably includes one or more selected from intestinal cells, goblet cells, enteroendocrine cells, Paneth cells, and M cells as the small intestinal epithelial cells, and it is particularly preferable that the small intestinal epithelial cells include all of intestinal cells, goblet cells, enteroendocrine cells, and Paneth cells. The presence of endoderm cells in the cell structure according to the present disclosure can be determined based on positive expression of an endoderm cell marker. An example of an intestinal cell marker is CDX2, an example of a goblet cell marker is MUC2, an example of an enteroendocrine cell marker is CGA, and an example of a Paneth cell marker is DEFA6. An example of an M cell marker is GP2. Other markers for intestinal epithelial cells include ECAD (E-cadherin), ZO-1, Na+ / K+-ATPase, and villin. In addition, definitive endoderm markers FOXA2, SOX17, or CXCR4 can also be used as markers for identifying endoderm cells. Furthermore, GATA4, GATA6, or T (Brachyury), which are markers for early endoderm and mesoderm, can also be used as markers for distinguishing endodermal cells.

[0027] The ectoderm forms the epidermis of the skin, the epithelium of the distal part of the male urethra, hair, nails, skin glands (including mammary glands and sweat glands), sensory organs (including the epithelium of the distal part of the oral cavity, pharynx, nose, and rectum, salivary glands), and lenses. Part of the ectoderm invaginates into a groove during development to form a neural tube, which is the source of neurons and melanocytes of the central nervous system such as the brain and spinal cord. It also forms the peripheral nervous system. Differentiation of pluripotent stem cells 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 β-TUBLIN, NESTIN, GALANIN, GCM1, GFAP, NEUROD1, OLIG2, SYNAPTPHYSIN, DESMIN, and TH.

[0028] Ectoderm cells that may be contained in the cell structure according to one or more embodiments of the present disclosure include cells that constitute the enteric nerve plexus. The presence of ectoderm cells in the cell structure according to one or more embodiments of the present disclosure can be determined based on positive expression of an ectoderm cell marker. Markers that can be used to distinguish ectoderm cells include the enteric nerve plexus marker PGP9.5 and the neural progenitor cell marker SOX1.

[0029] The mesoderm forms the body cavity and the mesothelium lining it, muscles, skeleton, skin dermis, connective tissue, heart, blood vessels (including vascular endothelium), blood (including blood cells), lymphatic vessels, spleen, kidneys, ureters, and gonads (testes, uterus, and gonadal epithelium). Differentiation of pluripotent stem cells into mesodermal cells can be confirmed by measuring the expression level of mesoderm-specific genes. Examples of mesoderm-specific genes include FLK-1, COL2A1, FLT1, HBZ, MYF5, MYOD1, RUNX2, and PECAM1.

[0030] Mesodermal cells that may be included in the cell structure of the present disclosure include smooth muscle cells and interstitial cells of Cajal. The presence of mesodermal cells in the cell structure of the present disclosure can be determined based on positive expression of mesodermal cell markers. As mesodermal cell markers, smooth muscle cell marker α-smooth muscle actin (SMA), and interstitial cell markers of Cajal CD34 and CKIT (in the case of double positivity) can be used. In addition, GATA4, GATA6, or T (Brachyury), which are markers for early endoderm and mesoderm, can also be used as markers for distinguishing mesodermal cells.

[0031] The cell structure according to one or more embodiments of the present disclosure further preferably comprises intestinal stem cells. The presence of intestinal stem cells can be determined by using as an indicator the positivity of intestinal stem cell markers OLFM4 and LGR5. Preferably, the cell construct according to one or more embodiments of the present disclosure further comprises serotonin positive enteroendocrine cells.

[0032] Preferably, the cell structure according to one or more embodiments of the present disclosure further comprises a transporter-positive cell, such as the intestinal oligopeptide transporter (PEPT1) and the ATP-binding cassette (ABC) transporters ABCB1 and ABCG2.

[0033] Preferably, the cell structure according to one or more embodiments of the present disclosure further comprises cystic fibrosis transmembrane conductance regulator (CFTR)-positive small intestinal epithelial cells. CFTR-positive small intestinal epithelial cells are involved in mucus secretion. That is, the cell structure having CFTR-positive small intestinal epithelial cells has a mucus secretion ability similar to that of the small intestine, specifically, an ability to produce mucin and antimicrobial peptides. The cell structure according to one or more embodiments of the present disclosure is preferably lymphocyte-free.

[0034] Preferably, the cell structure according to one or more embodiments of the present disclosure further comprises histamine H1 receptor positive cells.

[0035] The cell structure according to one or more embodiments of the present disclosure preferably includes endodermal cells on the outer surface side, and ectodermal cells and / or mesodermal cells on the inner surface side (i.e., the surface surrounding the lumen). The cell structure according to this embodiment includes small intestinal epithelial cells, in particular, as endodermal cells on the outer surface side. This embodiment is preferable because a substance on the outer surface side can be transported into the lumen via the small intestinal epithelial cells. Furthermore, in this embodiment, when the small intestinal epithelial cells on the outer surface are further transporter-positive, it is even more preferable because transport of a substance via the transporter is possible. In other words, the cell structure according to the present disclosure including transporter-positive small intestinal epithelial cells has a substance absorption ability similar to that of the intestine.

[0036] The cell structure according to one or more embodiments of the present disclosure preferably has microvilli and crypts formed on a surface comprising small intestinal epithelial cells.

[0037] More preferably, the cellular structure according to one or more embodiments of the present disclosure includes a mucosa and a submucosa. The mucosa of the small intestine is formed by an epithelium and a basement membrane. The epithelium of the small intestine is composed of two types of cells, absorptive enterocytes and secretory cells, including mucus-producing goblet cells, enteroendocrine cells, and Paneth cells.

[0038] In the above embodiment of the cell structure according to one or more embodiments of the present disclosure, the ectodermal cells and / or mesodermal cells present on the inner surface side are preferably ectodermal cells and mesodermal cells, and more preferably include nerve cells, smooth muscle cells and interstitial cells of Cajal. The nerve cells preferably constitute the enteric nerve plexus.

[0039] The cell structure according to one or more embodiments of the present disclosure preferably has the ability to perform contraction similar to peristalsis. Such a function is generated by the development of a neural network and smooth muscle. Peristalsis is thought to occur when pacemaker cells, Cajal cells, stimulate smooth muscle cells after receiving signals from nerve cells or hormones. In the following description, the ability to perform contraction similar to peristalsis is referred to as "peristaltic motility." The cell structure according to the present disclosure having peristaltic motility particularly preferably exhibits drug responsiveness similar to that of the intestine, in that the frequency of contraction increases with histamine treatment and decreases with atropine treatment. The cell structure according to one or more embodiments of the present disclosure having peristaltic motility that exhibits drug responsiveness similar to that of the intestine can be suitably used for evaluating the effect of a drug on intestinal peristalsis.

[0040] The cell structure according to one or more embodiments of the present disclosure is characterized in that it further comprises macrophages and / or monocytes. The cellular structure according to one or more embodiments of the present disclosure includes macrophages and / or monocytes, thereby providing functions more similar to those of the small intestine.

[0041] The cell structure according to one or more embodiments of the present disclosure preferably includes macrophages, thereby enabling the cell structure to have phagocytosis, ingesting substances outside the cell structure and decomposing the ingested substances within the macrophages.

[0042] The cell structure comprising monocytes according to one or more embodiments of the present disclosure can be cultured to allow the monocytes to mature into macrophages or dendritic cells.

[0043] In the cell structure according to one or more embodiments of the present disclosure, the surface surrounding the lumen (inner surface) contains TGF-β and interleukin-8 (IL-8). In small intestinal tissue in vivo, monocytes are stably maintained on the basement membrane via TGF-β and IL-8. The inner surface of the cell structure according to one or more embodiments of the present disclosure is considered to be an environment that is likely to retain monocytes and macrophages, which are mature monocytes, similar to small intestinal tissue in vivo.

[0044] The macrophages contained in the cell structure according to one or more embodiments of the present disclosure are preferably macrophages formed from monocytes, monoblasts, hematopoietic progenitor cells, or hematopoietic stem cells injected into the lumen, and more preferably from mature monocytes.

[0045] The monocytes contained in the cell structure according to one or more embodiments of the present disclosure are preferably monocytes injected into the lumen, but may also be mature monoblasts, hematopoietic progenitor cells, or hematopoietic stem cells injected into the lumen.

[0046] In a cell structure comprising macrophages according to one or more embodiments of the present disclosure, one or more, more preferably both, selected from macrophage markers IBA1 and HAM56 are expressed.

[0047] In a cellular structure including macrophages according to one or more embodiments of the present disclosure, the macrophages are preferably attached on the inner surface surrounding the lumen, so that substances taken into the lumen from the outside of the cellular structure are easily degraded by phagocytosis of the macrophages.

[0048] When the cell structure according to one or more embodiments of the present disclosure comprises monocytes, the monocytes may be suspended in the liquid (buffer or medium) that fills the lumen or may be attached to the inner surface.

[0049] The cell structure according to one or more embodiments of the present disclosure has a function similar to that of the small intestine and preferably has the ability to secrete cytokines, which may be secreted into the lumen of the sac-like cell structure or to the outside.

[0050] Examples of cytokines that can be secreted into the lumen by the cell structure according to one or more embodiments of the present disclosure include one or more selected from IL-10, IL-12p40, IL-12p70, IL-15, EGF, IL-1a, and CCL-4. Among these, IL-12p40, IL-12p70, IL-15, EGF, and IL-1a are cytokines that are not secreted into the lumen by a cell structure that has the same structure but does not have macrophages.

[0051] Examples of cytokines that can be secreted to the outside by the cell structure according to one or more embodiments of the present disclosure include one or more selected from IL-1ra, IL-7, IL-12p70, and VEGF. These cytokines are not secreted to the outside by a cell structure that has the same structure but does not have macrophages.

[0052] The cell constructs according to one or more embodiments of the present disclosure preferably express one or more selected from OLFM4, MUC2, LGR5, ECAD, CDX2, DEFA6, ChgA, GP2, villin and ZO-1.

[0053] The cell constructs according to one or more embodiments of the present disclosure also preferably express one or more selected from SMA, PGP9.5 and BMP2.

[0054] <2. Manufacturing method of cell structures> Also herein, A method for producing a cell structure containing an inner cavity, comprising endodermal cells, ectodermal cells, and mesodermal cells, and further comprising macrophages, the method comprising the steps of: Step 11 of injecting monocytes into a cell structure containing a lumen and including endodermal cells, ectodermal cells, and mesodermal cells; and Step 12: maturing the monocytes into macrophages in the cell structure into which the monocytes have been injected in step 11 A method is disclosed that includes:

[0055] According to this method, it is easy to produce the above-mentioned cell structure containing macrophages according to one or more embodiments of the present disclosure. In the following description, the cell structure containing an inner lumen and containing endodermal cells, ectodermal cells, and mesodermal cells into which monocytes are injected in step 11 may be referred to as a "precursor cell structure."

[0056] 2.1. Characteristics of precursor cell structures In the precursor cell structure, preferred embodiments of endodermal, ectodermal, and mesodermal cells and markers that can be used are as described for the cell structure comprising macrophages according to one or more embodiments of the present disclosure.

[0057] The precursor cell structure has similar characteristics to the cell structure containing macrophages according to one or more embodiments of the present disclosure, except that it does not contain macrophages. The precursor cell structure preferably contains endodermal cells on the outer surface side, and ectodermal cells and / or mesodermal cells on the lumen side (inside). It is particularly preferred that this precursor cell structure contains small intestinal epithelial cells as endodermal cells on the outer surface side. The precursor cell structure more preferably has developed microvilli and crypts on the outer surface containing small intestinal epithelial cells.

[0058] The precursor cell construct more preferably has a layered structure similar to the intestinal wall of the small intestine, including a mucosa and a submucosa, the characteristics of which are as described for the cell constructs of the present disclosure.

[0059] The precursor cell construct preferably has the ability to undergo contractile movements similar to peristalsis.

[0060] The precursor cell structure at the time when the monocytes are injected in step 11 may be, but is not limited to, a precursor cell structure that has been in place for, for example, 40 days or more, preferably 50 days or more since seeding.

[0061] <2.2.Process 11> In step 11, monocytes are injected into the precursor cell construct. The monocytes to be injected may be those isolated from a living body such as a human, or may be those induced to differentiate from pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, specific examples of which are as described above.

[0062] The monocytes to be injected are preferably mature monocytes at the time of injection, but may be precursor cells of monocytes such as monoblasts, hematopoietic progenitor cells, or hematopoietic stem cells.

[0063] The monocytes are preferably injected into the lumen of the precursor cell construct.

[0064] In step 11, it is preferable to immerse the precursor cell structure in a suitable liquid medium (buffer solution, culture medium, etc.) and, with the liquid medium present in and around the inner cavity of the precursor cell structure, inject a suspension of monocytes into the inner cavity using a syringe or the like.

[0065] When injecting monocytes, the precursor cell structure may be in a state of being suspended in a liquid medium, or in a state of being adhered to a cell culture substrate, which will be described later. The precursor cell structure suspended in a liquid medium can be held with tweezers or the like when injecting monocytes.

[0066] There is no particular limitation on the amount of monocytes injected in step 11. For example, 10,000 to 100,000 monocytes can be injected per precursor cell structure.

[0067] <2.3.Process 12> In step 12, the monocytes are allowed to mature into macrophages in the cell structure into which they were injected in step 11.

[0068] In step 12, the cell structure into which the monocytes have been injected in step 11 is cultured in an appropriate medium to mature the monocytes. In this case, it is preferable to use a medium containing a factor that promotes the maturation of monocytes, for example, a factor that stimulates leukocyte stem cells. An example of a factor that stimulates leukocyte stem cells is M-CSF (Invitrogen) used in the Examples. Other components of the medium are not particularly limited.

[0069] If monocytes are injected into the lumen of the precursor cell structure in step 11, then in step 12, the monocytes preferably engraft on the surfaces surrounding the lumen of the cell structure and mature into macrophages.

[0070] The time and temperature for maturing monocytes into macrophages in step 12 are not particularly limited, but examples of the time include 3 days to 25 days, and the temperature includes 25 to 38°C.

[0071] In step 12, it is not necessary for all monocytes to mature into macrophages, and some of the monocytes may remain as monocytes or may mature from monocytes into dendritic cells. Preferably, all of the infused monocytes mature into macrophages, or the majority mature into macrophages and the remainder remain as monocytes.

[0072] 2.4. Method for producing precursor cell structure The precursor cell structure used in step 11 is 21, seeding pluripotent stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface; Step 22: culturing the pluripotent stem cells to induce differentiation into a cell structure (precursor cell structure) that contains a lumen and includes endodermal cells, ectodermal cells, and mesodermal cells; The composition can be produced by a method comprising the steps of:

[0073] A preferred embodiment of the method for producing this precursor cell structure will now be described.

[0074] <2.4.1.Cell culture substrate> The cell culture substrate comprises a pattern of cell adhesive sites on a surface. The shape of the cell adhesion portion is not particularly limited, but can be, for example, a cell adhesion portion that encapsulates a non-cell adhesion portion, extends continuously or intermittently along the periphery of the non-cell adhesion portion, and surrounds the non-cell adhesion portion.

[0075] Other examples of the shape of the cell adhesion portion include polygons, including squares that do not include cell non-adhesive portions, circles, ellipses, and the like, with circles being preferred.

[0076] In the following description, the area including the cell adhesion portion where the cell structure is cultured by adhesion is referred to as the "cell culture portion."

[0077] The cell culture substrate includes one or more cell culture sections, preferably two or more cell culture sections, on the surface of the substrate. The shapes and dimensions of the two or more cell culture sections included in one substrate may be the same or different. The cell adhesion regions are typically arranged in islands among the non-cell adhesion regions.

[0078] When the cell culture section contains a non-cell-adhesive portion, the non-cell-adhesive portion contained in the cell-adhesive portion may be referred to as a "second non-cell-adhesive portion" or a "central portion" to distinguish it from a non-cell-adhesive portion (a first non-cell-adhesive portion described below) present in a portion other than the cell culture section.

[0079] In the following description, the portion of the cell culture substrate used in the present disclosure other than the cell adhesive portion and the cell non-adhesive portion may be referred to as the "support substrate." In other words, the cell culture substrate used in one or more embodiments of the present disclosure can be said to include a support substrate having a surface including a pattern of one or more cell adhesive portions.

[0080] <2.4.1.1. Support substrate, non-cell adhesive portion, and cell adhesive portion of cell culture substrate> The characteristics of the support substrate and the characteristics other than the shapes of the cell non-adhesive portion and the cell adhesive portion are described below.

[0081] The support substrate used for the cell culture substrate is not particularly limited as long as it is a support substrate formed of a material capable of forming a cell non-adhesive portion and a cell adhesive portion on its surface. Specifically, the support substrate may include inorganic materials such as glass, metal, ceramic, and silicon, and organic materials such as elastomers and plastics (e.g., polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, and vinyl chloride resin). In particular, it is preferable to use a glass substrate as the support substrate. The shape of the support substrate is not limited, and examples thereof include flat shapes such as flat plates, flat membranes, films, and porous membranes, and three-dimensional shapes such as cylinders, stamps, multi-well plates, and microchannels.

[0082] In the present disclosure, "cell adhesiveness" refers to the strength of adhering cells, i.e., the ease with which cells adhere. A "cell adhesive portion" refers to an area on a surface with good cell adhesiveness, and a "cell non-adhesive portion" refers to an area on a surface with poor cell adhesiveness. Therefore, when cells are seeded on a surface on which cell adhesive portions and cell non-adhesive portions are arranged in a predetermined pattern, the cells adhere to the cell adhesive portions but do not adhere to the cell non-adhesive portions, resulting in the cells being arranged in a pattern on the surface of the cell culture substrate.

[0083] The "cell adhesion portion" is defined as a portion to which cells to be actually cultured, preferably stem cells, adhere when seeded on the cell culture substrate, and the "cell non-adhesive portion" is defined as a portion to which cells to be actually cultured, preferably stem cells, do not adhere when seeded. When seeding cells on the cell culture substrate, the surface of the cell culture substrate may be coated with a protein or the like to enhance cell adhesion. Specific examples of "stem cells" are as described in this specification. The cell non-adhesive portion may be covered by cells that have adhered to and proliferated on the cell adhesion portion.

[0084] The cell adhesion spreading rate during actual cell culture can be used as an index for determining whether a region is a cell adhesion region or a cell non-adhesive region. The surface of a cell adhesion region having cell adhesive properties is preferably a surface with a cell adhesion spreading rate of 60% or more, and more preferably a surface with a cell adhesion spreading rate of 80% or more. A high cell adhesion spreading rate allows cells to be cultured efficiently. The cell adhesion spreading rate in the present disclosure is defined as the spreading rate when the seeding density is 4000 cells / cm 2 More than 30000 cells / cm 2 Cells to be cultured within this range are seeded on the surface to be measured and stored in an incubator at 37°C with a CO2 concentration of 5%. After 14.5 hours of culture, the adhesion rate is defined as the percentage of cells that have attached and spread out ({(number of attached cells) / (number of seeded cells)} x 100(%)).

[0085] In the above measurement, the cells are seeded on the measurement surface by suspending them in DMEM medium containing 10% FBS, and then slowly shaking the measurement surface on which the cells are seeded so that the cells are distributed as uniformly as possible. Furthermore, the cell adhesion spreading rate is measured after replacing the medium immediately before the measurement to remove unadhered cells. In the measurement of the cell adhesion spreading rate, the measurement points are those excluding points where the cell density is likely to be specific (for example, the center of a specified area where the cell density is likely to be high, and the periphery of a specified area where the cell density is likely to be low).

[0086] The cell adhesion portion may be a region where a cell adhesion layer is formed on the surface of the support substrate, or when the surface of the support substrate is cell adhesive (for example, the surface of a glass substrate), it may be a region where the surface of the support substrate is exposed, but is preferably a region where the cell adhesive surface of the support substrate is exposed. The cell non-adhesive portion may be a region where a cell non-adhesive layer is formed on the surface of the support substrate. The cell adhesion portion and the cell non-adhesive portion can be formed by various materials and methods. Preferably, the cell non-adhesive portion is a portion where the surface of the support substrate is covered with a cell non-adhesive layer such as a layer containing a hydrophilic organic compound such as a hydrophilic polymer. The average thickness of the cell non-adhesive layer constituting the cell non-adhesive portion is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, more preferably 1 nm to 10 μm, and most preferably 1.5 nm to 1 μm. If the average thickness is 0.8 nm or more, it is preferable because the adsorption of proteins and the adhesion of cells are less affected by the region of the support substrate that is not covered with the cell non-adhesive layer. In addition, if the average thickness is 500 μm or less, coating is relatively easy. In particular, when the cell non-adhesive layer is formed of a polyethylene glycol layer, the thickness of the layer can be 5 nm to 10 nm. Specific examples of hydrophilic organic compounds are described below. Particularly preferred embodiments of the method for forming cell adhesive sites and cell non-adhesive sites include the following two embodiments.

[0087] In the first embodiment, a cell non-adhesive layer is formed on the surface of the support substrate, and then a predetermined treatment is applied to a part of the cell non-adhesive layer to develop cell adhesiveness and form a cell adhesive portion. Specifically, a cell non-adhesive hydrophilic film containing a hydrophilic organic compound such as a hydrophilic polymer is formed on the surface of the support substrate as a cell non-adhesive layer, and then a part of the hydrophilic film, which is the cell non-adhesive layer, is selectively subjected to an oxidation treatment and / or decomposition treatment to modify the part into a cell adhesive portion having cell adhesiveness. In this embodiment, a cell non-adhesive hydrophilic film is formed, and then an oxidation treatment and / or decomposition treatment is applied to the part where cell adhesion is desired, thereby converting the part into a part having cell adhesiveness and forming the cell adhesive portion. In the cell culture substrate formed by the first embodiment, the non-cell-adhesive portion is a portion of the surface of the support substrate covered with a layer containing a hydrophilic organic compound such as a hydrophilic polymer, and the cell-adhesive portion is a portion of the surface of the support substrate exposed after the layer containing a hydrophilic organic compound such as a hydrophilic polymer has been removed by oxidation treatment and / or decomposition treatment, or a portion of the layer containing a hydrophilic organic compound such as a hydrophilic polymer that has been subjected to oxidation treatment and / or decomposition treatment and is covered with a layer (=cell adhesion layer) that has been modified to have cell adhesive properties.

[0088] The second form is a form in which the cell adhesion portion and the cell non-adhesive portion are formed depending on the density of the organic compound on the surface of the support substrate. In the cell culture substrate formed by the second form, the cell adhesion portion is a surface with a low density of hydrophilic organic compounds such as hydrophilic polymers (including the case where no hydrophilic organic compounds are included), and the cell non-adhesive portion is a surface with a high density of hydrophilic organic compounds such as hydrophilic polymers. The second form utilizes the fact that the surface of a support substrate containing a high density of hydrophilic organic compounds such as hydrophilic polymers has cell non-adhesive properties, whereas the surface of a support substrate with a low density of the compounds has cell adhesive properties. When a first region to which the compound is easily bound and a second region to which the compound is not easily bound are provided on the surface of the support substrate, and a film of the compound is formed on the surface of the substrate, the first region becomes a cell non-adhesive portion and the second region becomes a cell adhesive region. Alternatively, a part of the surface of the support substrate is selectively masked with a photoresist or the like, a film of the hydrophilic organic compound is formed on the unmasked region to form a cell non-adhesive portion, and then the masking is removed to expose the surface of the support substrate, thereby forming a cell adhesive portion.

[0089] In addition, the present invention is not limited to the above-mentioned form, and a support substrate having a cell-nonadhesive surface (which may be the surface of a cell-nonadhesive layer) may be prepared, and a part of the surface may be patterned and coated with a cell-adhesive protein such as collagen or fibronectin to form a cell-adhesive pattern. Alternatively, a support substrate having a cell-adhesive surface (which may be the surface of a cell-adhesive layer) may be prepared, and a part of the surface may be coated with a cell-nonadhesive resin such as silicone rubber (e.g., Keiju (registered trademark) manufactured by Mitsubishi Chemical), and the remaining part may be made into a cell-adhesive pattern. Alternatively, a support substrate having a conductive layer of a predetermined pattern on its surface may be prepared, and a cell-nonadhesive layer may be laminated on the surface of the support substrate, and the cell-nonadhesive layer covering the conductive layer may be peeled off by applying a voltage to the conductive layer, and the exposed conductive layer may be made into a cell-adhesive portion (specifically, see JP 2012-120443 A and JP 2013-179910 A).

[0090] Below, we will explain in order the above-mentioned first and second forms, in which cell adhesive portions and non-cell adhesive portions are formed on the surface of a support substrate to produce a cell culture substrate having a surface including cell adhesive portions and non-cell adhesive portions.

[0091] First, the first embodiment will be described.

[0092] In the first embodiment, a hydrophilic film containing a hydrophilic organic compound, preferably a hydrophilic polymer, is first provided as a cell non-adhesive layer on the surface of a support substrate. The hydrophilic film is a thin film having water-solubility or water-swellability, and is not particularly limited as long as it has cell non-adhesive properties before oxidation and / or decomposition, and the exposed surface of the support substrate after oxidation and / or decomposition, or the surface of the thin film modified by oxidation and / or decomposition, exhibits cell adhesive properties.

[0093] When the cell non-adhesive layer is a hydrophilic film formed by a hydrophilic organic compound, it is preferable to provide a binding layer between the surface of the support substrate and the hydrophilic film as necessary. The binding layer is preferably a layer containing a material having a functional group (binding functional group) capable of binding to the functional group of the organic compound of the hydrophilic film. Examples of combinations of the binding functional group of the material of the binding layer and the functional group of the hydrophilic organic compound include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. In each combination, either one may be the functional group on the binding layer side. In these methods, before coating with a hydrophilic organic compound, a binding layer is formed on the support substrate by a material having a predetermined functional group. In the cell non-adhesive layer, the water contact angle of the surface of the binding layer before forming a thin film of a hydrophilic organic compound is typically 45° or more, preferably 47° or more, when a silane coupling agent having an epoxy group at its terminal is used as the material having a binding functional group. Such a binding layer can be obtained by forming a coating of a material having a binding functional group on the surface of a supporting substrate.

[0094] Examples of the hydrophilic organic compound include hydrophilic polymers (including hydrophilic oligomers), water-soluble organic compounds, surface active substances, and amphiphilic substances, with hydrophilic polymers being particularly preferred.

[0095] Specific examples of hydrophilic polymers include polyalkylene glycols, zwitterionic polymers having phospholipid polar groups, polyacrylamides, polyacrylic acids, polymethacrylic acids, polyvinyl alcohols, polysaccharides, etc. These specific examples of hydrophilic polymers also include those in the form of derivatives. The molecular shape of the hydrophilic polymer can be linear, branched, dendrimers, etc.

[0096] Specific examples of the polyalkylene glycol include polyethylene glycol, polypropylene glycol, and copolymers of polyethylene glycol and polypropylene glycol, such as Pluronic F108 and Pluronic F127.

[0097] Specific examples of amphoteric polymers having a phospholipid polar group include poly(methacryloyloxyethyl phosphorylcholine) (=MPC polymer), copolymers of methacryloyloxyethyl phosphorylcholine and acrylic monomers, and the like. A specific example of polyacrylamide is poly(N-isopropylacrylamide). A specific example of polymethacrylic acid is poly(2-hydroxyethyl methacrylate). Specific examples of polysaccharides include dextran and heparin.

[0098] It is desirable that the surface of a support substrate provided with a non-cell-adhesive layer has high cell non-adhesive properties when covered with the non-cell-adhesive layer, and that after oxidation and / or decomposition treatment of the non-cell-adhesive layer, the exposed surface of the support substrate, or the surface of a layer formed by modifying the non-cell-adhesive layer by oxidation and / or decomposition treatment, exhibits cell adhesive properties.

[0099] As the hydrophilic polymer, polyethylene glycol (PEG) is particularly preferred. PEG contains at least an ethylene glycol chain (EG chain) consisting of one or more ethylene glycol units ((CH2)2-O), and may be linear or branched. The ethylene glycol chain is, for example, represented by the following formula: -((CH2)2-O) m - (m is an integer indicating the degree of polymerization) It refers to a structure represented by the following formula: m is preferably an integer of 1 to 13, and more preferably an integer of 1 to 10.

[0100] PEG also includes ethylene glycol oligomers. PEG also includes those into which functional groups have been introduced. Examples of functional groups include epoxy groups, carboxyl groups, N-hydroxysuccinimide groups, carbodiimide groups, amino groups, glutaraldehyde groups, and (meth)acryloyl groups. The functional groups are preferably introduced at the terminals, optionally via a linker. Examples of PEG into which functional groups have been introduced include PEG (meth)acrylate and PEG di(meth)acrylate.

[0101] The cell adhesion portion can be formed by subjecting a non-cell adhesion layer containing a hydrophilic organic compound formed on the surface of the support substrate to an oxidation treatment and / or decomposition treatment to expose the surface of the support substrate having cell adhesive properties, or by modifying the non-cell adhesion layer to convert it into a cell adhesion layer. In the present disclosure, the term "oxidation" is used in the narrow sense to mean a reaction in which an organic compound reacts with oxygen to increase the oxygen content compared to before the reaction.

[0102] In this disclosure, "decomposition" refers to a change in which the bonds of an organic compound are broken to produce two or more organic compounds from one type of organic compound. "Decomposition treatment" typically includes, but is not limited to, decomposition by oxidation treatment and decomposition by ultraviolet irradiation. When the "decomposition treatment" is decomposition involving oxidation (i.e., oxidative decomposition), the "decomposition treatment" and the "oxidation treatment" refer to the same treatment. In addition, decomposition and removal of the non-adhesive cell layer is also included in the "decomposition treatment".

[0103] Decomposition by UV irradiation refers to an organic compound absorbing UV light, going through an excited state, and then decomposing. When UV light is irradiated into a system in which an organic compound exists together with molecular species containing oxygen (oxygen, water, etc.), in addition to the UV light being absorbed by the compound and causing decomposition, the molecular species may become activated and react with the organic compound. The latter reaction can be classified as "oxidation." The reaction in which an organic compound is decomposed by oxidation caused by activated molecular species can be classified as "decomposition by oxidation" rather than "decomposition by UV irradiation."

[0104] As described above, the "oxidation treatment" and the "decomposition treatment" may overlap as operations, and it is not possible to clearly distinguish between the two. Therefore, in this specification, the term "oxidation treatment and / or decomposition treatment" is used.

[0105] Next, the second embodiment will be described.

[0106] In the cell culture substrate formed by the second embodiment, the cell adhesion portion of the surface of the support substrate is a surface having a low density of hydrophilic organic compounds such as hydrophilic polymers (including cases where no hydrophilic organic compounds are present), and the cell non-adhesive portion is a surface having a high density of hydrophilic organic compounds. That is, the density of the hydrophilic organic compounds differs between the cell adhesion portion and the cell non-adhesive portion. The higher the density, the more difficult it tends to be for cells to adhere. In the cell adhesion portion, the density of the hydrophilic organic compounds is low enough to allow cells to adhere. Preferred examples of the hydrophilic organic compounds and hydrophilic polymers are as described above for the first embodiment.

[0107] In the second embodiment, when the cell adhesion portion and the cell non-adhesion portion are formed by a hydrophilic film with controlled density, it is preferable to form a binding layer on the support substrate as necessary to enhance adhesion to the support substrate, and then form a hydrophilic film made of a hydrophilic organic compound. The binding layer is preferably a layer containing a material containing a functional group (binding functional group) capable of binding to a functional group of the hydrophilic organic compound. Examples of combinations of the functional group of the material of the binding layer and the functional group of the hydrophilic organic compound include epoxy group and hydroxyl group, phthalic anhydride and hydroxyl group, carboxyl group and N-hydroxysuccinimide, carboxyl group and carbodiimide, amino group and glutaraldehyde, etc. In each combination, either one may be the functional group on the binding layer side. In these methods, a binding layer is formed on the support substrate by a material having a predetermined functional group before coating with a hydrophilic material. The density of the material in the binding layer is an important factor that determines the binding force. The density can be easily evaluated using the contact angle of water on the surface of the binding layer as an index. The water contact angle was measured using CA-Z manufactured by Kyowa Interface Science Co., Ltd., 30 seconds after pure water was dropped from a microsyringe.

[0108] The density of the material having a binding functional group in the binding layer of the cell adhesion part is low. In the case where a silane coupling agent having an epoxy group at the end is used as the material having a binding functional group constituting the binding layer, the water contact angle of the surface of the binding layer before forming a thin film of a hydrophilic organic compound in the cell adhesion part is typically 10° to 43°, preferably 15° to 40°. Examples of the method for forming such a binding layer include a method in which a coating (binding layer) of a material having a binding functional group is formed on the surface of a support base material, and then the surface of the binding layer is oxidized and / or decomposed. Examples of the method for oxidizing and / or decomposing the binding layer surface include a method of irradiating the binding layer surface with ultraviolet light, a method of treating with a photocatalyst, and a method of treating with an oxidizing agent. The entire surface of the binding layer may be oxidized and / or decomposed, or may be partially treated. Partial treatment may be performed by using a mask such as a photomask or a stencil mask, or by using a stamp. In addition, the oxidation treatment and / or decomposition treatment may be performed by a direct drawing method such as a method using a laser such as an ultraviolet laser. Regarding the various conditions, the same conditions as those in the method of forming a cell adhesion site by oxidation and / or decomposition of a hydrophilic film can be applied. A thin film of a hydrophilic organic compound can be formed on the binding layer thus formed, thereby forming a cell adhesion site.

[0109] The density of the material having the binding functional group in the binding layer of the cell non-adhesive portion is high. In the case of using a silane coupling agent having an epoxy group at the end as the material having the binding functional group, the water contact angle of the surface of the binding layer before forming the thin film of the hydrophilic organic compound in the cell non-adhesive portion is typically 45° or more, preferably 47° or more. Such a binding layer is obtained by forming a coating of a material having a binding functional group on the surface of the support substrate. When the surface of the binding layer is partially oxidized and / or decomposed, the remaining part that is not treated becomes the binding layer having the water contact angle. A thin film of a hydrophilic organic compound is formed on the binding layer thus formed, thereby forming a cell non-adhesive layer.

[0110] In the second embodiment, a portion of the surface of the support substrate may be selectively masked with a photosensitive photoresist or the like, a film of the hydrophilic organic compound may be formed in the unmasked area to form a non-cell adhesive portion, and then the masking may be removed to expose the surface of the support substrate to form a cell adhesive portion.

[0111] Next, the characteristics of the cell adhesive portions and the cell non-adhesive portions formed in the above first or second embodiment, or by other methods, will be further described.

[0112] The carbon content of the cell adhesion portion (including the binding layer, if present) is preferably lower than the carbon content of the cell non-adhesion portion (including the binding layer, if present). Specifically, the carbon content of the cell adhesion portion is preferably 20 to 99% of the carbon content of the cell non-adhesion portion. This range is particularly suitable when the thickness of the hydrophilic organic compound layer contained in the cell adhesion portion and the cell non-adhesion portion (the sum of the thickness of the binding layer and the thickness of the hydrophilic film, if present) is 10 μm or less. The "carbon content (atomic concentration, %)" is defined as follows:

[0113] In addition, the percentage (%) of carbon bonded to oxygen in the carbon in the cell adhesion portion (including the binding layer if present) is preferably smaller than the percentage (%) of carbon bonded to oxygen in the carbon in the non-cell adhesion portion (including the binding layer if present). Specifically, the percentage (%) of carbon bonded to oxygen in the carbon in the cell adhesion portion is preferably 35 to 99% of the percentage (%) of carbon bonded to oxygen in the carbon in the non-cell adhesion portion. This range is particularly suitable when the thickness of the hydrophilic film (the sum of the thickness of the binding layer and the thickness of the hydrophilic film if a binding layer is present) is 10 μm or less. The "percentage (atomic concentration, %) of carbon bonded to oxygen" is defined as follows.

[0114] Methods for evaluating the hydrophilic organic compound layer (including the binding layer, if present) contained in the cell adhesion portion and the cell non-adhesion portion include contact angle measurement, ellipsometry, atomic force microscope observation, electron microscope observation, Auger electron spectroscopy, X-ray photoelectron spectroscopy, and various mass spectrometry. Among these methods, X-ray photoelectron spectroscopy (XPS / ESCA) is the most quantitative. This measurement method determines a relative quantitative value, which is generally calculated as an element concentration (atomic concentration, %). The X-ray photoelectron spectroscopy analysis method in the present disclosure will be described in detail below.

[0115] The "carbon amount" of the cell adhesion portion and the cell non-adhesion portion is defined as "the carbon amount obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectroscopy device." In addition, in the present disclosure, the "proportion of carbon bonded to oxygen" of the cell adhesion portion and the cell non-adhesion portion is defined as "the proportion of carbon bonded to oxygen obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectroscopy device." Specific measurements can be performed as described in JP 2007-312736 A.

[0116] <2.4.1.2. Preferred examples of shapes of cell adhesion parts> As described above, an example of the shape of the cell adhesion part is a polygon such as a square not including a cell non-adhesive part, a circle, an ellipse, etc., and a circle is preferable. In the case of a circle, the diameter can be preferably a diameter that satisfies the above-mentioned area range, and specifically, the diameter of the circle can be 350 μm or more, preferably 800 μm or more, preferably 1000 μm or more, preferably 1200 μm or more, more preferably 1500 μm or more, preferably 6000 μm or less, more preferably 4000 μm or less, even more preferably 3000 μm or less, and even more preferably 2000 μm or less. In this example, the cell culture part consists of only the cell adhesion part.

[0117] <2.4.2.Process 21> Step 21 is a step of seeding pluripotent stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface. Here, the preferred embodiments of the cell culture substrate and the pluripotent stem cells are as described above.

[0118] The cell culture substrate is preferably precoated with a precoat agent for the purpose of promoting adhesion of pluripotent stem cells to cell adhesion sites. The precoat agent is a component that is applied to the cell culture substrate in advance to promote adhesion of cells to cell adhesion sites. Examples of precoat agents include extracellular matrices (collagen, fibronectin, proteoglycan, laminin, vitronectin), gelatin, lysine, peptides, gel-like matrices containing these, serum, etc. By carrying out the precoat treatment, adhesion of pluripotent stem cells with low adhesiveness to cell adhesion sites can be promoted, and cell adhesion culture and differentiation induction can be effectively carried out.

[0119] Pluripotent stem cells can be cultured under conditions that maintain the undifferentiated state before seeding. The medium used for the culture is not particularly limited as long as it does not induce differentiation of stem cells, and examples of the medium include a medium containing leukemia inhibitory factor, which is known to have the property of maintaining the undifferentiated state of mouse embryonic stem cells and mouse induced pluripotent stem cells, and a medium containing basic FGF, which is known to have the property of maintaining the undifferentiated state of human iPS cells.

[0120] Step 21 and step 22 described later may be performed in a medium capable of growing pluripotent stem cells and inducing differentiation, and the medium is not particularly limited. As the basal medium used in step 21 and step 22 described later, Knockout DMEM (KDMEM) medium, DMEM medium, EMEM medium, MEM medium, DMEM-F12 medium, BME medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. may be used. Various growth factors, serum or serum substitutes, antibiotics, amino acids, etc. may be added to the medium. For example, 0.1 to 2% pyruvic acid, 0.1 to 2% non-essential amino acids, 0.1 to 2% penicillin / streptomycin, 0.1 to 1% glutamine, 0.1 to 2% β-mercaptoethanol, and 1 mM to 20 mM ROCK inhibitor (e.g., Y27632) may be added. Specific examples of the medium used in step 21 and step 22 described below include XF32 medium described in the Examples, and commercially available media such as StemFit (Ajinomoto Co., Ltd.), StemFlex (Life Technologies, Inc.), ReproFF (ReproCell, Inc.), etc. The medium may be a serum-containing medium or a serum-free medium containing a serum alternative component, but is preferably a serum-free medium containing a serum alternative component. The medium is preferably free of xenogeneic components.

[0121] The seeding density of pluripotent stem cells on the cell culture substrate may be according to a conventional method and is not particularly limited. In the present disclosure, the stem cells are seeded at a density of 3 × 10 4 cells / cm 2 It is preferable to sow at a density of 3 × 10 or more. 4 ~5×10 7 cells / cm 2 It is more preferable to sow at a density of 3 × 10 4 ~2.5×10 7 cells / cm 2 It is even more preferable to sow the seeds at a density of 100-200 μg / ml.

[0122] <2.4.3.Process 22> Step 22 is a step of culturing the pluripotent stem cells seeded in step 21 on the cell culture substrate to induce differentiation into a cell structure (precursor cell structure) that contains an inner lumen and includes endodermal cells, ectodermal cells, and mesodermal cells.

[0123] The medium that can be used in step 22 is as described above.

[0124] The culture temperature in step 22 is usually 37° C. It is preferable to use a CO2 cell culture device or the like to culture in an atmosphere with a CO2 concentration of about 5%.

[0125] The culture period in step 22 varies depending on the initial seeding density of the cells and the shape and size of the cell adhesion portion, but is preferably about 2 to 4 weeks. When stem cells are cultured on the cell culture substrate and induced to differentiate, 2 to 4 weeks after seeding, the sac-shaped precursor cell structures that have been induced to differentiate, contain a lumen, and contain endodermal cells, ectodermal cells, and mesodermal cells naturally float and detach, and can be collected. In addition, the detachment of the precursor cell structures from the cell culture substrate may be promoted using various techniques, such as mild enzyme treatment (e.g., Accutase or TrypLE) that does not destroy the sac-shaped precursor cell structures, EDTA treatment, spraying with a liquid such as a medium, and physical detachment with a scraper. The precursor cell structures may be used in step 11 while attached to a cell culture substrate.

[0126] The precursor cell structure obtained in step 22 preferably has a long axis length of 5 mm or more, more preferably 10 mm or more, when used in step 11. There is no particular upper limit to the long axis length of the precursor cell structure, but it is usually 50 mm or less.

[0127] Hereinafter, the present disclosure will be described with reference to specific experimental results, but the scope of the present disclosure is not limited to the scope of the experimental results. EXAMPLES

[0128] 1. Preparation of Cell Culture Substrate (First stage reaction) 58.5g of toluene and 20.25g of epoxy silane TSL8350 (manufactured by Momentive Performance Materials) were mixed, and a catalytic amount of triethylamine was added while stirring the mixture, and the mixture was further stirred at room temperature for several minutes. A 5-inch square glass substrate that had been UV-cleaned in advance was immersed in the above epoxy silane solution and left at room temperature for 20 hours to perform a surface treatment. The surface-treated glass substrate was then washed with ethanol, then washed with water, and dried. The average water contact angle of the substrate surface after drying was 56°. In this way, an epoxy silane-treated substrate was obtained.

[0129] (Second stage reaction) While stirring 45g of polyethylene glycol (molecular weight 400), a catalytic amount of concentrated sulfuric acid was slowly added, and the mixture was further stirred at room temperature for several minutes. The epoxysilane-treated substrate was immersed in the polyethylene glycol and reacted at 120°C for 30 minutes. After the reaction, the substrate was thoroughly washed with water and then dried. This resulted in the production of a glass substrate on which a hydrophilic thin film was formed.

[0130] (Patterning process) The photomask used was a 5-inch size photomask with a pattern in which multiple circular openings of the same size were formed. The photomask had a pattern in which 1.5 mm circular openings were formed, and the space between the openings, i.e., the shortest distance between the openings, was all 0.35 mm. The photomask was gently placed on the film-forming surface of the glass substrate on which the hydrophilic thin film had been formed, and a xenon excimer lamp (172 nm, 10 mW / cm2) was used to illuminate the back side of the mask. 2 The substrate was irradiated with vacuum ultraviolet light from a 500 nm UV light source for 1 minute. This oxidized the areas of the hydrophilic thin film surface that corresponded to the openings of the photomask. The substrate was cut into 5 cm squares and used for cell culture.

[0131] The shape of the cell adhesion regions in the obtained cell culture substrate was circular, the diameter of the cell adhesion regions was 1.5 mm, the space between the cell adhesion regions, i.e., the shortest distance between the cell adhesion regions, was 0.35 mm in all cases, and the percentage of the cell adhesion regions in the cell culture substrate was 51.8%.

[0132] 2. Culturing Intestinal Organoids 2.1. Cell lines Human ES cell (hESC) lines SEES1, SEES2, and SEES3 were established at the Department of Reproductive and Cellular Medicine, National Center for Child Health and Development (Akutsu H, et al. Regen. Ther. 2015;1:18-29). These ES cells were cultured in a commercially available serum-free DMEM (trade name: KnockOut TM 20% KnockOut in D-MEM (Thermo Fisher Scientific) TM The cells were maintained on γ-irradiated mouse embryonic fibroblast (MEF) feeder layers in medium supplemented with serum replacement (KnockOut Serum Replacement) (Life Technologies), 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-Strep), and 8 ng / ml recombinant human bFGF (all purchased from Life Technologies). Medium was changed every 2 days. Cells were passaged approximately once a week using enzymatic (dispase; Wako Pure Chemical Industries) or mechanical (EZPassage; Life Technologies) methods.

[0133] Human iPSCs (hiPSCs) were established at the Department 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 human fetal lung fibroblast MRC5 cell line using retroviral vectors (Makino H, et al. Exp Cell Res. 2009; 315(16):2727-2740; Nishino K, et al. PLoS ONE. 2010; 5(9):e13017; Toyoda M, et al. Genes Cells. 2011; 16(1):1-11). These hiPSCs were maintained on γ-irradiated MEF feeder layers in iPSellon medium (Cardio Incorporated) supplemented with 10 ng / ml bFGF.

[0134] 2.2. Formation of human intestinal organoids In previous studies, the inventors have established xeno-free (XF) conditions for establishing and growing human ES cells (Akutsu H, et al. Regen. Ther. 2015;1:18-29). TM D-MEM, 15% KnockOut TM The cells were stably maintained in XF hESC medium containing Knockout Serum Replacement 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 Systems), 200 ng / ml recombinant human IGF-1 (LONG R3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Life Technologies). The following three types of differentiation-inducing media were prepared. Note that "%" in the medium composition refers to volume % unless otherwise specified.

[0135] Differentiation-inducing medium 1 (sometimes referred to as "XF-KSR(-) medium" herein): 80%Knockout TM D-MEM, 20% KnockOut TM Knockout Serum Replacement (XF-KSR; Life Technologies) medium containing 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 0.055 mM β-mercaptoethanol, and 10 μM Y-27632.

[0136] Differentiation-inducing medium 2 (sometimes referred to herein as "XF hESC medium"): 85%Knockout TM D-MEM, 15% KnockOut TM The medium contained serum replacement (Knockout Serum Replacement) 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 Systems), 200 ng / ml recombinant human IGF-1 (LONG R3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Life Technologies).

[0137] Differentiation-inducing medium 3 (sometimes referred to as "XF-KSR medium" herein): 80%Knockout TM D-MEM, 20% KnockOut TM Medium containing serum replacement (Knockout Serum Replacement) XF CTS (XF-KSR; Life Technologies), 1 mM pyruvate, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, and 0.055 mM β-mercaptoethanol.

[0138] To generate intestinal organoids, undifferentiated hESCs or hiPSCs were dispersed using dispase and seeded on the cell culture substrate prepared by the above procedure, which was placed in a 90 mm culture dish and coated with 0.1% human recombinant type I collagen peptide (RCP) (Fujifilm). Other materials such as vitronectin (Life Technologies) may also be used in this protocol. The matrix-coated culture dish was pre-incubated at 37°C for 1 hour, the coating solution was removed, and the substrate was washed three times with PBS. 4 × 10 cells were seeded in 3 ml of medium. 6 The cells were seeded on the substrate and held for 10 minutes. After that, the medium was removed by aspiration, and 10 ml of fresh differentiation-inducing medium 1 was gently added. As described above, differentiation-inducing medium 1 contained the Rho-associated protein kinase inhibitor Y-27632 (Wako Pure Chemical Industries) and did not contain growth factors. hESCs or hiPSCs were cultured in differentiation-inducing medium 1 containing Y-27632 and not containing growth factors for one day, and then cultured in differentiation-inducing medium 3 containing no growth factors and Y-27632, and after 3 days, the differentiation-inducing medium 3 was replaced with differentiation-inducing medium 2 containing growth factors. Thereafter, the cells were cultured in differentiation-inducing medium 2 while appropriately replacing the medium. The differentiation-inducing medium 2 was replaced gently every 3 to 4 days. After 40 to 60 days, multiple organoids resembling the intestine were collected and cultured together in the differentiation-inducing medium 2 in a 60-mm Ultralow Adhesion plate (NOF Corporation). In another experiment, undifferentiated hESCs or hiPSCs were seeded on the substrate in the same manner and cultured in differentiation-inducing medium 1 until day 4, and the same operations were performed except that the medium was changed to differentiation-inducing medium 2 on day 4. Unless otherwise specified, the culture was performed in a stationary state in an incubator at 37°C with a CO2 concentration of 5%.

[0139] 2.3.Induction of differentiation into monocytes Undifferentiated hESCs or hiPSCs were dispersed using dispase and then immersed in StemFlex TM1x10 cells were suspended in medium (Gibco) supplemented with 1 mM rock inhibitor (Y27632; Calbiochem), 50 ng / mL VEGF (Peprotech EC Ltd.), 20 ng / mL SCF (Miltenyi Biotec Ltd.), and 50 ng / mL BMP-4 (Peprotech EC Ltd.). 5 The cells were dispensed at 1 / well into a low protein binding 96-well plate (Sumitomo Bakelite) and cultured for 4 days to produce embryoid bodies (EBs).

[0140] Next, the prepared EBs were TM Monocytes were obtained by culturing for 2 to 3 weeks in a medium containing 15 (Lonza) supplemented with 100 ng / mL M-CSF (Invitrogen), 25 ng / mL IL-3 (R&D), 2 mM glutamax (Invitrogen), 100 U / mL penicillin and 100 mg / mL streptomycin (Invitrogen), and 0.055 mM β-mercaptoethanol (Invitrogen).

[0141] 2.4. Injection of monocytes into the lumen of intestinal organoids Using differentiation-inducing medium 2 supplemented with 100 ng / mL M-CSF (Invitrogen), intestinal organoids prepared in 2.2 that were more than 60 days old after seeding were inoculated with 5x10 cells using a syringe (080401, 1 ml, Nipro) and an injection needle (minimally invasive microinjection needle, 453106A, Unisys). 6 Monocytes were microinjected at 15-80μl per ml depending on the size and volume of the intestinal organoids. Macrophage-resident intestinal organoids were produced by culturing in the same medium for 2-3 weeks.

[0142] In addition, intestinal organoids containing green fluorescent protein (GFP)-expressing macrophages were prepared using the same procedure as above, except that monocytes expressing GFP were injected as monocytes.

[0143] As a comparative experiment, intestinal organoids that had not been injected with monocytes were co-cultured with monocytes. One intestinal organoid that had been seeded for more than 60 days, prepared in 2.2, and 2.0x10 4 Monocytes at 10 cells / ml were cultured for 2 to 3 weeks.

[0144] 2.5. Quantitative RT-PCR analysis RNA was isolated from intestinal organoids using RNeasy Mini Kit (Qiagen), and 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 manufacturer's instructions. Quantitative RT-PCR was performed using SYBR Green PCR Master mix and QuantStudio 12K Flex Real-Time PCR System (Life Technologies) (n=3). Primer sequences are shown in the table below.

[0145] [Table 1]

[0146] After amplification, dissociation curves were obtained to confirm that each PCR product was amplified. GAPDH was used as a standard housekeeping gene. Using QuantStudio 12K Flex software v1.0, the mRNA expression level of the target gene was normalized to the mRNA expression level of GAPDH, and the relative expression level of the target gene mRNA was quantified. cDNA from small intestine tissue of a healthy adult (R1234226-50, BioChain Institute) was used as a positive control.

[0147] 2.6. Immunocytochemical analysis The intestinal organoids were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries) for 5 minutes at 4°C, permeabilized with 0.2% Triton X-100 for 2 minutes at room temperature, and further blocked with 5% normal serum in PBS for each antibody as necessary. The intestinal organoids after the above treatment were incubated overnight at 4°C with primary antibodies against the following antigens: The antigens were as follows: IBA1 (ab5076, 1:200, abcam); HAM56 (14-6548-93, 1:1000, Invitrogen); TGF-β (ab170874, 1:50, abcam); IL-8 (MAB208-100, R&D Systems); OLFM4 (NBP2-24535, 12 μg / ml, Novous Biologicals); GP2 (HPA016668, 1:1000, Sigma-Aldrich); Villin (sc-7672, 1:50, Santa Cruz Biotechnology); ZO-1 (40-2200, 1:100, Invitrogen); E-cadherin (610181, 1:50, BD Pharmingen; CDX2 (ab76541, 1:100), and PGP9.5 (ab8189, 1:10) (from Abcam); DEFA6 (HPA019462, 1:500) and SMA (A2547, 1:400) (from Sigma-Aldrich); and MUC2 (sc-7314, 1:50, Santa Cruz Biotechnology).

[0148] As secondary antibodies, Alexa 488- or Alexa 546-labeled anti-mouse, anti-rabbit, or anti-goat secondary antibodies (BD Biosciences) were used.

[0149] Cell nuclei were counterstained with DAPI.

[0150] Fluorescence was analyzed using an LSM 510 Meta Laser Scanning Confocal Microscope (Carl Zeiss Microscopy).

[0151] 2.7. Histochemical analysis Organoids were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. Every other section was mounted on a slide, stained with H&E, and analyzed. For Alcian Blue staining, the sections were incubated in 1% Alcian Blue in 3% acetic acid solution at pH 2.5 for 20 min, then incubated in 0.1% Nuclear Fast Red for 2 min, dehydrated in ethanol, and cleared with xylene.

[0152] Histochemical analysis of organoids was performed using sections stained with antibodies against IBA1 (ab5076, 1:200, Abcam), HAM56 (14-6548-93, 1:1000, Invitrogen), TGF-β (ab170874, 1:100, Abcam), CD14 (ab181470, 1:200, Abcam), CD163 (NB110-59935, 1:25, Novous Biologicals), and MHC II (ab55152, 1:200, Abcam). Polyclonal rabbit anti-mouse immunoglobulin-HRP (1:100; Dako) was used as secondary antibody.

[0153] 2.8. Electron Microscopy Electron microscopy of intestinal organoid samples was performed according to standard protocols (Tokai Electron Microscopy). In other words, intestinal 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, mounted on copper grids, and observed with a JEM-1200EX TEM (Jeol Ltd.) equipped with a Veleta CCD camera (Olympus).

[0154] 2.9. Isolation of macrophages using a cell sorter Wash with DPBS (1X) (Gibco) and place about 5 GFP-expressing macrophage-resident intestinal organoids prepared in 2.4 in a 1.5 ml tube, add 1 ml of Accumax, and then fragment as finely as possible using tweezers and ophthalmic scissors. After repeating the pipetting process 4 times every 5 minutes, the suspension is passed through a cell strainer and placed again in a 1.5 ml tube, and centrifuged at 3000g for 5 minutes using a TOMY centrifuge (TOMY). Remove the supernatant and resuspend in 1 ml of 2% FBS / PBS. Remove the supernatant and resuspend in 1 ml of 2% FBS / PBS. Use a cell sorter (SONY) to sort cells with high GFP expression.

[0155] 2.10. Phagocytosis assay To measure antigen uptake in intestinal organoids, we used pHrodo, which emits red fluorescence within acidic phagosomes. TM Red E. coli BioParticles (登録商標) Conjugate (Molecular Probes) was used. One GFP-expressing macrophage-resident intestinal organoid prepared in 2.4 was washed three times with DPBS (1X) (gibco) and then incubated with 500 μg / ml of the conjugate. After incubation at 37.0 °C for 1.5 hours, it was measured with a fluorescent microscope (BZ-X700, KEYENCE).

[0156] 2.11. Analysis of inflammatory cytokines Intestinal organoids (Minigut) and macrophage-resident intestinal organoids (MC-Minigut) were cultured for 72 hours in 1 ml of differentiation-inducing medium 2 and differentiation-inducing medium 2 plus M-CSF (Invitrogen), respectively, and the supernatant and contents were collected. The collected samples were subjected to a cytokine assay using MILLIPLEX MAP Human Cytokine / Chemokine Magnetic Bead Panel (Millipore) to measure inflammatory cytokines.

[0157] 2.12. Analysis of anti-inflammatory cytokines Intestinal organoids (Minigut) and macrophage-resident intestinal organoids (MC-Minigut) were cultured for 72 hours in 1 ml of differentiation-inducing medium 2 and differentiation-inducing medium 2 plus M-CSF (Invitrogen), respectively, and the supernatant and contents were collected. The collected samples were subjected to a cytokine assay using MILLIPLEX MAP Human Cytokine / Chemokine Magnetic Bead Panel (Millipore) to measure anti-inflammatory cytokines.

[0158] 3.Results 3.1. Sac-like cell structure before monocyte injection The sac-like cell structure (intestinal organoid) obtained in 2.2 before injection of monocytes contained the same cell types and tissues as those confirmed in the examples of Patent Document 3 and Non-Patent Document 1, and had peristaltic motility. The sac-like cell structure had a small intestinal epithelial cell layer including a villus layer on the outer surface side, and a multi-layer structure was confirmed to have a cell layer including smooth muscle cells, Cajal cells, and nerve cells on the inner surface side. The sac-like cell structure was spherical with a diameter of 5 mm or more, and had a structure containing a lumen. A schematic diagram of the obtained sac-like cell structure is shown in FIG. 1. As shown in the figure, the sac-like cell structure 10 is a sac-like object formed so that the wall part 11 of the multi-layer structure contains a lumen 12. The wall part 11 has a small intestinal epithelial cell layer including a villus layer on the outer surface 13 side, and a cell layer including smooth muscle cells, Cajal cells, and nerve cells on the inner surface 14 side.

[0159] 3.2. Expression of TGF-β and IL-8 The upper panel of Figure 2A, "Minigut," shows the results of immunofluorescent staining for TGF-β and IL-8 and DAPI staining of cell nuclei of iPS cell-derived intestinal organoids (sac-like cell structures) before injection of monocytes prepared in 2.2.

[0160] The lower panel of FIG. 2A, “Small intestine,” shows the results of immunofluorescent staining of TGF-β and IL-8 and DAPI staining of cell nuclei in small intestine tissue from a healthy adult (R1234226-50, BioChain Institute) (hereinafter referred to as “human small intestine tissue”). The left side of Figure 2B shows the results of quantitative RT-PCR analysis of TGF-β in human small intestine tissue (Small intestine), iPS cell-derived pouch-like cell structures (Minigut) before monocyte injection, and macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4. The vertical axis shows the relative expression level, with the expression level of TGF-β in human small intestine tissue set at 1. The right side of Figure 2B shows the results of quantitative RT-PCR analysis of IL-8 in human small intestine tissue (Small intestine), iPS cell-derived pouch-like cell structures (Minigut) before monocyte injection, and macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut). The vertical axis shows the relative expression level, with the expression level of IL-8 in human small intestine tissue set at 1.

[0161] The results shown in Figure 2A and Figure 2B indicate that intestinal organoids have high expression levels of TGF-β and IL-8, regardless of the presence or absence of macrophages. In intestinal tissue in vivo, TGF-β and IL-8 are thought to stably maintain immune cells in the basement membrane. It is suggested that intestinal organoids provide an environment similar to that in vivo.

[0162] 3.3. Identification of macrophages The upper left of Figure 3A shows the results of histochemical analysis of the macrophage marker IBA1 in the macrophage-internal iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4. The arrowheads indicate the expression of IBA1. The scale bar indicates 100 μm.

[0163] The lower left of Figure 3A shows the results of histochemical analysis of IBA1 in human small intestine tissue. IBA1 expression was confirmed throughout the tissue. The scale bar indicates 100 μm.

[0164] The upper right of Figure 3A shows the results of histochemical analysis of another macrophage marker, HAM56, in the macrophage-internalized iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4. The arrowheads indicate the expression of HAM56. The scale bar indicates 100 μm.

[0165] The lower right of Figure 3A shows the results of histochemical analysis of HAM56 in human small intestine tissue. HAM56 expression was confirmed throughout the tissue. The scale bar indicates 100 μm.

[0166] The top row of Figure 3B shows the results of immunofluorescence staining for GFP (first from the left), IBA1 (second from the left), and DAPI (third from the left) in the same area of ​​the GFP-expressing macrophage-internal iPS cell-derived intestinal organoid (MC(GFP+)-Minigut) prepared in 2.4. The right end shows the results of overlaying these. The arrowheads indicate the areas where GFP and DIBA1 were co-stained. The scale bar indicates 200 μm.

[0167] The bottom row of Figure 3B shows the results of immunofluorescence staining for GFP (first from the left), IBA1 (second from the left), and DAPI (third from the left) at the same location of the GFP-expressing macrophage-resident iPS cell-derived intestinal organoid (MC(GFP+)-Minigut) prepared in 2.4. The right end shows the overlapped results. The scale bar indicates 50 μm.

[0168] FIG. 3B shows that GFP-expressing macrophages colocalized with the macrophage marker IBA1.

[0169] Figure 3C shows the results of transmission electron microscopy of macrophages present on the surface surrounding the lumen of the GFP-expressing macrophage-containing iPS cell-derived intestinal organoids prepared in 2.4. The scale bar indicates 5 μm. Macrophages were confirmed to have attached to the wall.

[0170] The results in Figures 3A-C show that macrophage-resident intestinal organoids contain engrafted rather than floating macrophages and express the macrophage-specific markers IBA1 and HAM56.

[0171] 3.4. Phagocytosis by macrophages We confirmed that macrophage-resident intestinal organoids have phagocytosis characteristic of macrophages.

[0172] The upper part of Figure 4 shows the results of fluorescence microscopy of iPS cell-derived intestinal organoids containing GFP-expressing macrophages prepared in 2.4. Green fluorescence indicates GFP-expressing macrophages.

[0173] GFP-expressing macrophages-resident iPS cell-derived intestinal organoids were subjected to phagocytosis assay using pHrodo according to the method described in 2.10. TM Red E. coli BioParticles (登録商標) The conjugate was administered and incubated. When the conjugate was taken up by the phagocytosis of macrophages, it first entered the phagosome in the macrophage. Then, when the phagosome was fused with the lysosome to form the phagolysosome, the conjugate was degraded. The inside of the phagolysosome is an acidic environment. When the conjugate was degraded in the acidic environment, it was degraded by E. coli BioParticles. (登録商標) The conjugate emits red fluorescence. The lower part of Figure 4 shows an observation image of green fluorescence at the left edge, an observation image of red fluorescence at the center, and an image of the two images superimposed on the right edge. The green and red fluorescence were consistent, indicating that the GFP-expressing macrophages in the intestinal organoids phagocytosed the conjugate and degraded it in the phagolysosomes in an acidic environment.

[0174] In other words, it was confirmed that macrophage-resident intestinal organoids can take in external substances through the surface small intestinal epithelial cell layer, and can decompose the taken-up substances through phagocytosis by macrophages present on the luminal side.

[0175] 3.5. Isolation of macrophages using a cell sorter From the intestinal organoids containing GFP-expressing macrophages prepared in 2.4., cells highly expressing GFP were isolated using a cell sorter according to the procedure described in 2.9.

[0176] As outlined in Figure 5, the GFP-expressing macrophage-resident intestinal organoids prepared in 2.4 were dissociated into single cells, and fluorescence-activated cell sorting (FACS) was performed to separate GFP-positive intestinal organoid-resident macrophages (pGMACs).

[0177] 3.6. Detection of intestinal markers The upper left image of Figure 6 shows a superposition of immunofluorescent staining of CDX2 (intestinal cell marker) and villin (small intestinal epithelial cell marker) and DAPI staining of cell nuclei of the macrophage-resident iPS cell-derived intestinal organoid (MC-Minigut) prepared in 2.4. The scale bar indicates 100 μm.

[0178] The lower left image of Figure 6 is a superimposed image of immunofluorescence staining of ZO-1 (small intestinal epithelial cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei of the macrophage-resident intestinal organoid. The scale bar indicates 100 μm.

[0179] The upper right corner of Figure 6 shows a superimposition of immunofluorescence staining of CDX2 (intestinal cell marker) and MUC2 (goblet cell marker) and DAPI staining of cell nuclei of the macrophage-resident intestinal organoids. The scale bar indicates 100 μm.

[0180] The lower right image of Figure 6 shows a superposition of immunofluorescence staining of DEFA6 (Paneth cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei of the macrophage-resident intestinal organoid. The scale bar indicates 100 μm.

[0181] The results shown in FIG. 6 demonstrate that macrophage-resident intestinal organoids have the organization of small intestinal epithelium.

[0182] 3.7. Detection of gut-related markers The upper left image of Figure 7 shows the superposition of immunofluorescent staining of OLFM4 (intestinal stem cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei of the macrophage-resident iPS cell-derived intestinal organoid (MC-Minigut) prepared in 2.4. The scale bar indicates 100 μm. Green indicates OLFM4-positive cells.

[0183] The upper right image of Figure 7 is a superimposition of immunofluorescent staining of OLFM4 (intestinal stem cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei in human small intestine tissue (small intestine). The scale bar indicates 100 μm. The arrowheads indicate OLFM4-positive cells.

[0184] The lower left image of Figure 7 shows the macrophage-resident intestinal organoids, which are overlaid with immunofluorescence staining of GP2 (M cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei. The scale bar indicates 100 μm. The arrowheads indicate OLFM4-positive cells.

[0185] The lower right image of Figure 7 shows a superimposition of immunofluorescent staining of GP2 (M cell marker) and ECAD (small intestinal epithelial cell marker) and DAPI staining of cell nuclei in human small intestinal tissue. The scale bar indicates 100 μm. The arrowheads indicate OLFM4-positive cells.

[0186] The results shown in FIG. 7 demonstrate that macrophage-resident intestinal organoids contain intestinal stem cells and M cells.

[0187] 3.8. Quantitative analysis of expression of gut-related markers The expression levels of eight intestine-related markers in the macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4, the iPS cell-derived intestinal organoids (Minigut) prepared in 2.2 before injection of monocytes, and human small intestine tissue (Small intestine) were analyzed by quantitative RT-PCR. The expression levels of each marker in human small intestine tissue were expressed as a relative value, with the expression level in human small intestine tissue set at 1.

[0188] The markers whose expression levels were examined were LGR5 (intestinal stem cell marker), OLFM4 (intestinal stem cell marker), ECAD (small intestinal epithelial cell marker), CDX2 (intestinal cell marker), MUC2 (goblet cell marker), DEFA6 (Paneth cell marker), ChgA (enterocrine cell marker), and GP2 (M cell marker).

[0189] The measurement results are shown in Figure 8. The expression of OLFM4 and MUC2 was significantly increased in the macrophage-resident intestinal organoids (MC-Minigut) compared to the intestinal organoids (Minigut) before monocyte injection.

[0190] 3.9. Detection of smooth muscle and nerve markers The upper left image of Fig. 9 shows macrophage-resident iPS cell-derived intestinal organoid (MC-Minigut), the upper center image of iPS cell-derived intestinal organoid (Minigut), and the upper right image of human small intestine tissue (Small Intestine) are superimposed images of immunofluorescent staining of PGP9.5 (enteric nerve plexus marker) and SMA (smooth muscle marker) and DAPI staining of cell nuclei. The scale bar indicates 100 μm.

[0191] The expression levels of SMA (smooth muscle marker), PGP9.5 (enteric plexus marker), and BMP2 (intestinal macrophage marker) in the macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4., the iPS cell-derived intestinal organoids (Minigut) prepared in 2.2. before monocyte injection, and human small intestine tissue (Small intestine) were analyzed by quantitative RT-PCR. The expression levels of each marker in human small intestine tissue were expressed as relative values, with the expression level in human small intestine tissue set at 1. The measurement results for each marker are shown in the lower part of FIG. The above results shown in Figure 9 indicate that macrophage-inhabiting intestinal organoids have developed muscle and nerve tissues, similar to the intestinal organoids before injection of monocytes.

[0192] 3.10. Secretion of inflammatory cytokines For the macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4., and the iPS cell-derived intestinal organoids (Minigut) prepared in 2.2. before injection of monocytes, we analyzed cytokines in the intraluminal fluid (CF) of the intestinal organoids and in the culture supernatant (SF) of the intestinal organoids before the addition of LPS (lipopolysaccharide), which causes inflammation, and 24 hours after the addition of LPS.

[0193] Figure 10A shows the cytokine concentration in CF and SF of intestinal organoids (Minigut) before LPS addition. Figure 10B shows the cytokine concentration in CF and SF of intestinal organoids (Minigut) 24 hours after LPS addition.

[0194] Figure 10C shows the cytokine concentration in CF and SF of macrophage-inhabiting intestinal organoids (MC-Minigut) before LPS addition. Figure 10D shows the cytokine concentration in CF and SF of macrophage-inhabiting intestinal organoids (MC-Minigut) 24 hours after LPS addition.

[0195] The cytokines IL-10, IL-12p40, IL-12p70, IL-15, EGF, IL-1a, and CCL-4 were present at higher concentrations in the lumen (CF) of macrophage-resident intestinal organoids compared to intestinal organoids not injected with monocytes.

[0196] On the other hand, the culture supernatant (SF) of macrophage-resident intestinal organoids contained higher concentrations of the cytokines IL-1ra, IL-7, IL-12p70, and VEGF compared to intestinal organoids that had not been injected with monocytes.

[0197] 3.11. Secretion of anti-inflammatory cytokines For the macrophage-resident iPS cell-derived intestinal organoids (MC-MGUT) prepared in 2.4. and the iPS cell-derived intestinal organoids (MGUT) prepared in 2.2. before injection of monocytes, the concentration of the anti-inflammatory cytokine IL-10 in the fluid (CF) of the lumen of the intestinal organoids was analyzed before the addition of LPS (lipopolysaccharide), which causes inflammation, and 24 hours after the addition of LPS.

[0198] The results are shown in Figure 11A. Although no significant difference was observed, the IL-10 concentration tended to increase in the lumen fluid (CF) of macrophage-resident intestinal organoids (MC-MGUT) compared to intestinal organoids (MGUT) that were not injected with monocytes.

[0199] In addition, the gene expression levels of IL-10 in the macrophage-resident iPS cell-derived intestinal organoids (MC-Minigut) prepared in 2.4. and the iPS cell-derived intestinal organoids (Minigut) prepared in 2.2. before injection of monocytes were analyzed by quantitative RT-PCR before and 24 hours after LPS addition, as well as in human small intestinal tissue. The expression levels were expressed as relative values ​​with the expression level in human small intestinal tissue set to 1.

[0200] The results are shown in Figure 11B. In macrophage-resident intestinal organoids (MC-Minigut), the gene expression level of the anti-inflammatory cytokine IL-10 tended to increase compared to intestinal organoids (Minigut) that were not injected with monocytes.

[0201] 3.12. Observation of intestinal organoids co-cultured with monocytes The results of observing intestinal organoids co-cultured with monocytes without injecting monocytes in 2.4 are shown in Figures 12A, 12B, and 12C.

[0202] Figure 12A shows the results of histochemical analysis of CD14 (LPS receptor) in iPS cell-derived intestinal organoids co-cultured with monocytes (Mini-Gut(MCs+)), iPS cell-derived intestinal organoids before injection of monocytes prepared in 2.2 (Mini-Gut(MCs-)), and human small intestine tissue (Small intestine). No substantial difference was observed between Mini-Gut(MCs+) and Mini-Gut(MCs-).

[0203] Figure 12B shows the results of histochemical analysis of CD163 (scavenger receptor) in Mini-Gut (MCs+), Mini-Gut (MCs-), and small intestine. In Mini-Gut (MCs+), the presence of CD163 was observed, similar to that in small intestine.

[0204] Figure 12C shows the results of histochemical analysis of MHC II (antigen-presenting molecule) in Mini-Gut(MCs+), Mini-Gut(MCs-), and small intestine. No substantial difference was observed between Mini-Gut(MCs+) and Mini-Gut(MCs-).

[0205] These results showed that when intestinal organoids were co-cultured with monocytes, the expression of CD163 was increased in the intestinal organoids, but no significant difference was observed, indicating that injection of monocytes is effective in inducing macrophages in intestinal organoids.

Claims

1. A pouch-shaped intestinal organoid that contains a lumen, contains endodermal cells, ectodermal cells, and mesodermal cells, and contains macrophages, a small intestinal epithelial cell layer containing the endodermal cells on the outer surface side; a cell layer including the ectodermal cells and the mesodermal cells on the inner surface side; wherein the ectodermal cells and the mesodermal cells include smooth muscle cells, interstitial cells of Cajal and nerve cells; the macrophages are engrafted on the inner surface; and One or more selected from IBA1 and HAM56 are expressed; The pouch-shaped intestinal organoid.

2. The pouched intestinal organoid of claim 1, wherein the macrophages have phagocytosis.

3. The pouched intestinal organoid of claim 1 or 2, further comprising monocytes.

4. The pouch-shaped intestinal organoid according to any one of claims 1 to 3, which is derived from embryonic stem cells and / or artificial pluripotent stem cells.

5. (1) The endodermal cells express one or more genes selected from the group consisting of AFP, SERPINA1, SST, ISL1, IPF1, IAPP, EOMES, HGF, ALBUMIN, PAX4, and TAT, and / or the endodermal cells are positive for the expression of one or more markers selected from the group consisting of CDX2, MUC2, CGA, DEFA6, GP2, ECAD (E-cadherin), ZO-1, Na+ / K+-ATPase, villin, FOXA2, SOX17, CXCR4, GATA4, GATA6, and T (Brachyury); (2) The ectodermal cells express one or more genes selected from the group consisting of β-TUBLIN, NESTIN, GALANIN, GCM1, GFAP, NEUROD1, OLIG2, SYNAPT PHYSIN, DESMIN, and TH, and / or the ectodermal cells are positive for the expression of one or more markers selected from the group consisting of PGP9.5 and SOX1; and (3) The mesodermal cells express one or more genes selected from the group consisting of FLK-1, COL2A1, FLT1, HBZ, MYF5, MYOD1, RUNX2, and PECAM1, and / or the mesodermal cells are positive for the expression of one or more markers selected from the group consisting of α-smooth muscle actin (SMA), a combination of CD34 and CKIT, GATA4, GATA6, and T (Brachyury). The pouch-shaped intestinal organoid according to any one of claims 1 to 4.

6. A method for producing a pouch-shaped intestinal organoid that contains a lumen, contains endodermal cells, ectodermal cells, and mesodermal cells, and contains macrophages, comprising: A pouch-shaped intestinal organoid containing an inner lumen and containing endodermal cells, ectodermal cells, and mesodermal cells, a small intestinal epithelial cell layer containing the endodermal cells on the outer surface side; a cell layer including the ectodermal cells and the mesodermal cells on the inner surface side; wherein the ectodermal cells and the mesodermal cells include smooth muscle cells, interstitial cells of Cajal and nerve cells; Step 11, injecting monocytes into the pouched intestinal organoids; and Step 12: maturing monocytes into macrophages in the pouch-shaped intestinal organoid into which the monocytes have been injected in step 11 The method includes:

7. 21. Seeding pluripotent stem cells onto a cell culture substrate having a pattern of cell adhesion sites on its surface; The pluripotent stem cells are cultured to produce a pouch-shaped intestinal organoid that contains a lumen and contains endodermal cells, ectodermal cells, and mesodermal cells, a small intestinal epithelial cell layer containing the endodermal cells on the outer surface side; a cell layer including the ectodermal cells and the mesodermal cells on the inner surface side; wherein the ectodermal cells and the mesodermal cells include smooth muscle cells, interstitial cells of Cajal and nerve cells; Step 22 of inducing differentiation into the pouch-shaped intestinal organoid; The method according to claim 6, further comprising producing the pouch-shaped intestinal organoids used in step 11 by a method comprising:

8. (1) The endodermal cells express one or more genes selected from the group consisting of AFP, SERPINA1, SST, ISL1, IPF1, IAPP, EOMES, HGF, ALBUMIN, PAX4, and TAT, and / or the endodermal cells are positive for the expression of one or more markers selected from the group consisting of CDX2, MUC2, CGA, DEFA6, GP2, ECAD (E-cadherin), ZO-1, Na+ / K+-ATPase, villin, FOXA2, SOX17, CXCR4, GATA4, GATA6, and T (Brachyury); (2) The ectodermal cells express one or more genes selected from the group consisting of β-TUBLIN, NESTIN, GALANIN, GCM1, GFAP, NEUROD1, OLIG2, SYNAPT PHYSIN, DESMIN, and TH, and / or the ectodermal cells are positive for the expression of one or more markers selected from the group consisting of PGP9.5 and SOX1; and (3) The mesodermal cells express one or more genes selected from the group consisting of FLK-1, COL2A1, FLT1, HBZ, MYF5, MYOD1, RUNX2, and PECAM1, and / or the mesodermal cells are positive for the expression of one or more markers selected from the group consisting of α-smooth muscle actin (SMA), a combination of CD34 and CKIT, GATA4, GATA6, and T (Brachyury). The method according to claim 6 or 7.

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