SHEET-SHAPED CELL STRUCTURE, METHOD FOR PRODUCING SHEET-SHAPED CELL STRUCTURE, AND METHOD FOR PRODUCING SHEET-SHAPED CELL STRUCTURE

Abstract

To provide a sheet-like cell structure having a cell layer structure similar to an intestinal tract wall of a small intestine which is suitable for transplantation, and can be fabricated under a heterologous component absence condition, a processing method of a sheet-like cell structure, and a method for manufacturing a sheet-like cell structure.SOLUTION: A sheet-like cell structure 10 having an area of 20 mm2 or more, which includes an entodermal cell, an ectodermal cell, and a mesodermal cell, in which the structure includes the entodermal cell on a first surface 11 side, and includes the ectodermal cell and / or the mesodermal cell on a second surface 12 side. A method for manufacturing a sheet-like cell structure having an area of 20 mm2 or more, which includes an entodermal cell, an ectodermal cell, and a mesodermal cell. The method includes cutting out the sheet-like cell structure from a bag-shaped cell structure 20 including an entodermal cell, an ectodermal cell, and a mesodermal cell, which includes a lumen, and has the length in a longitudinal axis direction of 5 mm or more.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] This specification discloses a sheet-shaped cellular structure, a method for processing a sheet-shaped cellular structure, and a method for producing a sheet-shaped cellular structure. [Background technology]

[0002] Cell engineering has made it possible to form functional tissues that are three-dimensional and made up of a variety of cells. Cell sheets are a representative example of artificially produced tissues, and are actively used in the field of regenerative medicine, where they are transplanted into patients to promote healing and as tissue substitutes. 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). 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.

[0003] Patent Document 2 describes a method for producing a hollow organ permeated with blood vessels, comprising the steps of: a) transplanting human intestinal organoids (HIOs) into an immunodeficient organism, the HIOs being derived from human embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs), and during the transplantation step, the HIOs form mature intestinal tissue. This method requires in vivo maturation of the immunodeficient organism.

[0004] Patent Document 3 describes a method for constructing an intestinal-like cell mass equipped with an intramural nervous system, comprising the steps of (a) recovering undifferentiated embryonic stem cells, and inducing embryoid bodies by hanging drop culture of the recovered undifferentiated embryonic stem cells in a medium containing BDNF, and (b) attaching the embryoid bodies induced in step (a) onto a culture dish and further culturing them. The intestinal-like cell mass obtained by this method has peristaltic motility. The intestinal-like cell mass obtained by this method is small in size, on the μm scale.

[0005] Patent Document 4 describes a biocompatible tissue implant for repairing tissue injury or tissue defect. The biocompatible tissue implant includes a biological tissue slice having a shape suitable for transplantation into a tissue site, the tissue slice includes an effective amount of living cells, and the tissue slice has dimensions such that the living cells can migrate and proliferate from the tissue slice and integrate with the tissue at the tissue injury or tissue defect. Patent Document 4 describes that the biocompatible tissue implant includes an extracellular matrix material (ECM). However, Patent Document 4 does not describe the formation of the biocompatible tissue implant in a self-organizing manner by cell culture. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2014-236716 A [Patent Document 2] Special Publication No. 2017-532964 [Patent Document 3] JP 2006-239169 A [Patent Document 4] JP 2005-169112 A Summary of the Invention [Problem to be solved by the invention]

[0007] While conventional cell sheets and organoids have made it possible to partially mimic human tissues and organs, it is difficult in many aspects to completely replace human tissues and organs. This is due to the limited size of tissues that can be produced at the current technological level and the incomplete formation of cell layer structures. In particular, small intestinal epithelial cells are required for organoids that mimic the functions of the small intestine, but until recently, it was difficult to even culture small intestinal epithelial cells. For example, the intestine-like cell mass having peristaltic motility ability described in Patent Document 3 is very small in size (μm scale) and is not suitable for transplantation.

[0008] The biocompatible tissue implant described in Patent Document 4 is formed by arranging cells using cell engineering, and is not formed self-organically by cell culture, so the layered structure of the cells differs from the natural layered structure of tissue in the body.

[0009] Furthermore, the hollow organ having mature intestinal tissue and a network of blood vessels described in Patent Document 2 is produced through maturation in the body of a non-human animal, and is therefore not suitable for transplantation into humans.

[0010] The small intestine is a complex organ containing cells derived from three germ layers (endoderm, ectoderm, and mesoderm). The small intestine is 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, and enteric plexus derived from the ectoderm, and performs functions such as secretion, absorption, and peristalsis. Conventionally, no sheet-shaped cell structure suitable for transplantation that contains endodermal cells, ectoderm cells, and mesodermal cells like the small intestine has been provided.

[0011] Thus, the present specification discloses a sheet-shaped cell structure that is suitable for transplantation, has a cell layer structure similar to that of the intestinal wall of the small intestine, and can be prepared in the absence of xenogeneic components. [Means for solving the problem]

[0012] The present disclosure includes the following inventions. (1) A cell that contains endodermal, ectodermal, and mesodermal cells and has an area of ​​20 mm 2 The above sheet-like cell structure. (2) containing endodermal cells on the first surface side; A sheet-shaped cell structure according to (1), comprising ectodermal cells and / or mesodermal cells on the second surface side. (3) A sheet-shaped cell structure described in (1) or (2), comprising a small intestinal epithelial cell layer on the side of the first surface. (4) The sheet-shaped cell structure described in (3), further comprising a submucosa-like layer. (5) The sheet-shaped cell structure according to any one of (1) to (4), which is derived from embryonic stem cells or induced pluripotent stem cells. (6) The sheet-shaped cell structure described in any one of (1) to (5), which does not contain lymphocytes. (7) A sheet-shaped cell structure described in any one of (1) to (6), which has the ability to produce mucin and antimicrobial peptides. (8) A sheet-shaped cell structure described in any one of (1) to (7) above, which has peristaltic motility. (9) A sheet-shaped cell structure described in any one of (1) to (8), which does not contain any heterologous components. (10) placing the sheet-shaped cell structure on a flat surface; A sheet-shaped cell structure described in any of (1) to (9), wherein A / B is 0.003 or more, where A is the height of the lowest point on the surface opposite the flat surface of the sheet-shaped cell structure and B is the height of the highest point. (11) placing the sheet-shaped cell structure on a flat surface; A sheet-shaped cell structure described in any of (1) to (9), wherein when the height of the lowest point on the surface opposite the flat surface of the sheet-shaped cell structure is A and the height of the highest point is B, A / B is 0.40 or less. (12) A method for processing a sheet-shaped cell structure according to any one of (1) to (11), comprising the steps of: Placing the sheet-shaped cell structure in contact with a template surface; processing the sheet-shaped cell structure into a shape that conforms to the template surface; The method includes: (13) The mold surface is a flat surface, The method according to (12), wherein the sheet-shaped cell structure to be processed is the sheet-shaped cell structure according to (10). (14) The method according to (12), wherein the mold surface is a curved and / or uneven surface. (15) A cell comprising endodermal cells, ectodermal cells, and mesodermal cells, the cell having an area of ​​20 mm 2 The method for producing the sheet-shaped cell structure described above comprises the steps of: A method comprising cutting out a sheet-like cell structure from a pouch-like cell structure that contains an inner lumen and has a longitudinal length of 5 mm or more, the pouch-like cell structure comprising endodermal cells, ectodermal cells, and mesodermal cells. (16) The method according to (15), wherein the pouch-like cell structure contains endodermal cells on the outer surface side and ectodermal cells and / or mesodermal cells on the inner surface side. (17) The method according to (15) or (16), wherein the pouch-like cell structure comprises a small intestinal epithelial cell layer on the outer surface side. Effect of the Invention

[0013] The sheet-shaped cell structure according to one or more embodiments of the present disclosure is suitable for transplantation. The sheet-like cellular structure according to one or more embodiments of the present disclosure is easy to process. According to the method according to one or more embodiments of the present disclosure, a sheet-shaped cell structure suitable for transplantation can be produced. [Brief description of the drawings]

[0014] [Figure 1] Fig. 1 is a diagram for explaining a method for producing a sheet-shaped cell structure from a sac-shaped cell structure. Fig. 1A shows a sac-shaped cell structure 20 that contains endodermal cells, ectodermal cells, and mesodermal cells and contains a lumen 22. Fig. 1B shows a sheet-shaped cell structure 10 obtained by cutting out a part of a wall 21 of the sac-shaped cell structure 20. [Diagram 2] Figure 2 shows a photograph of a sheet-like cell structure obtained by cutting out the pouch-like cell structure. The scale bar indicates 300 μm. [Diagram 3] FIG. 3 shows a schematic cross section of a sheet-shaped cell structure 10 having a flat or less distorted shape. [Figure 4] FIG. 4 shows a schematic cross section of a sheet-shaped cell structure 10 with a large distortion. [Diagram 5]Figure 5 is a diagram illustrating a method for processing a distorted sheet-shaped cell structure 10 into a shape that conforms to a flat template surface 50. Figure 5A shows a state in which a distorted sheet-shaped cell structure 10 is placed in contact with a flat template surface 50. Figure 5B shows a state in which the distorted sheet-shaped cell structure 10 has been processed into a shape that conforms to the flat template surface 50 to form a flat sheet-shaped cell structure 10. [Figure 6] Figure 6 is a diagram illustrating a method for processing a flat sheet-like cell structure 10 into a shape that conforms to a distorted template surface 60. Figure 6A shows a flat sheet-like cell structure 10 placed in contact with a convexly distorted template surface 60. Figure 6B shows a distorted sheet-like cell structure 10 obtained by processing the flat sheet-like cell structure 10 into a shape that conforms to a convexly distorted template surface 60. [Figure 7] FIG. 7A shows an image of the sheet-shaped cellular structure contracting within the field of view in a video of the structure repeating peristaltic movements, and FIG. 7B shows an image of the structure expanding within the field of view. [Figure 8] Figure 8 shows a fluorescence microscopic image of a sheet-shaped cell structure cultured in D-MEM medium at 37°C for 5 days. Viable cells are stained fluorescently green with Calcein-AM solution, and dead cells are stained fluorescent red with PI solution. [Figure 9] Figure 9A shows the measurement results of the thickness of a hemispherical sheet-like cell structure. The horizontal axis shows the measurement points on the X axis at a measurement pitch of 0.03 mm, and the vertical axis shows the thickness (unit: mm). Figure 9B shows the measurement results of the thickness of a flat sheet-like cell structure. The horizontal axis shows the measurement points on the X axis at a measurement pitch of 0.01 mm, and the vertical axis shows the thickness (unit: mm). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] <1. Characteristics of the cell layer of the sheet-shaped cell structure> The sheet-shaped cell structure according to the present disclosure contains endodermal cells, ectodermal cells, and mesodermal cells and has an area of ​​20 mm 2 The present invention is characterized in that:

[0016] The area of ​​the sheet-shaped cell structure refers to the apparent area when the sheet-shaped cell structure is viewed in plan. 2 By having an area of ​​30 mm or more, it is possible to cover inflamed areas, wounds, lesions, sutured areas after surgery, etc., and transplantation is easy. 2 More than 50mm, preferably 50mm 2 More than 80mm, preferably 80mm 2 More preferably, 100 mm 2 More preferably, 150 mm 2 The upper limit of the area is not particularly limited, but is usually 2000 mm 2 The following is the result.

[0017] The shape of the sheet-shaped cell structure when viewed in a plan view is not particularly limited, but may be circular, such as a perfect circle or an ellipse, or polygonal, such as a triangle or a rectangle.

[0018] The sheet-shaped cell structure according to the present disclosure is preferably derived from pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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

[0024] Next, the characteristics of the cells in the sheet-shaped cell structure according to 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.

[0025] Endodermal cells that may be included in the sheet-shaped cell structure of the present disclosure include small intestinal epithelial cells. The sheet-shaped cell structure of the present disclosure preferably includes one or more small intestinal epithelial cells selected from intestinal cells, goblet cells, enteroendocrine cells, and Paneth 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 sheet-shaped cell structure of 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. Other examples of markers for intestinal epithelial cells include ECAD, 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.

[0026] 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.

[0027] Ectoderm cells that can be contained in the sheet-shaped cell structure of the present disclosure include cells that constitute the enteric nerve plexus. The presence of ectoderm cells in the sheet-shaped cell structure 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.

[0028] 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.

[0029] Mesodermal cells that may be contained in the sheet-shaped cell structure of the present disclosure include smooth muscle cells and interstitial cells of Cajal. The presence of mesodermal cells in the sheet-shaped cell structure of the present disclosure can be determined based on positive expression of mesodermal cell markers. As mesodermal cell markers, α-smooth muscle actin (SMA), a smooth muscle cell marker, and CD34 and CKIT (in the case of double positivity), interstitial cells of Cajal, 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.

[0030] The sheet-shaped cell structure of the present disclosure further preferably comprises intestinal stem cells. The presence of intestinal stem cells can be determined by using as an indicator a positive reaction to the intestinal stem cell marker LGR5. The sheet-shaped cell structure of the present disclosure preferably further comprises serotonin-positive enteroendocrine cells.

[0031] The sheet-shaped cell structure of the present disclosure preferably further contains transporter-positive cells. Examples of the transporter include intestinal oligopeptide transporter (PEPT1) and ATP-binding cassette (ABC) transporters ABCB1 and ABCG2.

[0032] The sheet-shaped cell structure of the present disclosure preferably 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, a sheet-shaped cell structure having CFTR-positive small intestinal epithelial cells is a cell that exhibits mucus secretion ability (secretion control) similar to that of the small intestine. CFTR-positive small intestinal epithelial cells have the ability to produce mucin and antimicrobial peptides. The sheet-shaped cell structure of the present disclosure preferably does not contain lymphocytes.

[0033] The sheet-shaped cell structure of the present disclosure preferably further contains histamine H1 receptor positive cells.

[0034] The sheet-shaped cell structure of the present disclosure preferably contains endodermal cells on the first surface side and ectodermal cells and / or mesodermal cells on the second surface side. The sheet-shaped cell structure according to this embodiment contains, in particular, small intestinal epithelial cells as endodermal cells on the first surface side. This embodiment is preferable because a substance on the first surface side can be transported to the second surface side via the small intestinal epithelial cells. In addition, in this embodiment, when the small intestinal epithelial cells on the first surface are further transporter-positive, it is even more preferable because transport of a substance via the transporter is possible. In other words, the sheet-shaped cell structure of the present disclosure containing transporter-positive small intestinal epithelial cells has a substance absorption ability similar to that of the intestine.

[0035] More preferably, the sheet-shaped cell structure of the present disclosure has microvilli and crypts formed on the surface containing small intestinal epithelial cells.

[0036] More preferably, the sheet-like cell structure of the present disclosure includes a mucosa and a submucosa-like layer. The mucosa of the small intestine is formed by epithelium and a basement membrane. The epithelium of the small intestine is composed of two types of cells, absorptive enterocytes and secretory cells, which include mucus-producing goblet cells, enteroendocrine cells, and Paneth cells. The submucosa-like layer is a layer of connective tissue connected to the mucosa, and includes interstitial cells of Cajal that are positive for CKIT or S-100 or both. In the submucosa-like layer, submucosal plexuses composed of interstitial cells of Cajal are formed, and functional peristalsis like that of the intestine is generated.

[0037] In the above-described embodiment of the sheet-shaped cell structure of the present disclosure, the ectodermal cells and / or mesodermal cells present on the second surface side preferably form lymphatic endothelium (mesoderm) in the submucosa and smooth muscle (mesoderm) that constitutes the mucosa.

[0038] The sheet-shaped cell structure of the present disclosure preferably has the ability to perform contraction movements similar to peristalsis. Such a function is generated by the development of nerve networks and smooth muscles. Peristalsis is thought to occur when pacemaker cells, Cajal cells, receive signals from nerve cells or hormones and then stimulate smooth muscle cells. In the following description, the ability to perform contraction movements similar to peristalsis is referred to as "peristaltic motility." The sheet-shaped cell structure of 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 sheet-shaped cell structure of the present disclosure having peristaltic motility and exhibiting drug responsiveness similar to that of the intestine can be suitably used for evaluating the effect of a drug on intestinal peristalsis.

[0039] <2. Manufacturing method> The sheet-shaped cell structure of the present disclosure can be produced by cutting out a pouch-shaped cell structure that contains an inner lumen and has a longitudinal length of 5 mm or more, and that contains endodermal cells, ectodermal cells, and mesodermal cells.

[0040] The sac-like cell structure corresponds to an intestinal organoid having a sac-like structure in which a small intestinal epithelial cell layer including a villus layer is arranged outward, as described in JP 2019-000014 A and Uchida et al., JCI Insight Vol. 2 e86492 2017. According to these documents, the sac-like cell structure is formed in a self-organizing manner and has a cell layer structure similar to the intestinal wall of the original small intestine, has a size of several centimeters, and can be produced under conditions in which no xenogeneic components are present. However, since it has a sac-like structure, it is not suitable for transplantation. In addition, since the lumen of the sac-like structure is filled with luminal fluid, tension is generated due to the difference in osmotic pressure between the luminal fluid and the culture solution, and the structure is changed to an unfavorable one. The sheet-shaped cell structure of the present disclosure can solve the above problems of the pouch-shaped cell structure.

[0041] <2.1. Characteristics of the sac-shaped cell structure> In the pouch-shaped cell structure, preferred embodiments of the endodermal cells, ectodermal cells, and mesodermal cells and markers that can be used are as described for the sheet-shaped cell structure of the present disclosure.

[0042] The sac-shaped cell structure preferably contains endodermal cells on the outer surface side, and ectodermal cells and / or mesodermal cells on the lumen side (inside). The sac-shaped cell structure according to this embodiment contains small intestinal epithelial cells, in particular, as endodermal cells on the outer surface side. This embodiment is preferable because substances on the outer surface side can be absorbed into the lumen via the small intestinal epithelial cells. Furthermore, in this embodiment, when the small intestinal epithelial cells on the outer surface side are further transporter positive, absorption of substances via the transporter is possible, which is even more preferable.

[0043] The pouch-like cell structure more preferably has microvilli and crypts developed on its outer surface containing small intestinal epithelial cells.

[0044] More preferably, the pouch-like cell structure has a layer structure similar to that of the intestinal wall of the small intestine, including a mucosa and a submucosa-like layer, the characteristics of which are as described for the sheet-like cell structure of the present disclosure. The sac-like cell structure preferably has the ability to undergo contractile movement similar to peristalsis.

[0045] <2.2. Manufacturing method of the pouch-shaped cell structure> Specific examples of the method for producing the sac-shaped cell structure include: 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 pouch-like cell structure that contains an inner lumen and has a longitudinal length of 5 mm or more and contains endodermal cells, ectodermal cells, and mesodermal cells; The method includes the steps of:

[0046] <2.2.1.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.

[0047] 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.

[0048] 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."

[0049] 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.

[0050] 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.

[0051] 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.

[0052] <2.2.1.1. 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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(%)).

[0057] 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).

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] First, the first embodiment will be described.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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".

[0075] 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."

[0076] 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.

[0077] Next, the second embodiment will be described.

[0078] 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.

[0079] 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.

[0080] 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 treatment and / or decomposition treatment 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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:

[0085] 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.

[0086] 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.

[0087] 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.

[0088] <2.2.1.2. 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.

[0089] <2.2.2.Process 21> Step 21 of the method for producing a cell structure according to the present disclosure 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] <2.2.3.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 pouch-like cell structure containing an inner lumen and having a longitudinal length of 5 mm or more, the pouch-like cell structure containing endodermal cells, ectodermal cells, and mesodermal cells.

[0095] The medium that can be used in step 22 is as described above.

[0096] 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%.

[0097] 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 induced sac-like cell structures, which contain a lumen, are 5 mm or longer in the long axis direction, and contain endodermal cells, ectodermal cells, and mesodermal cells, naturally float and detach, and can be collected. In addition, the detachment of the sac-like 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-like cell structures, EDTA treatment, spraying with a liquid such as a medium, and physical detachment with a scraper.

[0098] The pouch-shaped cell structure obtained in step 22 has a length in the long axis direction of 5 mm or more, more preferably 10 mm or more, when detached from the cell culture substrate. The upper limit of the long axis length of the pouch-shaped cell structure is not particularly limited, but is usually 50 mm or less.

[0099] <2.3. Method for producing sheet-shaped cell structures from pouch-shaped cell structures> The sheet-shaped cell structure of the present disclosure can be produced by cutting it out from the pouch-shaped cell structure. It is generally known that processing of cellular structures is destructive and leads to the loss of functional structures. This is because the extracellular skeleton (ECM) created in vitro is generally very fragile. As a result of processing, the balance between the tension of the intracellular skeleton and the ECM can easily be lost, resulting in a collapsed structure such as aggregation, or proteases can destroy the ECM due to cell death, resulting in the loss of the original functional structure. In fact, cell sheet engineering has attempted to maintain functional structures by layering ECM between cell layers. The sheet-shaped cell structure of the present disclosure was able to survive for a long period of time while maintaining the functional multi-layer structure derived from the pouch structure. Surprisingly, the sheet-shaped cell structure of the present disclosure exhibited peristaltic motility for at least 5 days even when incubated in phosphate buffered saline (PBS), and it is believed that the functional structure was maintained for 5 days in PBS.

[0100] This manufacturing method will be described with reference to FIG. 1A shows a pouch-like cell structure 20 in which a wall 21 containing endodermal cells, ectodermal cells, and mesodermal cells forms a pouch so as to enclose a lumen 22. The wall 21 preferably has a layered structure in which an outer surface 11 and an inner surface 12 can be distinguished, and more preferably contains endodermal cells on the outer surface 11 side, and ectodermal cells and / or mesodermal cells on the inner surface 12 side. More preferred embodiments of the endodermal cells, ectodermal cells, and mesodermal cells are as described above.

[0101] A portion of the wall 21 of the pouch-like cell structure 20 can be cut out to produce the sheet-like cell structure 10 shown in FIG. 1B. The first surface 11 of the sheet-like cell structure 10 is the outer surface 11 of the pouch-like cell structure 20, and the second surface 12 is the inner surface 11 of the pouch-like cell structure 20. FIG. 2 shows a photograph of the sheet-like cell structure obtained by cutting out the pouch-like cell structure.

[0102] Surprisingly, in the examples of the present specification, it was confirmed that a sheet-shaped cell structure 10 obtained by cutting out a portion of the wall 21 of a bag-shaped cell structure 20 can retain peristaltic motility for at least five days.

[0103] The sheet-shaped cell structure is easier to transplant than the pouch-shaped cell structure.

[0104] In addition, since the lumen of the sac-like cell structure is filled with luminal fluid, tension may be generated due to the difference in osmotic pressure between the luminal fluid and the culture fluid, which may cause the structure to change to an undesirable one. Furthermore, if the osmotic pressure of the luminal fluid is higher than that of the surrounding culture fluid, the sac-like cell structure may expand, inhibiting peristaltic movement. In contrast, the sheet-like cell structure is not affected by the surrounding culture fluid and is likely to retain its shape, and its peristaltic movement is less likely to be inhibited.

[0105] The means for cutting the sheet-shaped cell structure 10 from the pouch-shaped cell structure 20 is not particularly limited.

[0106] For example, an incision can be made in a part of the wall 21 of the bag-shaped cell structure 20 extending from the outer surface 11 to the inner surface 12, and the incision can be widened as necessary to separate a part of the wall 21 of the bag-shaped cell structure 20 from the surrounding area to obtain a sheet-shaped cell structure 10.

[0107] The incision in the wall 21 of the sac-like cell structure 20 can be formed by destroying the wall 21 of the sac-like cell structure 20 by mechanical means such as a blade, needle, osmotic pressure, etc., or by decomposing the extracellular matrix, separating intercellular bonds, or destroying cells by means of electricity, heat, chemicals, enzyme treatment, etc.

[0108] The incision may be a hole large enough to insert a tool such as tweezers, or may be a linear incision. The linear incision is preferably a incision that will become the peripheral portion of the sheet-shaped cell structure 10.

[0109] The method of widening the incision is not particularly limited. For example, the incision can be widened by holding two points on either side of the wall 21 of the bag-shaped cell structure 20 in which the incision is formed with an appropriate jig and pulling the jig apart. An example of the jig is tweezers, but is not particularly limited. When holding two points of the wall 21 with a jig, each point of the wall 21 may be held by being sandwiched between the jigs, or each point of the wall 21 may be held by being adhered to the jig.

[0110] <3. Characteristics of the shape of sheet-shaped cell structures and processing> The sheet-shaped cellular structure of the present disclosure may be flat or have a small amount of distortion, or may have a distorted shape.

[0111] An example of a sheet-like cellular structure of the present disclosure having a flat or less distorted shape is shown in Fig. 3. When the sheet-like cellular structure 10 shown in Fig. 3 is placed on a flat surface 30 with the second surface 12 facing the flat surface 30, the height of the lowest position 112 on the first surface 11 facing the opposite side of the flat surface 30 of the sheet-like cellular structure 10 is A and the height of the highest position 111 is B, and for example, A / B is 0.40 or less. Here, the height B of the position 111 refers to the distance from the flat surface 30 to the position 111 along the normal direction of the flat surface 30. The height A of the position 112 refers to the distance from the flat surface 30 to the position 112 along the normal direction of the flat surface 30. The sheet-shaped cell structure of the present disclosure, which is flat or has little distortion as shown in FIG. 3, is preferable because it can easily cover inflamed areas, wounds, lesions, sutures after surgery, and the like.

[0112] The flat or less distorted sheet-shaped cell structure of the present disclosure can be obtained by cutting out a flat or less distorted portion of the wall of a pouch-shaped cell structure. Also, as described below, a distorted sheet-shaped cell structure can be obtained by processing it so that it fits the flat or less distorted template surface.

[0113] An example of a sheet-shaped cellular structure of the present disclosure having a distorted shape is shown in Figure 4. When the sheet-shaped cellular structure 10 shown in Figure 4 is placed on a flat surface 30 with the second surface 12 facing the flat surface 30, A / B, defined in the same manner as in the embodiment shown in Figure 3, is, for example, 0.003 or more.

[0114] The sheet-shaped cell structure of the present disclosure having a distorted shape as shown in FIG. 4 is suitable for covering inflamed areas, wounds, lesions, sutures after surgery, and the like on distorted surfaces.

[0115] The distorted sheet-shaped cell structure of the present disclosure can be obtained by cutting out a distorted portion of the wall of a sac-shaped cell structure. It can also be obtained by processing the sheet-shaped cell structure so that it conforms to the distorted template surface. An example of a distorted sheet-shaped cell structure is a hemispherical fragment obtained by dividing a sac-shaped cell structure into two.

[0116] The shape of the sheet-shaped cell structure of the present disclosure can be changed by processing.

[0117] A further embodiment of the present disclosure is a method for fabricating the sheet-shaped cellular structure of the present disclosure, comprising: Placing the sheet-shaped cell structure in contact with a template surface; processing the sheet-shaped cell structure into a shape that conforms to the template surface; The present invention relates to a method comprising the steps of:

[0118] The method of processing the sheet-like cell structure into a shape that conforms to the mold surface is not particularly limited. For example, the sheet-like cell structure may be urged toward the mold surface and pressed against the mold surface to form a shape that conforms to the mold surface. One example of the method of urging the sheet-like cell structure toward the mold surface is a method in which, with the sheet-like cell structure placed on the mold surface, centrifugal force is applied to the sheet-like cell structure in a direction perpendicular to the mold surface and toward the mold surface. Another example of the method of urging the sheet-like cell structure toward the mold surface is a method in which, with the sheet-like cell structure placed on the mold surface, another mold that faces the mold surface is brought close to the mold surface from the side of the sheet-like cell structure, so as to sandwich the sheet-like cell structure and press the sheet-like cell structure against the mold surface. The mold surface may be a flat surface, or may be a surface that is partially or entirely curved and / or uneven. A preferred embodiment of the processing method will now be described with reference to FIGS.

[0119] In the embodiment shown in Figure 5, the distorted sheet-like cell structure 10 is placed in contact with a flat template surface 50 (Figure 5A), and then the distorted sheet-like cell structure 10 is processed into a shape that conforms to the flat template surface 50 to obtain a flat sheet-like cell structure 10 (Figure 5B).

[0120] In the embodiment shown in Figure 6, a flat sheet-like cellular structure 10 is placed in contact with an upwardly convexly curved template surface 60 (Figure 6A), and then the flat sheet-like cellular structure 10 is processed into a shape that conforms to the curved template surface 60 to obtain a curved sheet-like cellular structure 10 (Figure 6B). The sheet-shaped cell structure processed into a shape conforming to the surface of the template may be used for transplantation as is, or may be further cultured after processing.

[0121] <4. Transplantation of sheet-shaped cell structures> The sheet-shaped cell structure of the present disclosure can be used for transplantation into the bodies of humans or non-human animals, particularly for covering inflamed areas, wounds, lesions, sutures after surgery, the abdominal cavity, defective intestinal tissue, etc. in organs.

[0122] In particular, in an embodiment in which the sheet-shaped cell structure of the present disclosure has the ability to secrete mucus, specifically the ability to produce mucin and antimicrobial peptides, it is preferable because it is highly effective in protecting the fusion site. The sheet-shaped cell structure of the present disclosure can protect the healed area by adhering it to an inflamed area, a wounded area, a lesion, a sutured area after surgery, or the abdominal cavity, preferably.

[0123] The sheet-shaped cell structure of the present disclosure can preferably be allowed to adhere to an inflamed area, a wound, a lesion, a sutured area after surgery, or the abdominal cavity, thereby suppressing leakage from or into the hemorrhage site, or invasion into the hemorrhage site.

[0124] The sheet-shaped cell structure of the present disclosure can preferably be allowed to adhere to an inflamed area, a wound, a lesion, a sutured area after surgery, or the abdominal cavity, thereby providing cells or a scaffold for cells necessary for healing of the adherent area.

[0125] The sheet-shaped cell structure of the present disclosure can preferably be allowed to adhere to an inflamed area, a wound, a lesion, a sutured area after surgery, or the abdominal cavity, thereby preventing the adhesion of the adhesion area to a heterotopic site.

[0126] The sheet-shaped cell structure of the present disclosure can be preferably allowed to adhere to an inflamed area, a wound, a lesion, a sutured area after surgery, or the abdominal cavity, thereby alleviating increased inflammation at the adherent area. The sheet-shaped cellular structure of the present disclosure can preferably be caused to heal at suture sites after surgery, thereby preventing postoperative complications. The sheet-shaped cell structure of the present disclosure can preferably be transplanted into defective intestinal tissue to substitute for intestinal function.

[0127] The sheet-shaped cell structure of the present disclosure can be held with tweezers or the like and attached to a desired transplant site in either open surgery or laparoscopic surgery. When transplanting to a site where transplantation is difficult by laparoscopic surgery or the like, the sheet-shaped cell structure of the present disclosure may be transplanted in a state where it is integrated with a support, or may be transplanted while being held by a carrier such as a wire. The sheet-shaped cell structure of the present disclosure can be fixed to a biocompatible support and transplanted together with the support.

[0128] When the sheet-shaped cell structure of the present disclosure is transplanted into a living body, a biocompatible adhesion aid such as fibrin may be applied to the sheet-shaped cell structure and / or the transplant site. The sheet-shaped cell structure and the transplant site may also be sutured together.

[0129] When attaching the sheet-shaped cell structure of the present disclosure to a transplantation site, for example, the sheet-shaped cell structure of the present disclosure may be placed on the transplantation site in a spread-out state using tweezers, or the sheet-shaped cell structure of the present disclosure may be placed on the transplantation site and then spread out.

[0130] Next, a preferred transplantation method will be described in an embodiment in which the sheet-shaped cell structure of the present invention comprises endodermal cells, such as a small intestinal epithelial cell layer, on the first surface side and ectodermal cells and / or mesodermal cells on the second surface side.

[0131] When the sheet-shaped cell structure according to the embodiment is held on a carrier for maintaining the sheet structure, it is preferable to attach the first surface to the carrier and transplant the second surface to a transplant site. In this case, it is preferable that the adhesive force between the carrier and the sheet-shaped cell structure is weaker than the adhesive force between the living body and the sheet-shaped cell structure. The surface of the carrier may be subjected to a protein adsorption inhibition treatment such as a hydrophobic treatment.

[0132] The sheet-shaped cell structure according to the embodiment is preferably transplanted in a direction that matches the layer structure of the transplant site. For example, when transplanted into the inner wall of the intestine, the first surface is placed facing the inside of the lumen. 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

[0133] <Example 1> A sheet-shaped intestinal organ-like cell structure was produced as follows. The method is described below. (Preparation of cell culture substrate) A cell culture substrate was prepared, which included a cell adhesion portion consisting of a circular pattern with a diameter of 1500 μm, which is a region formed on a glass substrate by oxidative decomposition of a layer of polyethylene glycol 400, and a cell non-adhesive portion, which is a region where the surface of the glass substrate is coated with polyethylene glycol 400 outside the circular pattern of the cell adhesion portion. The cell culture substrate includes a plurality of cell adhesion portions consisting of the circular patterns formed at intervals of 300 to 500 μm. In the following description, the cell adhesion portion consisting of a circular pattern is referred to as a "circular cell adhesion portion."

[0134] The cell culture substrate was prepared according to the procedure described in Japanese Patent No. 5070565. The outline of the procedure is described below.

[0135] (First stage reaction) 39.0g of toluene, 0.48g of epoxy silane TSL8350 (GE Toshiba Silicone), and 0.97g of triethylamine were mixed and stirred at room temperature for 10 minutes. A 10cm square glass substrate that had been UV-cleaned was immersed in this silane solution with the cleaned surface facing upwards. After leaving it at room temperature for 16 hours, the substrate was washed with ethanol and water and dried with a nitrogen blower. This resulted in the formation of a thin film containing epoxy groups on the surface of the glass substrate.

[0136] (Second stage reaction) 25μl of concentrated sulfuric acid was added drop by drop to 50g of polyethylene glycol (PEG400) with an average molecular weight of 400 while stirring. After stirring for several minutes, the entire amount was transferred to a glass dish. The above substrate was immersed in the mixture and reacted at 80℃ for 20 minutes. After the reaction, the substrate was thoroughly washed with water and dried with nitrogen blow. This resulted in the formation of a uniform hydrophilic thin film on the glass surface.

[0137] (Oxidation treatment) A photomask was prepared by coating the entire surface with a titanium oxide photocatalyst. The photomask used was a 5-inch size photomask with multiple openings formed at intervals of 300 to 500 μm in shape corresponding to the circular cell adhesion parts of the above dimensions, and with openings of approximately 1.5 cm in width around the periphery. The illuminance of the exposure machine was measured in advance at a wavelength of 350 nm to serve as a guide for setting the exposure time. The photocatalyst layer of this photomask was brought into contact with the hydrophilic thin film on the surface of the substrate, and the substrate was placed in the exposure machine so that light was irradiated from the photomask side. The illuminance at a wavelength of 350 nm was 20 mW / cm 2 The substrate was exposed to a mercury lamp for 50 seconds to partially decompose the hydrophilic thin film on the substrate surface by oxidation. The substrate was cut into pieces of 5 cm x 5 cm and used as a cell adhesion substrate. Before use in cell culture, the cell culture substrate was sterilized with EOG for 22 hours.

[0138] The cell culture substrate was placed on the bottom of a 10 cm Petri dish (Falcon) and coated with vitronectin (Life Technologies) diluted 1 / 100 in phosphate-buffered saline (PBS) at room temperature for at least 30 minutes, and then washed three times with PBS before use.

[0139] (culture) The National Center for Child Health and Development (NCHD) has established a human iPS cell line, Edom iPS cells, by transiently expressing the four Yamanaka factors in cells obtained from menstrual blood using a Sendai virus vector (PLOS Genet. 2011 May; 7(5): e1002085. Published online 2011 May 26. doi: 10.1371 / journal.pgen.1002085PMCID: PMC3102737). Edom iPS cells were pre-grown in StemFit medium (Ajinomoto) in a cell culture dish coated with vitronectin (Vitronectin, Life Technologies) diluted 1 / 100 with PBS in the same manner as above. The grown cells were detached from the dish by treating with EDTA solution (Life Technologies) diluted to 0.5 mM with PBS.

[0140] The Edom iPS cells were detached and collected and placed on the cell culture substrate at 1×10 7 The cells were seeded and cultured for 45 days. The culture was performed according to the procedure described in Uchida et al., JCI Insight Vol. 2 e86492 2017. The day after seeding, the cell culture substrate with the cells attached was transferred to a 10 cm diameter cell culture dish (Falcon), and the medium was maintained at about 15 mL, and the medium was replaced every two days.

[0141] The composition of the xenogeneic component-free medium (referred to as XF32 medium) used is as follows, and was prepared by mixing each of the components (content concentrations were taken from Uchida et al., JCI Insight Vol. 2 e86492 2017). Knockout DMEM (KDMEM; ThermoFisher Scientific): Approximately 85% by volume of the total medium XenoFree-Knockout Serum Replacement (XF-KSR; ThermoFisher Scientific): Approximately 15% by volume of the total medium Basic Fibroblast Growth Factor (bFGF; ThermoFisher Scientific): 20ng / mL Insulin growth factor-I (IGF-I; Nichirei): 200ng / mL Hereglin (Fujifilm Wako Pure Chemical Industries): 10ng / mL Glutamax-I (L-alanyl-L-glutamine; ThermoFisher Scientific): 2mM Non Essential Amino Acid Solution (NEAA; ThermoFisher Scientific): 0.1 mM for all non-essential amino acids Sodium Pylvate (ThermoFisher Scientific): 1mM Streptomycin (ThermoFisher Scientific): 50U / mL Penicillin (ThermoFisher Scientific) 50μg / mL

[0142] During the culture, the cells proliferated on the circular cell adhesion sites and formed sac-like (spherical) cell structures, which naturally detached and floated 3 to 4 weeks after seeding. After detachment, the cells were cultured in suspension for up to 45 days after seeding.

[0143] The pouch-like cell structure (intestinal organoid) contained the same cell types and tissues as those confirmed in JP 2019-000014 A and Uchida et al., JCI Insight Vol. 2 e86492 2017, and had peristaltic motility. The pouch-like cell structure had a small intestinal epithelial cell layer including a villus layer on the outer surface side, and a multi-layered structure was confirmed with a cell layer (submucosa-like layer) including smooth muscle cells, Cajal cells, and nerve cells on the inner surface side. The pouch-like cell structure was spherical with a diameter of about 5 mm and had a structure containing a lumen.

[0144] The pouch-like cell structures (intestinal organoids) were harvested into cell culture dishes, medium was added, and spontaneous peristaltic movement was observed by video recording for 40 minutes.

[0145] Next, the collected intestinal organoids were divided into hemispherical pieces on a cell culture dish using a scalpel, and hemispherical pieces were obtained. The hemispherical pieces were distorted hemispherical shapes of the cell sheets, and are referred to as sheet-like cell structures in the following description. The peristaltic motility of the sheet-shaped cell structure was confirmed by video. As a result, it was confirmed that the sheet-shaped cell structure obtained by dividing the bag-shaped cell structure had peristaltic motility. The sheet-like cell structure is circular with a diameter of 5.23 mm and an area of ​​23.82 mm. 2 It was.

[0146] <Example 2> It was confirmed that the sheet-shaped cell structure is excellent for detecting structural changes. The method is described below. The video of the peristaltic movement of the sheet-shaped cell structure filmed in Example 1 was used to digitize the area and determine the maximum and minimum values ​​using Image J. An image of the sheet-shaped cell structure at the point where it was most contracted within the field of view (most contracted image within the field of view) is shown in Figure 7A, and an image of the sheet-shaped cell structure at the point where it was most expanded within the field of view (expanded image within the field of view) is shown in Figure 7B. The results are shown below. It can be seen that the sheet-shaped cell structure exhibits a wide range of structural changes, making these changes easy to grasp visually.

[0147] [Table 1]

[0148] <Example 3> We verified whether the sheet-shaped cell constructs could be used for transplantation or evaluation after processing, using a 5-day standard as a guideline for domestic distribution. The method used is described below.

[0149] The sheet-shaped cell structure obtained in Example 1 was cultured in D-MEM medium at 37° C. for 5 days, and then transferred to a different culture dish and washed three times with PBS.

[0150] After washing, the sheet-shaped cell structure was immersed in a mixture of 1 ml of PBS, 1 μl of 1 mg / ml Calcein-AM solution, and 1 μl of 1 mg / ml propidium iodide (PI) solution, and incubated in an incubator for 15 minutes to perform double staining.

[0151] Next, the sheet-shaped cell structure was subjected to fluorescent observation to observe live cells stained with green fluorescence by Calcein-AM solution and dead cells stained with red fluorescence by PI solution.

[0152] The image of the sheet-shaped cell structure observed by a fluorescence microscope is shown in Figure 8. In the sheet-shaped cell structure, most of the cells were live cells that showed green fluorescence, indicating that the constituent cells survived for at least five days after processing. Furthermore, it was shown that the sheet-shaped cell structure had peristaltic motility for at least five days after processing. This result indicates that the sheet-shaped cell structure can retain its function as an intestinal organoid for five days after processing.

[0153] <Example 4> As shown in Figure 5A, the hemispherical sheet-like cell structure 10 obtained in Example 1 was placed on a flat template surface 50 with the lumen side (the side of the second surface 12) facing the flat template surface 50, and centrifugal force was applied to the sheet-like cell structure 10 in a direction perpendicular to the template surface 50 and toward the template surface, thereby processing the sheet-like cell structure 10 into a shape that conforms to the flat template surface 50, thereby obtaining a flat sheet-like cell structure 10.

[0154] The three-dimensional structures of the hemispherical and flat sheet-like cell structures were measured using the NH-3SPs "ultra-precise non-contact three-dimensional measuring device" (Mitaka Kohki Co., Ltd.).

[0155] For the measurements, the hemispherical sheet-like cell structure and the flat sheet-like cell structure were placed on a dish with the lumen side facing down, and the measurement range was from the edge to the center in a planar view. After fixing the Y axis, the structure was scanned in the X axis direction to obtain thickness data on the Z axis (unit: mm). Measurements of the hemispherical sheet-like cell structure were taken immediately after it was divided into two.

[0156] Figure 9A shows the measurement results of the thickness of a hemispherical sheet-like cell structure. The horizontal axis shows the measurement points on the X axis at a measurement pitch of 0.03 mm, and the vertical axis shows the thickness (unit: mm). The minimum thickness value A of the hemispherical sheet-like cell structure was 0.00277 mm, the maximum thickness value B was 0.8754 mm, and A / B was 0.00316. Figure 9B shows the measurement results of the thickness of a flat sheet-like cell structure. The horizontal axis shows the measurement points on the X axis at a measurement pitch of 0.01 mm, and the vertical axis shows the thickness (unit: mm). The minimum thickness value A of the flat sheet-like cell structure was 0.00766 mm, the maximum thickness value B was 0.01568 mm, and A / B was 0.488. It should be noted that in A and B of FIG. 9, the measurement values ​​at the measurement points where an error occurred in the detection result are not shown.

Claims

1. A 20 mm2 cell culture medium containing endodermal, ectodermal and mesodermal cells ２ The above sheet-shaped cell structure, wherein the sheet-shaped cell structure is derived from embryonic stem cells or induced pluripotent stem cells.

2. containing endodermal cells on the first surface side; The sheet-shaped cell structure according to claim 1 , comprising ectodermal cells and / or mesodermal cells on the second surface side.

3. The sheet-shaped cell structure according to claim 2 , comprising a small intestinal epithelial cell layer on the side of the first surface.

4. The sheet-shaped cellular structure according to claim 3, further comprising a submucosa-like layer.

5. The sheet-shaped cell structure according to any one of claims 1 to 4, which does not contain lymphocytes.

6. The sheet-shaped cell structure according to any one of claims 1 to 5, which has the ability to produce mucin and antimicrobial peptides.

7. The sheet-shaped cell structure according to any one of claims 1 to 6, which has peristaltic motility.

8. The sheet-shaped cell structure according to any one of claims 1 to 7, which does not contain any xenogeneic components.

9. The sheet-shaped cell structure is placed on a flat surface, A sheet-shaped cell structure described in any one of claims 1 to 8, wherein when the height of the lowest point on the surface opposite the flat surface of the sheet-shaped cell structure is A and the height of the highest point is B, A / B is 0.003 or more.

10. The sheet-shaped cell structure is placed on a flat surface, A sheet-like cell structure described in any one of claims 1 to 9, wherein when the height of the lowest point on the surface opposite the flat surface of the sheet-like cell structure is A and the height of the highest point is B, A / B is 0.40 or less.

11. A method for processing the sheet-shaped cell structure according to any one of claims 1 to 10, comprising the steps of: Placing the sheet-shaped cell structure in contact with a template surface; processing the sheet-shaped cell structure into a shape that conforms to the template surface; The method includes:

12. The mold surface is a flat surface, The method according to claim 11, wherein the sheet-shaped cellular structure subjected to processing is the sheet-shaped cellular structure described in claim 9.

13. The method of claim 11 , wherein the mold surface is a curved and / or uneven surface.

14. A 20 mm2 cell culture medium containing endodermal, ectodermal and mesodermal cells ２ The method for producing the sheet-shaped cell structure described above comprises the steps of: The sheet-shaped cell structure is derived from embryonic stem cells or induced pluripotent stem cells, and The method comprises: A sheet-like cell structure is cut out from a sac-like cell structure that contains an inner lumen and has a longitudinal length of 5 mm or more and contains endodermal cells, ectodermal cells, and mesodermal cells. The method comprising:

15. The method according to claim 14 , wherein the pouch-like cell structure comprises endodermal cells on the outer surface side and ectodermal cells and / or mesodermal cells on the inner surface side.

16. The method according to claim 14 or 15, wherein the pouch-like cell structure comprises a small intestinal epithelial cell layer on the outer surface side.

17. A method according to any one of claims 14 to 16, wherein the sheet-shaped cell structure has peristaltic motility.

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