Feeder cells, cell sheet, production method for feeder cells and cell sheet, and method for maintaining or proliferating cells using feeder cell

Abstract

To provide feeder cells and a method for producing the same, for maintaining or proliferating, by bidimensional culturing, cells for which maintenance and proliferation by bidimensional culturing is considered difficult.SOLUTION: Feeder cells are prepared by proliferating and isolating fibroblast-like cells derived from a bowel structure that is produced from pluripotent stem cells and that includes endoderm-derived cells and mesoderm-derived cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to feeder cells for maintaining or proliferating cells, cell sheets containing the feeder cells and methods for producing them, and methods for maintaining or proliferating cells using the feeder cells.

Background Art

[0002] In recent years, primary cultured cells obtained from living organisms, pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), and cells differentiated from pluripotent stem cells have been widely used in fields such as basic research, regenerative medicine, and drug discovery.

[0003] In these fields, there is a demand for techniques for inducing the differentiation of pluripotent stem cells to proliferate cells having specific properties, or for organizing a plurality of cells having such specific properties to form tissues and organs. Conventionally, such techniques have been actively studied. For example, a technique has been proposed in which intestinal epithelial cells are induced to differentiate from pluripotent stem cells, and the differentiated intestinal epithelial cells are maintained and proliferated by three-dimensional culture to form an intestinal structure (for example, Patent Document 1).

[0004] On the other hand, various attempts have been made regarding two-dimensional culture of intestinal epithelial cells, but a method for two-dimensional culture for maintaining and proliferating intestinal epithelial cells has not yet been found.

[0005] By the way, in order to induce the differentiation of pluripotent stem cells into cells having specific properties or to promote the maintenance and proliferation of the cells after differentiation induction, cells that produce and supply specific factors (for example, differentiation induction factors, etc.), that is, feeder cells, may be used (for example, Patent Document 2).

[0006] However, even when culturing intestinal epithelial cells two-dimensionally by using feeder cells, a technique for maintaining or proliferating them has not been known. Therefore, there has been a demand for finding a practical method for two-dimensionally culturing and maintaining or proliferating cells such as intestinal epithelial cells, which have been considered difficult to maintain and proliferate by two-dimensional culturing.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Under such circumstances, there has been a demand for finding a practical method for two-dimensionally culturing and maintaining or proliferating cells such as intestinal epithelial cells, which have been considered difficult to maintain and proliferate by two-dimensional culturing.

[0009] Therefore, the present disclosure provides feeder cells for two-dimensionally culturing and maintaining or proliferating cells such as intestinal epithelial cells, which have been considered difficult to maintain and proliferate by two-dimensional culturing, and a method for producing such feeder cells. Further, the present disclosure provides a cell sheet containing the above-described feeder cells.

Means for Solving the Problems

[0010] Embodiments of the present disclosure relate to the following [1] to

[28] .

[0011] [1] A method for producing feeder cells for maintaining or proliferating cells, comprising: (A) Prepare an intestinal structure produced from pluripotent stem cells and containing endoderm-derived cells and mesoderm-derived cells; (B) Culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure; and (C) Isolating the fibroblast-like cells to obtain feeder cells A method comprising the above steps. [2] The intestinal structure is produced by a method comprising: (a) Prepare a cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and cell non-adhesion regions surrounding each of the cell adhesion regions; (b) Seeding pluripotent stem cells on the cell culture substrate; and (c) Culturing the seeded pluripotent stem cells in a medium The method according to [1], wherein the intestinal structure is produced by the above method. [1] The method according to [1]. [3](D) The method according to [1] or [2], further comprising the step of proliferating the isolated fibroblast-like cells. [4] The method according to [1] or [2], wherein in step (B), the intestinal structure is cultured in a culture vessel and at least a part of the intestinal structure is in contact with the culture vessel. [5] The method according to [2], wherein the culturing period in step (c) is 60 days or more. [6] Feeder cells produced by the method according to [1] or [2]. [7] Fibroblast-like cells that are positive for PDGFRA and CD81 and express Foxl1 and GREM1. [8] The fibroblast-like cells according to [7], which are positive for PDGFRA and CD81, negative for CD34, and express Foxl1 and GREM1. [9] Fibroblast-like cells according to [7], which are derived from pluripotent stem cells or from a culture of an intestinal structure produced from pluripotent stem cells and containing endoderm-derived cells and mesoderm-derived cells.

[10] The intestinal structure is (a) Preparing a cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and cell non-adhesion regions surrounding each of the cell adhesion regions; (b) Seeding pluripotent stem cells onto the cell culture substrate; and (c) Culturing the seeded pluripotent stem cells in a medium which is produced by a method comprising: The fibroblast-like cells described in [9].

[11] The fibroblast-like cells described in [9], wherein the pluripotent stem cells are of human origin.

[12] The feeder cells described in [6] or the fibroblast-like cells described in [7], which are feeder cells for maintaining or proliferating at least one cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

[13] The feeder cells described in [6] or the fibroblast-like cells described in [7], which are feeder cells for maintaining or proliferating the cells by planar culture.

[14] The feeder cells described in [6] or the fibroblast-like cells described in [7], which are feeder cells capable of subculturing the cells three or more times.

[15] A cell sheet comprising the feeder cells described in [6] or the fibroblast-like cells described in [7].

[16] A co-culture comprising at least one cell selected from the group consisting of small intestinal epithelial cells and hepatocytes and the feeder cells described in [6] or the fibroblast-like cells described in [7].

[17] The co-culture described in

[16] , wherein the number of subcultures of the cells is three or more.

[18] A cell sheet comprising the co-culture described in

[16] .

[19] The cell sheet described in

[18] , which is circular or substantially circular with a diameter of 1 cm or more.

[20] The cell sheet described in

[18] , which comprises a layer of at least one cell selected from the group consisting of small intestinal epithelial cells and hepatocytes.

[21] The cell sheet described in

[20] , which further comprises a layer of the feeder cells. A graft material comprising the cell sheet described in

[22] ,

[15] or

[18] .

[23] A method for maintaining or proliferating cells, the method comprising culturing the cells in the presence of feeder cells described in [6] or fibroblast-like cells described in [7].

[24] The method according to

[23] , wherein the culturing of the cells is planar culturing.

[25] The method according to

[23] or

[24] , wherein the cells are at least one cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

[26] The method according to

[23] or

[24] , wherein the cells and the feeder cells are derived from the same individual.

[27] The method according to

[23] or

[24] , wherein the cells and the feeder cells are derived from different individuals.

[28] Cells that express at least one gene selected from the group consisting of DCN, ACTA2, and NOG, and that express the genes of ADAMTS19, ANO1, BLID, LOC100507053, LHX8, SFRP4, AHRR, SLC14A1, FMO3, PZP, ABCG4, EYA2, TMEM92-AS1, ANO10, EGFL6, SNCAIP, LGSN, PCDHB15, and KRTAP1-5. [Effect of the Invention]

[0012] According to the present disclosure, feeder cells for two-dimensionally culturing and maintaining or growing cells such as intestinal epithelial cells, which are considered difficult to maintain and grow by two-dimensional culturing, and a method for producing such feeder cells can be provided. Further, according to the present disclosure, a cell sheet containing the feeder cells as described above can be provided. Furthermore, since the above-described feeder cells have good feeder ability even when the passage number is repeated 10 or more times, they can be widely spread without immortalization, and the culture system using the feeder cells can be expected to serve as a standard model of a culture system closer to the human body than commonly used feeder cells such as mouse embryonic fibroblasts (MEF) and immortalized feeder cells. In addition, the feeder cells have adhesiveness to a general culture surface, show expression of CD90, and are present in the positional relationship where mesenchymal stem cells originally exist within the organoid, so it can be easily inferred that they are a type of mesenchymal stem cell, and a therapeutic effect by transplantation of the feeder cells can also be expected.

Brief Description of the Drawings

[0013]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0014] As used herein, the term "feeder cell" refers to a cell that is co-cultured with a target cell or a pre-differentiated cell thereof, etc. in order to prepare culture conditions that promote the maintenance and / or proliferation of the target cell, and refers to a cell different from the target cell. In the present disclosure, the feeder cells may be seeded simultaneously with the target cells, or may be seeded before the seeding of the target cells. In one embodiment, the feeder cells are seeded before the seeding of the target cells to form a layer of feeder cells (i.e., a feeder layer). The target cells and the feeder cells may be cultured so as to be in contact with each other, or may be cultured so as not to be in contact with each other. In one embodiment, the target cells and the feeder cells are cultured such that at least a part of the target cells is in contact with the feeder cells. Note that the proliferation of the feeder cells may be suppressed by known methods such as irradiation with radiation (e.g., γ-rays) or treatment with an antibiotic (e.g., mitomycin C).

[0015] As used herein, the term "pluripotent stem cell" refers to a cell having so-called differentiation pluripotency. As used herein, the terms "pluripotency" and "differentiation pluripotency" refer to the ability of a cell to differentiate into all germ layers (i.e., ectoderm, mesoderm, and endoderm) that make up a living body. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), induced pluripotent stem cells (iPS cells), stem cells derived from adult tissues or umbilical cord blood or umbilical cord, and the like.

[0016] As used herein, the term "fibroblast-like cell" refers not only to so-called fibroblasts, but also to cells having a flattened or spindle-shaped morphology differentiated from fibroblasts, such as myofibroblasts. The origin of the fibroblast-like cells is not particularly limited. For example, the fibroblast-like cells can be cells derived from the above-described pluripotent stem cells or cells derived from the intestinal structures described below. Further, the fibroblast-like cells can be cells exhibiting a specific gene expression profile described below. It should be noted that the "fibroblast-like cells" in the present disclosure do not include the above-described various naturally occurring cells, but are cells obtained by subjecting naturally occurring cells or artificially obtained cells to artificial manipulation, that is, artificially obtained cells (artificial cells).

[0017] [Method for preparing feeder cells] According to one aspect of the present disclosure, there is provided a method for preparing feeder cells (hereinafter, also referred to as "feeder cells of the present disclosure") for maintaining cells such as intestinal epithelial cells, which are considered difficult to be maintained and proliferated by two-dimensional culture. The feeder cells of the present disclosure can be prepared by a method including the following steps (A) to (C) (hereinafter, also referred to as "preparation method of the present disclosure"): (A) A step of preparing an intestinal structure prepared from pluripotent stem cells and containing endoderm-derived cells and mesoderm-derived cells, (B) A step of culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure, and (C) A step of isolating fibroblast-like cells to obtain feeder cells. Hereinafter, each of the steps (A) to (C) will be described in detail.

[0018] <Step (A)> In step (A), an intestinal structure prepared from pluripotent stem cells and containing endoderm-derived cells and mesoderm-derived cells is prepared. The intestinal structure is a structure containing endoderm-derived cells and mesoderm-derived cells and having at least one function equivalent or similar to that of the intestine.

[0019] The intestinal structure is not particularly limited as long as it has the above-described cell composition and functions, and can be obtained, for example, by inducing the differentiation of pluripotent stem cells. In one embodiment, the intestinal structure used in the production method of the present disclosure can be produced by a method including the following steps (a) to (c).

[0020] (Step (a)) Step (a) is a step of preparing a cell culture substrate including a substrate, a plurality of isolated cell adhesion regions (cell-adhesive regions) formed on the substrate, and cell non-adhesion regions (cell-non-adhesive regions) surrounding each of the cell adhesion regions.

[0021] As used herein, "cell adhesiveness" means the strength of cell adhesion, that is, the ease of cell adhesion. Therefore, the "cell adhesion region" means a region with relatively high cell adhesiveness, and the "cell non-adhesion region" means a region with relatively low cell adhesiveness. Specifically, cell adhesiveness means the degree to which cell adhesion and spreading occur due to the chemical properties, physical properties, etc. on the substrate (substrate surface). In a cell adhesion region with relatively high cell adhesiveness, cell adhesion is likely to occur, and in a cell adhesion region with relatively low cell adhesiveness, cell adhesion is less likely to occur. Therefore, when cells are seeded on a substrate on which the cell adhesion region and the cell non-adhesion region are patterned as described above, cells are likely to adhere to the cell adhesion region and less likely to adhere to the cell non-adhesion region, so that the seeded cells will be arranged in a pattern on the substrate.

[0022] Cell adhesion can be determined using the cell adhesion and spreading rate when culturing cells seeded on a substrate and arranged in a pattern as an index. The higher the cell adhesion and spreading rate, the more efficiently the cells can be cultured. The cell adhesion and spreading rates in the cell adhesion region and the cell non-adhesion region are not particularly limited as long as the effects of the present disclosure are achieved. For example, they can be 60% or more and less than 60%, respectively. The cell adhesion and spreading rate in the cell adhesion region is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. On the other hand, the cell adhesion and spreading rate in the cell non-adhesion region is preferably less than 40%, more preferably 5% or less, and even more preferably 2% or less. The cell adhesion and spreading rate is defined as the ratio of the cells that have adhered and spread ({(number of cells adhered to the measurement target surface) / (number of cells seeded on the measurement target surface)}×100(%)) when the cells to be cultured within the range of a seeding density of 4,000 cells / cm 2 or more and less than 30,000 cells / cm 2 are seeded on the measurement target surface, stored in an incubator at a temperature of 37°C and a CO2 concentration of 5%, and cultured for 14.5 hours.

[0023] Seeding of cells onto the measurement target is performed by suspending the cells in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, adding the cell suspension onto the measurement target, and then gently shaking the measurement target to which the cells have been added so that the cells are distributed as uniformly as possible. Furthermore, the measurement of the cell adhesion and spreading rate is performed after removing the cells that have not adhered to the measurement target by changing the medium immediately before the measurement. In the measurement of the cell adhesion and spreading rate, the locations excluding the locations where the cell density is likely to be specific (for example, the center of a predetermined region where the density is likely to be high, the periphery of a predetermined region where the density is likely to be low) are used as the measurement locations.

[0024] In the present disclosure, the base material used for producing the intestinal structure is not particularly limited as long as the effects of the present disclosure are achieved. For example, the cell non-adhesive region is formed by immobilizing polyethylene glycol on the base material, and the cell adhesive region is a base material formed by subjecting at least a part of the polyethylene glycol immobilized on the base material to an oxidation treatment and / or a decomposition treatment. Such a base material can be produced, for example, by forming a thin film of polyethylene glycol (PEG) on the entire surface of the base material, and then subjecting the region where cell adhesion is desired to an oxidation treatment and / or a decomposition treatment to impart cell adhesiveness to form a cell adhesive region. The portion where neither the oxidation treatment nor the decomposition treatment is performed becomes the cell non-adhesive region where PEG is immobilized.

[0025] The base material is not particularly limited as long as it is formed of a material capable of forming a PEG thin film on its surface. Examples of the material of the base material include inorganic materials such as metals, glass, ceramics, and silicon, and organic materials typified by elastomers and plastics (for example, 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, vinyl chloride resin, etc.). The shape of the base material is also not particularly limited, and examples thereof include flat shapes such as flat plates, flat films, films, and porous membranes, and three-dimensional shapes such as cylinders, stamps, multi-well plates, and microchannels. In one embodiment, a film-shaped base material is used. The thickness of the film-shaped base material is not particularly limited, but is usually 0.1 to 1,000 μm, preferably 1 to 500 μm, and more preferably 10 to 200 μm.

[0026] Polyethylene glycol (PEG) contains at least an ethylene glycol chain (EG chain) composed of one or more ethylene glycol units (-(CH2)2-O-). The ethylene glycol chain may be linear or branched. The ethylene glycol chain is, for example, represented by the following formula: -((CH2)2-O)m - (m represents an integer of the degree of polymerization) refers to the structure represented by. m is not particularly limited as long as the effects of the present disclosure are achieved, but is preferably 1 to 13, more preferably 1 to 10.

[0027] PEG includes ethylene glycol oligomers. Further, PEG includes those having a functional group introduced therein. Examples of the functional group include an epoxy group, a carboxyl group, an N-hydroxysuccinimide group, a carbodiimide group, an amino group, a glutaraldehyde group, a (meth)acryloyl group, and the like. The functional group is preferably introduced at the end of the PEG chain via a linker as needed. Examples of PEG having a functional group introduced therein include PEG (meth)acrylate, PEG di(meth)acrylate, and the like.

[0028] The average thickness of the PEG thin film formed on the substrate is not particularly limited, but is preferably 0.8 nm to 500 μm, more preferably 0.8 nm to 100 μm, still more preferably 1 nm to 10 μm, and particularly preferably 1.5 nm to 1 μm. When the average thickness of the PEG thin film is 0.8 nm or more, in the adsorption of proteins and the adhesion of cells, it is less likely to be affected by the regions not covered by the PEG thin film on the substrate surface. On the other hand, when the average thickness is 500 μm or less, the coating can be performed relatively easily. Furthermore, by making the thickness of the PEG thin film a certain value or more, cell non-adhesiveness decreases, and the adhesion and spreading of cells to regions other than the cell adhesion region can be suppressed. Also, by making the thickness of the PEG thin film a certain value or less, factors necessary for cell survival contained in the culture solution can reach cells close to the substrate within the cell adhesion region.

[0029] As methods for forming a PEG thin film on a substrate, there can be mentioned a method of directly adsorbing PEG onto the surface of the substrate, a method of directly coating PEG onto the surface of the substrate, a method of performing a crosslinking treatment after coating PEG onto the surface of the substrate, a method of forming an underlayer on the surface of the substrate to enhance the adhesion between the substrate and the PEG thin film and then coating PEG, a method of forming a polymerization initiation point on the surface of the substrate and then polymerizing PEG, etc. In a preferred embodiment, the formation of the PEG thin film on the substrate is carried out by forming an underlayer on the substrate and then coating PEG.

[0030] The underlayer on the surface of the substrate can be formed, for example, by the method described in JP-A-2012-175983 or the like. In a preferred embodiment, the underlayer on the surface of the substrate can be formed by immobilizing a silane coupling agent having a functional group capable of reacting with the hydroxyl group at the end of PEG or the introduced functional group to form a covalent bond, or a functional group convertible to such a functional group, on the substrate surface. Examples of such functional groups include (meth)acryloyl group, (1H-imidazol-1-yl)carbonyl group, succinimidyl oxycarbonyl group, glycidyl group, epoxy group, aldehyde group, amino group, thiol group, carboxyl group, azide group, cyano group, active ester group (1H-benzotriazol-1-yloxycarbonyl group, pentafluorophenyloxycarbonyl group, p-nitrophenyloxycarbonyl group, etc.), halogenated carbonyl group, isocyanate group, maleimide group, etc. Among these functional groups, preferred functional groups are (meth)acryloyl group, glycidyl group, and epoxy group.

[0031] For example, when a silane coupling agent having a methacryloyl group at the terminal (methacryloyl silane) is used, the water contact angle of the surface of the substrate to which methacryloyl silane is added is typically 45° or more, preferably 47° or more, more preferably 48° or more, and even more preferably 50° or more. In the present disclosure, the water contact angle refers to the water contact angle measured at 23°C. When the surface of the substrate has the water contact angle as described above, PEG can be immobilized at a sufficient density.

[0032] The density of PEG immobilized on the substrate and cell adhesiveness can be easily evaluated using the water contact angle on the surface of the substrate as an index. Specifically, the lower the water contact angle on the surface of the substrate, the higher the density of PEG present on the surface of the substrate, and as a result, a region with low cell adhesiveness (i.e., a cell non-adhesive region) is formed. That is, for example, when the water contact angle of the surface of the substrate after immobilizing PEG on the surface of the substrate is, for example, 48° or less, preferably 40° or less, and more preferably 30° or less, it is considered that PEG is present on the surface of the substrate at a sufficient density and the cell adhesiveness is sufficiently low.

[0033] In the present disclosure, the oxidation treatment of polyethylene glycol for forming a cell adhesion region refers to a reaction in which an organic compound, that is, PEG reacts with oxygen and the oxygen content becomes higher than before the reaction. Further, in the present disclosure, the decomposition treatment of polyethylene glycol for forming a cell adhesion region refers to a reaction in which the bond of an organic compound, that is, PEG is cleaved. Examples of the "decomposition treatment" typically include decomposition by oxidation, decomposition by ultraviolet irradiation, etc., but are not limited thereto. When the "decomposition treatment" is decomposition accompanied by oxidation (i.e., oxidative decomposition), the "decomposition treatment" and the "oxidation treatment" refer to the same treatment.

[0034] Decomposition by ultraviolet irradiation means that PEG absorbs ultraviolet light and decomposes through an excited state. When ultraviolet light is irradiated into a system in which PEG and molecular species containing oxygen (e.g., oxygen, water, etc.) coexist, in addition to the absorption of ultraviolet light by PEG and subsequent decomposition, the molecular species containing oxygen may be activated and react with PEG. In this case, the latter reaction is classified as "oxidation". And the reaction in which PEG decomposes due to oxidation by the activated molecular species is classified as "decomposition by oxidation" rather than "decomposition by ultraviolet irradiation". Thus, "oxidation" and "decomposition" may overlap as operations, and there may be cases where they cannot be clearly distinguished. In this specification, such cases are also expressed as "oxidation and / or decomposition".

[0035] Examples of the method of oxidation and / or decomposition include a method of subjecting a PEG thin film to ultraviolet irradiation treatment, a method of photocatalytic treatment, a method of treatment with an oxidizing agent, etc. When partially oxidizing and / or decomposing a PEG thin film, it can be carried out using a mask or stamp such as a photomask or a stencil mask. Also, oxidation and / or decomposition can be carried out by a direct drawing method such as a method using a laser such as an ultraviolet laser.

[0036] When performing ultraviolet irradiation treatment, it is preferable to use a lamp that emits ultraviolet light in the VUV region to the UV-C region, such as a mercury lamp that emits ultraviolet light with a wavelength of 185 nm or 254 nm or an excimer lamp that emits ultraviolet light with a wavelength of 172 nm, as a light source. When performing photocatalytic treatment, it is preferable to use a light source that emits ultraviolet light with a wavelength of 365 nm or less, and more preferably a light source that emits ultraviolet light with a wavelength of 254 nm or less. As the photocatalyst, preferably a titanium oxide photocatalyst, a titanium oxide photocatalyst activated with metal ions or metal colloids, etc. are used. Also, as the oxidizing agent, organic acids and inorganic acids can be used without particular limitation, but since highly concentrated acids are difficult to handle, they are preferably diluted to a concentration of 10% or less before use. The optimal ultraviolet treatment time, photocatalytic treatment time, and oxidizing agent treatment time can be appropriately determined according to various conditions such as the ultraviolet intensity of the light source used, the activity of the photocatalyst, the oxidizing power and concentration of the oxidizing agent.

[0037] In one embodiment, the carbon content of the cell adhesion region (including the underlying layer if present) is preferably lower than the carbon content of the cell non-adhesion region (including the underlying layer if present). Specifically, the carbon content of the cell adhesion region is preferably 20 to 99% of the carbon content of the cell non-adhesion region. Also, the value of the percentage (%) of carbon bonded to oxygen among the carbon in the cell adhesion region (including the underlying layer if present) is preferably a smaller value than the value of the percentage (%) of carbon bonded to oxygen among the carbon in the cell non-adhesion region (including the underlying layer if present). Specifically, the value of the percentage (%) of carbon bonded to oxygen among the carbon in the cell adhesion region is preferably 35 to 99% of the value of the percentage (%) of carbon bonded to oxygen among the carbon in the cell non-adhesion region. This is because when the seeded cells are arranged in a pattern on the substrate, the cell adhesiveness increases with an increase in the ultraviolet exposure amount, but if the adhesiveness is high at the time of cell recovery, it becomes difficult to peel off the cells and recovery becomes difficult.

[0038] In this specification, the "carbon content" of the cell adhesion region is defined as the "carbon content obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectrometer", and the "percentage of carbon bonded to oxygen" is defined as the "percentage of carbon bonded to oxygen obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectrometer".

[0039] In the substrate used in the present disclosure, the area of each cell adhesion region is larger than 0.785 mm 2 and preferably larger than 1.0 mm 2 or more, more preferably larger than 1.2 mm 2 or more, still more preferably larger than 1.5 mm 2 or more, most preferably larger than 1.7 mm 2 or more, and preferably smaller than 25 mm 2 or less, more preferably smaller than 15 mm 2 or less, still more preferably smaller than 10 mm 2 or less, most preferably smaller than 5 mm 2 or less, and is pattern-formed so as to be in the following range. Pluripotent stem cells are 0.785 mm2 By culturing while adhering to a cell adhesion region having a larger area, cells can be cultured while remaining adhered without detaching, and the induction of differentiation into intestinal structures is promoted. Also, pluripotent stem cells are cultured on a cell adhesion region having an area of 25 mm 2 By culturing while adhering to a cell adhesion region having an area of 25 mm or less, differentiation into intestinal structures can be effectively induced.

[0040] The shape of each cell adhesion region is not particularly limited. For example, it can be a polygon such as a quadrilateral, a circle, an ellipse, etc. Among them, a circle is preferable, and the diameter is larger than 1.0 mm, preferably 1.2 mm or more, more preferably 1.5 mm or more, preferably 6 mm or less, more preferably 4 mm or less, even more preferably 3 mm or less, and particularly preferably 2 mm or less. In one substrate, the plurality of cell adhesion regions may have the same area and / or shape as each other, or may be different, but it is preferable that both the area and the shape are the same.

[0041] Also, in the substrate, each cell adhesion region is surrounded by a cell non-adhesion region, that is, they are isolated from each other, and are preferably separated from each other by 0.75 mm or more, more preferably 1.5 mm or more. That is, the shortest distance between the cell adhesion regions (in the case of a circle, the distance between the centers of the two circles is the value obtained by adding the above value to the sum of the respective radii) is preferably 0.75 mm or more, more preferably 1.5 mm or more. By isolating each cell adhesion region by a certain distance or more, the cells in each cell adhesion region are cultured uniformly at a constant interval without forming cell-cell junctions with the cells in other cell adhesion regions, and an experimental system with high reproducibility can be constructed.

[0042] The proportion of the cell adhesion regions in the substrate is usually 5 to 80%, preferably 20 to 70%, more preferably 40 to 60%. Note that this proportion is calculated based on the entire substrate excluding the bottom surface of the dish even when the substrate is placed in a dish (petri dish) or the like. By ensuring that the amount of cells in the culture medium is above a certain level, cell death can be prevented, and by keeping it below a certain level, depletion of factors necessary for survival and the resulting damage to the cells can be prevented.

[0043] In addition, it is preferable that each cell adhesion region is arranged regularly at a certain interval, for example, in a grid pattern, with the same pitch in both the vertical and horizontal directions. By making the paracrine effect of the products from the cells in each cell adhesion region constant, the influence on differentiation can be made constant.

[0044] For example, a pattern having a plurality of circular cell adhesion regions can be formed by using a photomask having a plurality of circular openings, arranging the glass substrate on which the PEG thin film is formed so as to face the photomask, irradiating ultraviolet rays from the side of the photomask, and subjecting the region corresponding to the opening of the photomask in the PEG thin film to an oxidation treatment.

[0045] The substrate used in the present disclosure is preferably pre-coated for the purpose of promoting the adhesion of pluripotent stem cells to the cell adhesion regions. The pre-coating treatment can be performed by coating the substrate with an extracellular matrix (for example, collagen, fibronectin, proteoglycan, laminin, vitronectin, etc.), gelatin, lysine, peptide, a gel matrix containing them, serum, or the like. By pre-coating the substrate, the adhesion of pluripotent stem cells with low adhesiveness (for example, ES cells, iPS cells, etc.) to the cell adhesion regions can be promoted, and cell adhesion culture and differentiation induction can be effectively performed.

[0046] Similarly, for the purpose of promoting the adhesion of pluripotent stem cells to the cell adhesion region, it is preferable to seed feeder cells before seeding the pluripotent stem cells and culture them for about 24 hours, and then culture the pluripotent stem cells on the feeder cells. The feeder cells can be used without particular limitation as long as they are commonly used in the art, and examples thereof include fibroblasts. The feeder cells are seeded at a density of less than 1.26×10 5 cells / cm 2 , preferably at a density of less than 6.3×10 4 cells / cm 2 , and preferably at a density of 3.15×10 4 cells / cm 2 or more.

[0047] The above-mentioned precoat treatment and seeding of feeder cells may be performed independently or in combination. Preferably, either the precoat treatment or the seeding of feeder cells is performed.

[0048] (Step (b)) Step (b) is a step of seeding pluripotent stem cells on the cell culture substrate provided with the cell adhesion region and the cell non-adhesion region prepared in the above-mentioned step (a).

[0049] Examples of the pluripotent stem cells seeded on the cell culture substrate include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), induced pluripotent stem cells (iPS cells), stem cells derived from adult tissues, umbilical cord blood, or umbilical cords. These pluripotent stem cells may be used alone or in combination of two or more. In a preferred embodiment, ES cells and iPS cells are used alone as the pluripotent stem cells.

[0050] The ES cells used in the present disclosure are preferably ES cells derived from mammals. For example, ES cells derived from rodents such as mice or primates such as humans can be used. In a preferred embodiment, ES cells derived from mice or humans are used. ES cells refer to a stem cell line created from the inner cell mass belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development. In vitro, they can be proliferated almost infinitely while maintaining pluripotency to differentiate into theoretically all tissues. As ES cells, for example, in order to facilitate confirmation of the degree of their differentiation, cells into which a reporter gene has been introduced near the Pdx1 gene can be used. For example, an ES cell line derived from 129 / Sv in which the LacZ gene has been integrated into the Pdx1 locus, or ES cell line SK7 having a GFP reporter transgene under the control of the Pdx1 promoter can be used. Alternatively, ES cell line 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.

[0051] The iPS cells used in the present disclosure are pluripotent cells obtained by reprogramming somatic cells. Regarding the generation of induced pluripotent stem cells, multiple groups including the group of Professor Shinya Yamanaka of Kyoto University, the group of Rudolf Jaenisch of the Massachusetts Institute of Technology, the group of James Thomson of the University of Wisconsin, and the group of Konrad Hochedlinger of Harvard University have been successful. For example, International Publication WO2007 / 069666 describes nuclear reprogramming factors for somatic cells containing gene products of the Oct family genes, Klf family genes, and Myc family genes, and nuclear reprogramming factors for somatic cells containing gene products of the Oct family genes, Klf family genes, Sox family genes, and Myc family genes. Further described is a method for producing induced pluripotent stem cells by nuclear reprogramming of somatic cells, which includes a step of contacting the somatic cells with the above nuclear reprogramming factors.

[0052] The type of somatic cells used here is not particularly limited, and any somatic cells can be used. That is, in this specification, somatic cells include all cells other than germ cells among the cells constituting a living body, and may be differentiated somatic cells or undifferentiated stem cells. The origin of somatic cells may be any of mammals, birds, fish, reptiles, and amphibians, and is not particularly limited, but is preferably a mammal (for example, rodents such as mice, and primates such as humans), and particularly preferably a mouse or a human. Also, when using human somatic cells, any somatic cells of a fetus, newborn, or adult can be used. Examples of somatic cells include, for example, fibroblasts (for example, skin fibroblasts), epithelial cells (for example, gastric epithelial cells, liver epithelial cells, alveolar epithelial cells), endothelial cells (for example, blood vessels, lymphatic vessels), nerve cells (for example, neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (for example, skeletal muscle cells, smooth muscle cells, cardiomyocytes), hepatocytes, non-parenchymal liver cells, adipocytes, osteoblasts, cells constituting the periodontal tissue (for example, periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), cells constituting the kidney, eye, ear, etc.

[0053] iPS cells refer to stem cells that have the ability to self-renew over a long period under predetermined culture conditions (for example, under the conditions for culturing ES cells), and also have the ability to differentiate into ectoderm, mesoderm, and endoderm under predetermined differentiation induction conditions. Also, the iPS cells in the present disclosure may be stem cells that have the ability to form teratomas when transplanted into test animals such as mice.

[0054] In order to produce iPS cells from somatic cells, first, at least one type of reprogramming gene is introduced into the somatic cells. A reprogramming gene is a gene that encodes a reprogramming factor having the action of reprogramming somatic cells into iPS cells. Specific examples of combinations of reprogramming genes include, but are not limited to, the following combinations. (i) Oct gene, Klf gene, Sox gene, Myc gene (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 large T gene (iv) Oct gene, Klf gene, Sox gene

[0055] The pluripotent stem cells before seeding on the cell culture substrate shall be those maintained undifferentiated using an undifferentiation-inducing medium. Switch to a differentiation-inducing medium before and after seeding on the surface of the cell culture substrate, seed on the surface of the cell culture substrate, and allow the cells to grow until confluent within the cell adhesion region as it is. The target differentiation-induced cells can be obtained by subjecting the resulting cell aggregate to an enzyme treatment.

[0056] The undifferentiation-inducing medium used in the present disclosure is not particularly limited as long as it is a medium that does not induce differentiation of pluripotent stem cells. For example, a medium containing leukemia inhibitory factor known to have the property of maintaining the undifferentiated state of mouse ES cells and mouse iPS cells, a medium containing basic FGF known to have the property of maintaining the undifferentiated state of human iPS cells, etc. can be mentioned. The differentiation-inducing medium is not particularly limited as long as it is a medium that induces differentiation of pluripotent stem cells. For example, a serum-containing medium, a serum-free medium containing known components having properties to substitute for serum, etc. can be mentioned. Depending on the type of cells used, MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. can be used.

[0057] Various growth factors, antibiotics, amino acids, etc. may be added to the above-mentioned various culture media. For example, 0.1-2% pyruvic acid, 0.1-2% non-essential amino acids, 0.1-2% penicillin / streptomycin, 0.1-1% glutamine, 0.1-2% β-mercaptoethanol, 1-20 mM ROCK inhibitor (e.g., Y27632) may be added. Humoral factors may be added to the differentiation induction medium, but according to the method for inducing differentiation of intestinal structures in the present disclosure, intestinal structures can be induced to differentiate without adding humoral factors. Therefore, in one embodiment, cells are cultured using a differentiation induction medium that does not contain humoral factors.

[0058] The method of seeding pluripotent stem cells on a cell culture substrate is not particularly limited and can be seeded by a method commonly used when seeding cells. In the present disclosure, pluripotent stem cells are, for example, 1.2×10 5 cells / cm 2 less than, preferably 3×10 4 cells / cm 2 or less, preferably 1.5×10 4 cells / cm 2 or more, and are seeded at this density.

[0059] (Step (c)) Step (c) is a step of culturing the pluripotent stem cells seeded on the cell culture substrate in the medium to obtain intestinal structures.

[0060] The culture of pluripotent stem cells is usually carried out at 37°C. Preferably, the culture is carried out in an atmosphere with a CO2 concentration of about 5% using a CO2 cell culture device or the like.

[0061] The medium used in this step (c) is not particularly limited as long as it can induce the differentiation of pluripotent stem cells, and can be appropriately selected according to the type of pluripotent stem cells. Examples of the medium include MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. In addition, these media may be added with serum or properties that substitute for serum as necessary.

[0062] The culture period after seeding pluripotent stem cells on the cell culture substrate is preferably 60 days or more, more preferably 80 days or more, still more preferably 100 days or more, and preferably 210 days or less, more preferably 140 days or less. On the 60th day after seeding pluripotent stem cells on the cell culture substrate, it has been found that the intestinal differentiation markers Villin and 5TH are detected, and furthermore, a sac-like structure is confirmed and peristaltic-like movements are observed. The intestinal structure obtained by steps (a) to (c) contains at least endoderm-derived cells and mesoderm-derived cells, but may also contain other cells.

[0063] As described above, through each of the steps (a) to (c), an intestinal structure can be obtained that is prepared from pluripotent stem cells and contains endoderm-derived cells and mesoderm-derived cells. The obtained intestinal structure is subjected to the subsequent step (B).

[0064] <Step (B)> In step (B), the intestinal structure prepared in the above-described step (A) is cultured to proliferate fibroblast-like cells derived from the intestinal structure.

[0065] The culture form of the intestinal structure is not particularly limited as long as the effects of the present disclosure can be achieved. It may be a form in which cells are cultured by adhering to a culture vessel (so-called adherent culture), or a form in which cells are cultured without adhering to a culture vessel (so-called suspension culture). In a preferred embodiment, the intestinal structure is cultured in a state where at least a part of the intestinal structure is in contact with the culture vessel. The culture vessel and the intestinal structure may be in constant contact or may be in intermittent contact.

[0066] In one embodiment, the intestinal structure is suspended and cultured in a culture vessel, and by appropriately adjusting the amount of the culture medium added to the culture vessel, at least a part of the culture vessel (for example, the bottom surface, inner wall, etc.) is brought into contact with the intestinal structure. For example, when using a dish as the culture vessel, a small amount of the culture medium is added to the dish, and the intestinal structure is suspended and cultured so that at least a part of the intestinal structure contacts the bottom surface of the dish.

[0067] The culture medium used in this step (B) is not particularly limited as long as it can induce the differentiation and proliferation of fibroblasts. Examples include MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. In addition, serum or properties substituting for serum may be added to these culture media as necessary.

[0068] The culture of the intestinal structure is usually carried out at 37°C. Preferably, it is cultured in an atmosphere with a CO2 concentration of about 5% using a CO2 cell culture device or the like.

[0069] The culture of the intestinal structure is preferably carried out until the fibroblast-like cells derived from the intestinal structure become confluent or almost confluent. The culture period can be appropriately set according to the growth rate of the fibroblast-like cells, the shape and size of the culture vessel used, etc. The culture period of the intestinal structure can be, for example, 1 to 6 days, 6 to 12 days, 12 to 72 days, etc.

[0070] In this step (B), in addition to the above-described fibroblast-like cells, cells having properties of intestinal epithelial cells (hereinafter also referred to as "intestinal epithelial cell-like cells") may proliferate. Thus, the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B) have properties different from those of intestinal epithelial cells obtained by culturing organoids starting from conventional stem cells. For example, it has been shown by the inventors that in the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B), the cell division-related protein Ki-67, which is a proliferation cell marker, is locally present. Here, generally, intestinal epithelial cells are known to be generated only by differentiating from stem cells that are positive for Lgr5 (Leucine-rich repeat-containing G-protein-coupled receptor 5), which is known as an intestinal epithelial stem cell, and it is generally known that differentiated intestinal epithelial cells do not proliferate. Therefore, in the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B) as described above, the local presence of Ki-67, which is a proliferation cell marker, can be said to suggest that the intestinal epithelial cell-like cells divide to generate new intestinal epithelial cell-like cells. That is, the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B) are considered to have specific properties in that they may proliferate independently of conventional Lgr5-positive stem cells. In addition, as the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B), various types of cells responsible for the functions of the small intestine, such as secretory cells confirmed by the expression of specific mucin proteins and absorptive epithelial cells confirmed by the expression of specific transporters, can be obtained.

[0071] The fibroblast-like cells (i.e., the feeder cells of the present disclosure) and the intestinal epithelial cell-like cells derived from the intestinal structure obtained by this step (B) may be used in combination with each other or each may be used alone.

[0072] <Step (C)> In step (C), the fibroblast-like cells derived from the intestinal structure proliferated in the above-described step (B), i.e., the feeder cells of the present disclosure, are isolated.

[0073] The isolation of fibroblast-like cells can be carried out by the methods commonly used when isolating fibroblasts. That is, it can be carried out by steps such as known enzyme treatment, scraping with a scraper, etc. These steps may be carried out alone or in combination of two or more.

[0074] In one embodiment, the fibroblast-like cells obtained by this step (C) express at least one fibroblast marker selected from the group consisting of vimentin and CD90. In a preferred embodiment, the fibroblast-like cells obtained by this step (C) express at least one gene selected from the group consisting of ACTA2, SOX9, and DCN.

[0075] In one embodiment, in step (C), in addition to isolating fibroblast-like cells derived from the intestinal structure described above, isolation of intestinal epithelial cell-like cells derived from the intestinal structure is performed. Specifically, isolation of intestinal epithelial cell-like cells derived from the intestinal structure can be performed according to the following procedure. First, the medium containing the intestinal structure after isolating fibroblast-like cells as described above is made into an environment in which only intestinal epithelial cell-like cells can survive. For example, by adding antibiotics such as puromycin and penicillin to the medium at a concentration of 100 μg / ml, an environment is created in which only intestinal epithelial cell-like cells having the activity to metabolize these antibiotics (drug-metabolizing enzyme: CYP) can survive. Next, dead cells are removed from the medium to isolate intestinal epithelial cell-like cells. When hepatocytes are present in the medium, the hepatocytes can survive by adding the antibiotics as described above, but since hepatocytes can be easily discriminated under a microscope as a population of multinucleated cells, intestinal epithelial cell-like cells can be isolated by removing the hepatocytes under a microscope. Also, since hepatocytes are not stained by immunostaining using an antibody against CDX2, intestinal epithelial cell-like cells can also be isolated by discriminating and removing hepatocytes using such an antibody. Since intestinal epithelial cell-like cells adhere to each other with strong cell-cell adhesion, when intestinal epithelial cell-like cells form a layered structure with other cells, the intestinal epithelial cell-like cells can be isolated by peeling them off from other cells using forceps or the like.

[0076] <Step (D)> The method for producing feeder cells of the present disclosure may further include a step (step (D)) of proliferating the fibroblast-like cells isolated in step (C) in addition to the above-described steps (A) to (C).

[0077] The culture of the fibroblast-like cells isolated in step (C) can be performed by a method commonly used for culturing fibroblasts.

[0078] The medium used in this project (D) is not particularly limited as long as it can proliferate fibroblasts. For example, MEM medium, BME medium, DMEM medium, DMEM-F12 medium, αMEM medium, IMDM medium, ES medium, DM-160 medium, Fisher medium, F12 medium, WE medium, RPMI1640 medium, etc. can be mentioned. In addition, serum or properties substituting for serum may be added to these media as necessary.

[0079] The culture of fibroblast-like cells is usually carried out at 37°C. Preferably, it is cultured in an atmosphere with a CO2 concentration of about 5% using a CO2 cell culture device or the like.

[0080] The culture of fibroblast-like cells is preferably carried out until the fibroblast-like cells become confluent or almost confluent. The culture period can be appropriately set according to the growth rate of fibroblast-like cells, the shape and size of the culture vessel used, etc. The culture period of fibroblast-like cells can be, for example, 3 to 7 days, 7 to 12 days, etc.

[0081] [Feeder cells] According to one aspect of the present disclosure, fibroblast-like cells (i.e., the feeder cells of the present disclosure) are provided for maintaining cells such as intestinal epithelial cells, which are considered difficult to maintain and proliferate by two-dimensional culture. In one embodiment, the feeder cells of the present disclosure exhibit a specific gene expression profile. In one embodiment, the feeder cells of the present disclosure are characterized by expressing each of the genes for platelet-derived growth factor receptor (PDGFRA), CD81, forkhead box L1 (Foxl1), and gremlin-1 (GREM1). Further, in another embodiment, the feeder cells of the present disclosure are characterized by expressing each of the genes for PDGFRA, CD81, and GREM1 and not expressing the CD34 gene. In one preferred embodiment, the feeder cells of the present disclosure are characterized by expressing each of the genes for PDGFRA, CD81, Foxl1, and GREM1 and not expressing the CD34 gene. According to these feeder cells of the present disclosure, cells such as intestinal epithelial cells, which are considered difficult to maintain and proliferate by two-dimensional culture, can be cultured two-dimensionally to be maintained or proliferated.

[0082] Conventionally, research has been conducted on feeder cells for maintaining cells such as intestinal epithelial cells, which are considered difficult to maintain and proliferate by two-dimensional culture, in mice. However, feeder cells that can maintain cells such as intestinal epithelial cells alone (i.e., have complete feeder ability) have not been found so far, and currently, the maintenance of cells such as intestinal epithelial cells is achieved by using a combination of multiple types of feeder cells. In addition, typical feeder cells found conventionally include mouse Trophocyte, Telocyte, etc. It is known that Trophocyte expresses each of the genes PDGFRA, CD81, and CD34, but does not express the Foxl1 gene. Also, it is known that Telocyte expresses each of the genes PDGFRA and Foxl1, but does not express each of the genes CD81 and CD34. Therefore, cells that express each of the genes PDGFRA, CD81, Foxl1, and GREM1, or cells that express each of the genes PDGFRA, CD81, and GREM1 and do not express the CD34 gene, like the feeder cells of the present disclosure, have not been found in the first place, and the fact that such cells have excellent feeder ability for cells such as intestinal epithelial cells is a surprising discovery that has never been known conventionally.

[0083] In one embodiment, the feeder cells of the present disclosure can be obtained by undergoing the above-described steps (A) to (C) and optionally step (D). The feeder cells of the present disclosure can two-dimensionally culture and maintain or proliferate cells that have conventionally been considered difficult to maintain and proliferate by two-dimensional culture. In particular, the feeder cells of the present disclosure exhibit feeder ability for target cells over long-term culture (for example, passage 10 times or more) compared to conventional feeder cells.

[0084] The cells to which the feeder cells of the present disclosure are applied (hereinafter also referred to as "target cells") are not particularly limited, but are preferably cells that are considered difficult to maintain and proliferate by two-dimensional culture as described above. More specifically, they are preferably cells that are prone to undergo rapid dedifferentiation due to stress caused by passage or culture period during two-dimensional culture. Examples of such target cells include small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, chondrocytes, and the like. In one preferred embodiment, the target cells are small intestinal epithelial cells and hepatocytes. Therefore, in one embodiment, the feeder cells of the present disclosure are feeder cells for maintaining or proliferating each of the above-described cells by two-dimensional culture (plane culture). That is, the feeder cells of the present disclosure can suppress the loss of the properties of the target cells and allow them to proliferate as long as the target cells maintain their original properties even when the target cells are passaged multiple times (for example, 3 times or more) by two-dimensional culture. In the present disclosure, the feeder cells and the target cells may be derived from the same individual (the feeder cells are derived from pluripotent stem cells derived from a specific individual, and the target cells are derived from pluripotent stem cells or natural cells derived from the same individual), or may be derived from different individuals (the feeder cells are derived from pluripotent stem cells derived from a specific individual, and the target cells are derived from pluripotent stem cells or natural cells derived from another individual of the same species). Note that the "natural cells" refer to cells that exist in an individual and have not been subjected to artificial manipulation (that is, "natural"), and specifically include small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, chondrocytes, etc. collected from humans.

[0085] In one embodiment, the target cells are cells derived from pluripotent stem cells, preferably small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, chondrocytes derived from pluripotent stem cells, and more preferably small intestinal epithelial cells, hepatocytes derived from pluripotent stem cells.

[0086] In a preferred embodiment, the target cells are intestinal epithelial cell-like cells collected from the intestinal structure described above, and particularly preferably, they are intestinal epithelial cell-like cells collected from the same intestinal structure prepared in step (A) to obtain the feeder cells of the present disclosure. Thus, since both the feeder cells and the intestinal epithelial cell-like cells of the present disclosure are derived from the same intestinal structure produced from the same pluripotent stem cells, there are advantages as described below. As a first advantage, since the origins of the feeder cells and the intestinal epithelial cell-like cells are the same pluripotent stem cells, the safety of each can be easily confirmed. Specifically, before differentiating and inducing each of the feeder cells and the intestinal epithelial cell-like cells, it is only necessary to confirm the safety of the pluripotent stem cells from which they are derived, and the safety of the feeder cells and the intestinal epithelial cell-like cells after differentiation and induction can be confirmed. In addition, since the origins of the feeder cells and the intestinal epithelial cell-like cells are the same pluripotent stem cells, no components of different origins enter after differentiation and induction. Therefore, when using one or both of these cells after differentiation and induction, there is no problem of contamination with components of different origins, and in particular, in medical applications, they can be used as so-called "xeno-free" cells as they are.

[0087] In one embodiment, the intestinal epithelial cell-like cells collected from the intestinal structure described above express at least one marker of intestinal epithelial cells selected from the group consisting of CDX2, ECAD, ZO-1, and cytokeratin AE1 / AE3. In a preferred embodiment, the intestinal epithelial cell-like cells collected from the intestinal structure described above express at least one marker of intestinal epithelial cells selected from the group consisting of CDX2 and Villin.

[0088] When culturing target cells using the feeder cells of the present disclosure, the passage number of the feeder cells used is not particularly limited, and for example, it can be 2 or more times, 3 or more times, 5 or more times, 7 or more times, 10 or more times, 12 or more times, 15 or more times, etc. On the other hand, the passage number of the feeder cells used can be, for example, 20 or less times, 15 or less times, 12 or less times, etc.

[0089] The feeder cells of the present disclosure can be cultured alone to proliferate only the feeder cells. Furthermore, by co-culturing the feeder cells of the present disclosure with specific target cells, both the feeder cells and the target cells can be proliferated together.

[0090] By culturing the feeder cells of the present disclosure alone, a culture (cell population) containing the feeder cells of the present disclosure or a culture consisting of the feeder cells of the present disclosure can be obtained. Specifically, by two-dimensionally culturing the feeder cells of the present disclosure alone, a culture having a two-dimensional shape (e.g., sheet-like) containing the feeder cells of the present disclosure or a culture having a two-dimensional shape (e.g., sheet-like) consisting of the feeder cells of the present disclosure can be obtained. In this specification, with respect to "culture" and "co-culture", "containing" a specific cell means that cells other than the specific cell can be included in the culture, and "consisting of" a specific cell means that cells other than the specific cell are not intentionally included in the culture.

[0091] In addition, as described above, the feeder cells of the present disclosure can maintain or proliferate target cells, particularly cells that are considered difficult to maintain and proliferate by two-dimensional culture, by two-dimensional culture. Therefore, by culturing target cells using the feeder cells of the present invention (i.e., co-culturing the feeder cells of the present disclosure and the target cells), a co-culture having a two-dimensional shape (e.g., sheet-like) containing the feeder cells of the present invention and the target cells, or a co-culture having a two-dimensional shape (e.g., sheet-like) composed of the feeder cells of the present disclosure and the target cells can be obtained. In particular, by using the feeder cells of the present disclosure, cells that have conventionally been considered difficult to maintain and proliferate by two-dimensional culture can be two-dimensionally maintained or proliferated, so that even such cells can be obtained as a co-culture having a two-dimensional shape (e.g., sheet-like). For example, it can be obtained as a co-culture containing a layer of feeder cells and a layer of target cells. Further, by separating and removing some or all of the feeder cells of the present disclosure from such a co-culture, a culture containing target cells, or a culture composed of target cells can be obtained. Specifically, the feeder cells of the present disclosure are formed into a sheet shape, and on the sheet, target cells that are considered difficult to maintain and proliferate by two-dimensional culture are co-cultured to be maintained or proliferated, thereby obtaining a co-culture of the target cells having a two-dimensional shape (e.g., sheet-like). Then, by separating and removing some or all of the feeder cells of the present disclosure from the obtained co-culture, a culture having a two-dimensional shape containing target cells, or a culture having a two-dimensional shape composed of target cells can be obtained. On the other hand, by separating and removing some or all of the target cells from the co-culture obtained as described above, a culture having a two-dimensional shape containing the feeder cells of the present disclosure, or a culture having a two-dimensional shape composed of the feeder cells of the present disclosure can also be obtained.

[0092] In one embodiment, the target cells are cells such as intestinal epithelial cells (e.g., small intestinal epithelial cells, large intestinal epithelial cells, etc.), hepatocytes, chondrocytes, etc., that are considered difficult to maintain and proliferate by two-dimensional culture, and are preferably intestinal epithelial cells.

[0093] The separation of the feeder cells and the target cells of the present disclosure contained in the co-culture can usually be carried out using known methods used in the field of cell culture. In one embodiment, when the co-culture contains the feeder cells of the present disclosure and intestinal epithelial cell-like cells, the intestinal epithelial cell-like cells are isolated from the co-culture using the method disclosed in WO 2019 / 131938 pamphlet.

[0094] In one embodiment, such co-cultures of the feeder cells and the target cells of the present disclosure, co-cultures containing the target cells, and co-cultures containing the feeder cells can all be used as so-called cell sheets. A cell sheet is a thin membrane produced by culturing cells in a sheet shape, and has advantages such as being able to be transplanted into a living tissue without using sutures and being able to be transplanted in a short time. Taking advantage of such characteristics, it is used for the treatment of various diseases and tissue regeneration. Therefore, in one embodiment, the cell sheet is used for the treatment of diseases and as a transplantation material for tissue regeneration.

[0095] As described above, since the feeder cells of the present disclosure can maintain or proliferate target cells by two-dimensional culture, a co-culture having a two-dimensional shape such as a cell sheet can be easily produced. Further, by appropriately setting the size, shape, culture time, etc. of the culture vessel for co-culturing the feeder cells and target cells of the present disclosure, a cell sheet having a desired size and two-dimensional shape can be produced. Conventionally, for cells such as intestinal epithelial cells, it has been difficult to maintain and proliferate them by two-dimensional culture, so it has not been easy to produce a co-culture having a two-dimensional shape such as a cell sheet mainly containing these cells. Therefore, for example, when producing a large cell sheet or the like, it has been necessary to use a plurality of cell sheets having a relatively small two-dimensional shape that can be produced, combined with each other. On the other hand, by using the feeder cells of the present disclosure, even such cells can be maintained or proliferated by two-dimensional culture, and their size and shape can be appropriately set. Therefore, as described above, a large cell sheet can be produced without combining a plurality of cell sheets. For example, by using the feeder cells of the present disclosure, a huge cell sheet such as a circle, ellipse, or polygon (e.g., quadrilateral, etc.) having a diameter of 1 cm or more, 2 cm or more, 3 cm or more, 5 cm or more, 10 cm or more can be produced as a single cell sheet without combining a plurality of cell sheets. Thus, the ability to produce a large cell sheet without combining a plurality of cell sheets for cells that have conventionally been difficult to maintain and proliferate by two-dimensional culture can be said to be one of the excellent effects brought about by the feeder cells of the present disclosure. For example, in Crohn's disease, the disease state is judged based on the size (diameter) of the ulcer, and when the diameter is 2 cm or more, it is judged that healing is difficult. Therefore, it can be said that efficiently producing such a huge cell sheet as a single cell sheet can greatly contribute to the treatment of Crohn's disease and the like for which treatment with a cell sheet is effective.

[0096] In addition, a method is conventionally known in which progenitor cells at a stage prior to differentiating into specific cells are prepared from pluripotent stem cells or the like, proliferated in a growth medium by two-dimensional culture, and then promoted to differentiate by a differentiation medium to obtain a structure composed of functional cells. Here, when the target cells are cells that are likely to undergo rapid dedifferentiation due to stress caused by passage or culture period during two-dimensional culture, dedifferentiation occurs during the differentiation promotion stage by the differentiation medium, and usually, the properties of the target cells cannot be maintained over a long period of time. That is, a structure in which the properties of the target cells are maintained over a long period of time cannot be obtained. However, by using the feeder cells of the present disclosure, such dedifferentiation during the differentiation promotion stage can be suppressed, and the properties of the target cells can be maintained over a long period of time. Therefore, the feeder cells of the present disclosure can also be suitably used in applications such as structures (for example, transplantation materials, drug discovery tools, etc.) that require maintaining the properties of the target cells over a long period of time.

[0097] In addition, the feeder cells of the present disclosure can be prepared so as not to contain components derived from different species (so-called under xeno-free conditions). Therefore, when used as a transplantation material such as a cell sheet as described above, the risk due to components derived from different species during transplantation can be eliminated. In addition, as shown in the examples, since the primary intestinal epithelial cells collected from humans could be proliferated using the feeder cells of the present disclosure as a scaffold, it is presumed that transplantation of the feeder cells of the present disclosure alone can contribute to rapid autologous tissue regeneration.

[0098] In addition, the feeder cells of the present disclosure are considered to have various functions in addition to the function as a feeder cell as described above. Therefore, the applications of the feeder cells of the present disclosure are not limited to the applications as such feeder cells, and they can also be used in applications where functions different from those of the feeder cells are expected.

[0099] For example, according to one aspect of the present disclosure, there is provided a pharmaceutical composition comprising the feeder cells (fibroblast-like cells) of the present disclosure, or a co-culture of the feeder cells (fibroblast-like cells) and various cells such as small intestinal epithelial cells and hepatocytes. Here, the pharmaceutical composition refers to a preparation obtained by preparing the feeder cells (fibroblast-like cells) and co-cultures of the present disclosure as an oral preparation or a parenteral preparation according to a conventional method. For formulation, acceptable additives for formulation may be used in combination. Examples of acceptable additives for formulation include excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, coloring agents, fragrances, buffering agents, antioxidants, pH adjusters, and the like. When the pharmaceutical composition is an oral preparation, it can take the form of solid preparations such as tablets, powders, fine granules, granules, capsules, pills, sustained-release preparations, and liquid preparations such as solutions, suspensions, and emulsions. When the pharmaceutical composition is a parenteral preparation, it can take the form of injections, suppositories, sheet-like patches, suspension for application, sprays, and the like.

[0100] The dosage (application amount) of the pharmaceutical composition is not particularly limited as long as the effects of the present disclosure are achieved, and can be appropriately adjusted according to the age, health status, body weight, etc. of the administration (application) subject. For example, when the feeder cells of the present disclosure are used as a sheet-like patch for treating mucosal ulcers in a subject, the patch is applied so as to completely cover the ulcer part and its peripheral part. When the ulcer is large, a plurality of patches are combined and applied so as to completely cover the ulcer part and its peripheral part.

[0101] The feeder cells (fibroblast-like cells) and co-cultures are preferably continuously ingested (administered) over a long period of time in order to better exert their effects, and the ingestion (administration) period can be, for example, 1 to 6 weeks, 1 to 12 weeks, 2 to 10 weeks, 4 to 10 weeks, 4 to 12 weeks, and the like. As used herein, "continuously" means continuously ingesting (administering) a determined amount of the pharmaceutical composition every day.

[0102] According to another aspect of the present disclosure, there is provided a method for treating a disease of a subject, which includes administering an effective amount of feeder cells (fibroblast-like cells) or co-cultures to the subject. The above method may be a non-therapeutic method or a therapeutic method.

[0103] In the method for treating a disease, the dosage and administration period of feeder cells (fibroblast-like cells) or co-cultures are not particularly limited as long as the effects of the present disclosure are achieved, and can be appropriately adjusted according to the type of disease, the age, health status, body weight, etc. of the administration subject.

[0104] According to another aspect of the present disclosure, there is provided a method for treating a disease of a subject, which includes applying the above-described cell sheet to the subject. The above method may be a non-therapeutic method or a therapeutic method.

[0105] In the method for treating a disease, the shape and size of the cell sheet are not particularly limited as long as the effects of the present disclosure are achieved, and can be appropriately adjusted according to the type of disease, the type of organ or tissue to which it is applied, the shape and size of the part to which it is applied, etc.

[0106] According to another aspect of the present disclosure, there is provided the use of the feeder cells (fibroblast-like cells) or co-cultures of the present disclosure for the manufacture of a composition for treating a disease.

[0107] In each of the above aspects, examples of diseases that can be treated using the feeder cells (fibroblast-like cells), co-cultures, and cell sheets of the present disclosure include diseases accompanied by ulcers in the digestive tract such as Crohn's disease and ulcerative colitis, and complications associated with digestive tract surgery such as after colon cancer surgery.

[0108] In each of the above-described aspects, the cell sheet containing the feeder cells (fibroblast-like cells) of the present disclosure, the cell sheet containing the feeder cells (fibroblast-like cells) and the co-culture, and the cell sheet containing the co-culture can be used for transplantation into the body of a human or non-human animal, particularly for covering inflamed parts, wound parts, lesion parts, suture parts after surgery, the abdominal cavity, and defective intestinal tissues, etc. in organs and the like.

[0109] In particular, when the cell sheet containing the feeder cells (fibroblast-like cells) of the present disclosure (i.e., the cell sheet of the present disclosure) has mucus-secreting ability, specifically mucin-secreting ability, it is preferable because it has a high effect of protecting the applied part (for example, the healing part in ulcers, etc.) of the cell sheet of the present disclosure. The cell sheet of the present disclosure can preferably protect the applied part by applying it to an inflamed part, a wound part, a lesion part, a suture part after surgery, the abdominal cavity, etc. and allowing it to heal.

[0110] The cell sheet of the present disclosure can preferably suppress leakage from the healing part, leakage into the healing part, or invasion into the healing part by applying it to an inflamed part, a wound part, a lesion part, a suture part after surgery, or the abdominal cavity and allowing it to heal.

[0111] The cell sheet of the present disclosure can preferably provide cells and a scaffold for cells necessary for the healing of the healing part by applying it to an inflamed part, a wound part, a lesion part, a suture part after surgery, or the abdominal cavity and allowing it to heal.

[0112] The cell sheet of the present disclosure can preferably prevent adhesion of the healing part to ectopic sites by applying it to an inflamed part, a wound part, a lesion part, a suture part after surgery, or the abdominal cavity and allowing it to heal.

[0113] The cell sheet of the present disclosure can preferably suppress immune reactions such as suppression of infiltration of blood cell-derived cells into the digestive tract tissue due to the body's own immune reaction by applying it to an inflamed part, a wound part, a lesion part, a suture part after surgery, or the abdominal cavity and allowing it to heal.

[0114] The cell sheet of the present disclosure can preferably alleviate enhanced inflammation at the site of healing by being adhered to an inflamed area, a wound area, a lesion area, a suture area after surgery, or the abdominal cavity. The cell sheet of the present disclosure can preferably prevent postoperative complications by being adhered to a suture area after surgery. The cell sheet of the present disclosure can preferably replace intestinal function by being transplanted into a damaged intestinal tissue.

[0115] The cell sheet of the present disclosure can be held by forceps or the like and attached to a desired transplantation site in any of endoscopic surgery, laparotomy, laparoscopic surgery, etc. When transplanting to a site where transplantation is difficult in laparoscopic surgery or the like, the cell sheet of the present disclosure may be transplanted in a state integrated with a support, or may be held and transplanted by a carrier such as a wire. The cell sheet of the present disclosure can be fixed to a biocompatible support and transplanted together with the support.

[0116] When transplanting the cell sheet of the present disclosure into a living body, a bioadhesive auxiliary substance having biocompatibility such as fibrin may be applied to the cell sheet of the present disclosure and / or the transplantation site. Also, the cell sheet of the present disclosure and the transplantation site may be sutured.

[0117] When attaching the cell sheet of the present disclosure to a transplantation site, for example, the cell sheet of the present disclosure may be spread with forceps and placed on the transplantation site for attachment, or the cell sheet of the present disclosure may be spread after being placed on the transplantation site. Also, the cell sheet of the present disclosure can be attached by pressing it against the transplantation site with a jig having a balloon structure.

[0118] Another aspect of the present disclosure relates to the following [1] to

[28] . [1] A method for producing feeder cells for maintaining or proliferating cells, comprising: (A) A step of preparing an intestinal structure produced from pluripotent stem cells and containing endoderm-derived cells and mesoderm-derived cells, (B) A step of culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure, and (C) A step of isolating the fibroblast-like cells to obtain feeder cells A method comprising the same. [2] The intestinal structure is (a) A step of preparing a cell culture substrate comprising a substrate, a plurality of isolated cell adhesion regions formed on the substrate, and a cell non-adhesion region surrounding each of the cell adhesion regions, (b) A step of seeding pluripotent stem cells on the cell culture substrate, and (c) A step of culturing the seeded pluripotent stem cells in a medium It is produced by a method comprising the same, The method according to [1]. [3](D) The method according to [1] or [2], further comprising a step of proliferating the isolated fibroblast-like cells. [4] In the step (B), the intestinal structure is cultured in a culture vessel, and at least a part of the intestinal structure is in contact with the culture vessel. The method according to any one of [1] to [3]. [5] The method according to [2], wherein the culture period in the step (c) is 60 days or more. [6] Feeder cells produced by the method according to any one of [1] to [5]. [7] Fibroblast-like cells that are positive for PDGFRA and CD81 and express Foxl1 and GREM1. [8] The fibroblast-like cells according to [7], which are positive for PDGFRA and CD81, negative for CD34, and express Foxl1 and GREM1. [9] Fibroblast-like cells according to [7] or [8], which are derived from pluripotent stem cells or are produced from pluripotent stem cells, and are derived from a culture of an intestinal structure containing endoderm-derived cells and mesoderm-derived cells.

[10] The intestinal structure is (a) A step of preparing a cell culture substrate comprising a substrate, a plurality of isolated cell adhesion regions formed on the substrate, and a cell non-adhesion region surrounding each of the cell adhesion regions, (b) A step of seeding pluripotent stem cells on the cell culture substrate, and (c) culturing the seeded pluripotent stem cells in a medium which is prepared by a method comprising the fibroblast-like cells described in [9]. The fibroblast-like cells according to [9] or

[10] , wherein the pluripotent stem cells are of human origin. The feeder cells according to [6] or the fibroblast-like cells according to any one of [7] to

[11] , which are feeder cells for maintaining or proliferating at least one cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes. The feeder cells according to [6] or the fibroblast-like cells according to any one of [7] to

[12] , which are feeder cells for maintaining or proliferating the cells by planar culture. The feeder cells according to [6] or the fibroblast-like cells according to any one of [7] to

[13] , which are feeder cells capable of subculturing the cells three or more times. A cell sheet comprising the feeder cells according to [6] or the fibroblast-like cells according to any one of [7] to

[14] . A co-culture of at least one cell selected from the group consisting of small intestinal epithelial cells and hepatocytes and the feeder cells according to [6] or the fibroblast-like cells according to any one of [7] to

[14] . The co-culture according to

[16] , wherein the number of subcultures of the cells is three or more times. A cell sheet comprising the co-culture according to

[16] or

[17] . The cell sheet according to

[18] , which is circular or substantially circular with a diameter of 1 cm or more. The cell sheet according to

[18] or

[19] , which comprises a layer of at least one cell selected from the group consisting of small intestinal epithelial cells and hepatocytes. The cell sheet according to any one of

[18] to

[20] , which further comprises a layer of the feeder cells. A transplantation material comprising the cell sheets according to

[15] and any one of

[18] to

[21] .

[23] A method for maintaining or proliferating cells, comprising the step of culturing the cells in the presence of the feeder cells described in [6] or fibroblast-like cells described in any one of [7] to

[14] .

[24] The method according to

[23] , wherein the culturing of the cells is a planar culture.

[25] The method according to

[23] or

[24] , wherein the cells are at least one kind of cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

[26] The method according to any one of

[23] to

[25] , wherein the cells and the feeder cells are derived from the same individual.

[27] The method according to any one of

[23] to

[26] , wherein the cells and the feeder cells are derived from different individuals.

[28] Cells that express at least one gene selected from the group consisting of DCN, ACTA2, and NOG, and express the genes of ADAMTS19, ANO1, BLID, LOC100507053, LHX8, SFRP4, AHRR, SLC14A1, FMO3, PZP, ABCG4, EYA2, TMEM92-AS1, ANO10, EGFL6, SNCAIP, LGSN, PCDHB15, and KRTAP1-5.

Example

[0119] Hereinafter, the present disclosure will be described in more detail with reference to examples, but the present disclosure is not limited to these examples.

[0120] [Example 1] Preparation of cell culture substrate 39.0 g of toluene and 13.5 g of methacryloylsilane TSL8370 (manufactured by GE Toshiba Silicone) were mixed, and 450 μl of triethylamine was added while stirring. After stirring at room temperature for several minutes as it was, the whole amount was transferred to a glass dish. A 5 cm square glass substrate washed with UV was immersed herein and left at room temperature for 16 hours. Thereafter, the glass substrate was washed with ethanol and water and dried by nitrogen blowing. Thereby, a thin film containing a methacryloyl group was formed on the surface of the glass substrate.

[0121] 10 g of polyethylene glycol diacrylate (PEGdA, manufactured by Aldrich) was dissolved with 0.1 g of a polymerization initiator 2,2'-dimethoxy-2-phenyl-acetophenone (DMPA, manufactured by Aldrich) at room temperature. This was spin-coated onto the methacryloylated substrate at 1500 rpm for 5 seconds. Then, immediately, ultraviolet rays were irradiated onto the entire surface of the substrate for 3 seconds under a nitrogen atmosphere. Subsequently, post-baking was performed at 160 °C for 10 minutes. This PEGdA-coated substrate was immersed in water overnight, then washed with water and dried. The average dry film thickness was 0.33 μm.

[0122] A photomask having a pattern with a plurality of circular openings with a diameter of 1.5 mm was used with a 5-inch size. The space between the openings of the photomask, that is, the shortest distance between the openings, was all 0.35 mm.

[0123] The mask was gently placed on the PEGdA surface of the PEGdA-coated substrate, and vacuum ultraviolet rays using a xenon excimer lamp (172 nm, 10 mW / cm 2 ) as a light source were irradiated for 1 minute. Thereby, the region corresponding to the opening of the photomask on the surface of the PEGdA film was oxidized. Next, the substrate was cut into 2.5 cm squares and used as a cell culture substrate.

[0124] The shape of the cell adhesion region in the obtained cell culture substrate was circular, its diameter was 1.5 mm, and the space between the cell adhesion regions, that is, the shortest distance between the cell adhesion regions, was all 0.35 mm.

[0125] [Example 2] Preparation of intestinal structure Pluripotent stem cells (SEES-2 cells, an ES cell line established by the National Center for Child Health and Development, Japan) were placed on the surface of the cell culture substrate prepared in Example 1 at about 1 × 10 7 cells / cm 2Seeds were sown and cultured for 60 days in an incubator under the conditions of 37 °C and a CO2 concentration of 5% for induction of differentiation. For the culture of pluripotent stem cells, XF32 medium was used. After 60 days of differentiation induction, the cell suspension was embedded in a gel using a cell suspension gelation reagent (iPGell, manufactured by Genostaff Co., Ltd.), and the gel-embedded culture was cut to prepare sections of the culture. The composition of the XF32 medium used for the culture of pluripotent stem cells is shown in Table 1 below.

Table 1

[0126] The sections of the obtained culture were immunostained using antibodies against Villin and 5TH, which are intestinal differentiation markers. As a result, Villin and 5TH were detected in the cell adhesion regions, and it was confirmed that a pouch-like structure (i.e., intestinal structure) was formed (Figure 1). Furthermore, since it was confirmed that the pouch-like structure exhibited peristaltic movement, it was confirmed that cells derived from the endoderm, mesoderm, and ectoderm necessary for peristaltic movement were present. In addition, as a result of observation with a stereomicroscope, peristaltic-like movements in the pouch-like structure were also observed, and the presence of Cajal cells, which are ectoderm, intestinal epithelial cells, which are endoderm, and smooth muscle, which is mesoderm, was confirmed.

[0127] [Example 3] Maintenance and proliferation of fibroblast-like cells and intestinal epithelial cells derived from intestinal structures 1 The intestinal structures prepared in Example 2 were transferred to another adhesive culture dish and cultured for 17 days under the conditions in an incubator at 37 °C and a CO2 concentration of 5% to proliferate fibroblast-like cells and intestinal epithelial-like cells derived from the intestinal structures. For the culture of the intestinal structures, XF32 medium was used. In culturing the intestinal structures, the amount of the medium was adjusted so that at least a part of it was always in contact with the bottom surface of the culture vessel.

[0128] Only the obtained fibroblast-like cells were subcultured by changing the medium twice a week and passaging once a week using D-MEM supplemented with 10% FBS, and a large stock of passage 7 was obtained. The obtained stock was divided into two, and each was stored in a -80°C freezer or in the gas phase of a tank containing liquid nitrogen. The stock stored in the tank containing liquid nitrogen was transferred to a -80°C freezer and then used. These fibroblast-like cells and intestinal epithelial cell-like cells were co-cultured for 5 days. The co-culture was performed by first culturing the fibroblast-like cells in D-MEM supplemented with 10% FBS for about 1 week, and then seeding the intestinal epithelial cell-like cells on the fibroblast-like cells. For the culture after seeding the intestinal epithelial cell-like cells, ESTEM-HE medium (GlycoTechnica) containing R-spondin 1 and Wnt3a was used. After culturing for 21 days (3 passages), the co-culture of the intestinal epithelial cell-like cells and the passage 7 fibroblast-like cells cultured for 1 week and replaced every passage was immunostained with antibodies against CDX2, a marker of intestinal epithelial cells, and Vimentin, a marker of fibroblasts. As a result, CDX2 and Vimentin were detected (Figure 2). In Figure 2, the lower left photo shows the immunostaining photo of CDX2, the upper right is the staining photo with DAPI, the lower right is the immunostaining photo using the antibody against Vimentin, and the upper left is the merged photo of the above three photos. Also, in each photo of Figure 2, the scale bar indicates 100 μm. From the photos in Figure 2, it was shown that when fibroblast-like cells and intestinal epithelial cell-like cells were co-cultured, the intestinal epithelial cell-like cells could be maintained and proliferated. Since Vimentin is a marker of mesoderm, and intestinal epithelial cells with the expression of CDX2 and Villin are considered to be derived from endoderm, Figures 1 and 2 can be said to support the presence of each cell derived from endoderm and mesoderm in the intestinal structure.

[0129] On the one hand, the obtained intestinal epithelial cell-like cells were subcultured alone for 14 days with two passages. The ESTEM-HE medium was used for the culture. After culturing for 14 days (two passages), the culture was immunostained with an antibody against CDX2, which is a marker for intestinal epithelial cells. As a result, CDX2 was detected only in extremely limited cells (Figure 3). In Figure 3, the lower left photograph shows the immunostaining photograph of CDX2, the upper right is the staining photograph with DAPI, and the upper left is the photograph obtained by merging the above two photographs. Also, in each photograph of Figure 3, the scale bar indicates 100 μm. From the photographs in Figure 3, it was shown that when the intestinal epithelial cell-like cells were cultured alone, the intestinal epithelial cell-like cells could not be maintained and the cells having such properties could not proliferate.

[0130] [Example 4] Maintenance and proliferation of fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures 2 Fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, the intestinal epithelial cell-like cells at passage 12 and the fibroblast-like cells at passage 7 were co-cultured two-dimensionally for 7 days. The ESTEM-HE medium was used for the culture. The co-culture was embedded in an IP gel. The co-culture embedded and fixed in the IP gel was sliced to obtain a section of the co-culture, and the obtained section was immunostained with an antibody against CDX2, which is a marker for intestinal epithelial cells. As a result, CDX2 was detected (Figure 4). In the photograph of Figure 4, the scale bar indicates 50 μm.

[0131] [Example 5] Cell polarity of fibroblast-like cells and intestinal epithelial cells derived from intestinal structures Fibroblast-like cells and intestinal epithelial cell-like cells derived from the intestinal structure prepared by the same method as in Example 3 were co-cultured. Specifically, the passage 12 intestinal epithelial cell-like cells and passage 7 fibroblast-like cells were two-dimensionally co-cultured for 7 days. The ESTEM-HE medium was used for the culture. The co-culture was embedded in an IP gel. The co-culture embedded and fixed in the IP gel was sliced to obtain a section of the co-culture, and the obtained section was immunostained with an antibody against Villin, which is a marker for intestinal epithelial cells. As a result, Villin was detected (Fig. 5). Furthermore, it was confirmed that Villin was localized on the luminal side (i.e., the Apical side) of the intestinal epithelium in the living body (Fig. 5). This is the same as the localization of Villin in the intestinal epithelium of the living body. In Fig. 5, the upper left photo shows the immunostaining photo of Villin, the upper right is the staining photo by DAPI, and the lower left is the photo obtained by merging the above two photos. Also, in each photo of Fig. 5, the scale bar indicates 50 μm in all cases.

[0132] [Example 6] Maintenance of Intestinal Epithelial Cells by Fibroblast-like Cells Derived from Intestinal Structure Passage 7 fibroblast-like cells derived from the intestinal structure prepared by the same method as in Example 3 were co-cultured with primary intestinal epithelial cells collected from humans. The culture was carried out by first culturing the fibroblast-like cells in D-MEM supplemented with 10% FBS for about 1 week, and then seeding the intestinal epithelial cells on the fibroblast-like cells. The ESTEM-HE medium was used for the culture after seeding the intestinal epithelial cells. Microscopic photos of the cells at each time point of passage 2 (day 5 of culture) and passage 4 (day 4 of culture) are shown in Figs. 6 and 7, respectively. On the other hand, co-culture of primary intestinal epithelial cells collected from humans was carried out under the same conditions as described above, except that mouse embryonic fibroblasts (MEFs), which are commonly used feeder cells, were used instead of the fibroblast-like cells derived from the intestinal structure as feeder cells. Microscopic photos of the cells at each time point of passage 2 (day 5 of culture) and passage 4 (day 4 of culture) are shown in Figs. 8 and 9, respectively. In each photo of Figs. 6 to 9, the scale bar indicates 100 μm in all cases.

[0133] From the comparison between FIGS. 6 and 7, it was shown that primary intestinal epithelial cells collected from humans can be maintained and proliferated by fibroblast-like cells derived from intestinal structures. In particular, it is noteworthy that primary intestinal epithelial cells that are not intestinal epithelial cells derived from intestinal structures can be maintained and proliferated.

[0134] On the other hand, from the comparison between FIGS. 8 and 9, it was shown that primary intestinal epithelial cells collected from humans cannot be maintained and proliferated by feeder cells that are not fibroblast-like cells derived from intestinal structures. In particular, in FIG. 9, the cells have clearly changed to a flat shape, which is a typical change in the case of dedifferentiation.

[0135] [Example 7] Maintenance of hepatocytes by fibroblast-like cells derived from intestinal structures Fibroblast-like cells at passage 7 derived from intestinal structures prepared by the same method as in Example 3 were co-cultured with hepatocytes. The culture was performed by first culturing the fibroblast-like cells in D-MEM supplemented with FBS to a concentration of 10 for about one week, and then seeding the hepatocytes on the fibroblast-like cells. ESTEM-HE medium was used for the culture after seeding the hepatocytes. A microscopic photograph of the cells on the 8th day after the start of the culture is shown in FIG. 10.

[0136] Since polygonal cells (arrows) characteristic of stem cells were observed in the center of the photograph in FIG. 10, it was shown that hepatocytes can be maintained and proliferated by fibroblast-like cells derived from intestinal structures.

[0137] Also, usually, for the culture of hepatocytes, an appropriate coating of the culture vessel and feeder cells such as mouse embryonic fibroblasts (MEFs) are required. However, it was shown that hepatocytes can be cultured without the need for these by using the feeder cells of the present disclosure. This result suggests that the feeder cells of the present disclosure are excellent as a culture substrate, and also suggests an application of isolating and using only the extracellular matrix (ECM) produced by the feeder cells of the present disclosure by means such as decellularization treatment.

[0138] [Example 8] Gene analysis 1 of fibroblast-like cells derived from intestinal structures For fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures prepared by the same method as in Example 3, gene expression of the intestine and site-specific cells constituting the intestine was analyzed. Furthermore, gene expression with various types of fibroblasts in vivo was analyzed. The results are shown in Fig. 11. In Fig. 11, "RYU" and "LONG" represent the gene expression of intestinal epithelial cell-like cells and fibroblast-like cells derived from intestinal structures prepared by the same method as in Example 3, respectively. Also, "human_SI" represents gene expression based on total RNA of the human small intestine, and DuSMFs, DuSPFs, ILSMFs, and ILSPFs represent the gene expression of fibroblasts near the mucosa and fibroblasts near the serosa in the duodenal submucosa, duodenal subperitoneum, ileal submucosa, and ileal subperitoneum, respectively.

[0139] From the results shown in Fig. 11, it was shown that fibroblast-like cells derived from intestinal structures express all of the genes expressed by the intestine and site-specific cells constituting the intestine shown in the figure. Also, it was shown that fibroblast-like cells derived from intestinal structures have a gene expression pattern that is significantly different from that of various types of fibroblasts in vivo with respect to the genes expressed by the intestine and site-specific cells constituting the intestine. From these results, it was suggested that fibroblast-like cells derived from intestinal structures, unlike various types of fibroblasts in vivo, can assist in the maintenance and / or proliferation of cells such as intestinal epithelial cells constituting the intestine.

[0140] [Example 9] Gene analysis 2 of fibroblast-like cells derived from intestinal structures For fibroblast-like cells and intestinal epithelial cell-like cells derived from the intestinal structure prepared by the same method as in Example 3, gene expression of the intestine and site-specific cells constituting the intestine was analyzed. Furthermore, gene expression based on the total RNA of the human small intestine was analyzed. The results are shown in Fig. 12-1. In Fig. 12-1, "RYU01", "RYU02", and "RYU03" all represent the gene expression of intestinal epithelial cell-like cells derived from the intestinal structure prepared by the same method as in Example 3. Also, "LONG01" represents the gene expression of fibroblast-like cells derived from the intestinal structure prepared by the same method as in Example 3. Also, "human_SI" represents the gene expression based on the total RNA of the human small intestine.

[0141] From the results shown in Fig. 12-1, it was shown that fibroblast-like cells derived from the intestinal structure express the genes NOG, HGF, WNT5A, and GREM1, which are genes expressed by site-specific cells constituting the intestine and the intestine shown in the figure. Here, NOG, WNT5A, and GREM1 are known to act as factors for maintaining intestinal epithelial cells (see Hans Clevers et al., Gastroenterology, 2012;143:1518-1529, “Redundant Sources of Wnt Regulate Intestinal Stem Cells and Promote Formation of Paneth”). Also, HGF is known to contribute to the maintenance of the homeostasis of colonic epithelial cells. Therefore, from these results, it was suggested that fibroblast-like cells derived from the intestinal structure may assist in the maintenance and / or proliferation of cells such as intestinal epithelial cell-like cells constituting the intestine.

[0142] Also, for fibroblast-like cells and intestinal epithelial cell-like cells derived from the intestinal structure prepared by the same method as in Example 3, gene expression of the WNT family, which are various signal transduction proteins, was analyzed. The results are shown in Fig. 12-2. In Fig. 12-2, "LONG01" represents the gene expression of fibroblast-like cells derived from the intestinal structure prepared by the same method as in Example 3. Also, "human_SI" represents the gene expression based on the total RNA of the human small intestine.

[0143] From the results shown in FIG. 12-2, it was shown that fibroblast-like cells derived from the intestinal structure have a difference in the expression pattern of WNT family genes compared to the human small intestine. From this result, it was suggested that fibroblast-like cells derived from the intestinal structure are different from human small intestine, that is, natural fibroblasts.

[0144] [Example 10] Hierarchical clustering by comprehensive gene analysis of fibroblast-like cells derived from intestinal structures Hierarchical clustering by comprehensive gene expression analysis was performed on fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures prepared by the same method as in Example 3. Furthermore, hierarchical clustering by comprehensive gene expression analysis was also performed on primary intestinal epithelial cells collected from humans. The results are shown in FIG. 13. In FIG. 13, "Ryu cells" and "Long cells" mean intestinal epithelial cell-like cells and fibroblast-like cells derived from intestinal structures, respectively.

[0145] From the results shown in FIG. 13, in Ryu cells, which are intestinal epithelial cells co-cultured with Long cells, which are fibroblast-like cells derived from intestinal structures, no correlation was found between the passage number and the gene expression pattern in comparison with human primary intestinal epithelial cells (human primary intestinal epithelial cells). From this result, it is strongly suggested that in Ryu cells, which are intestinal epithelial cell-like cells co-cultured with Long cells, which are fibroblast-like cells derived from intestinal structures, dedifferentiation due to the passage number has not occurred (that is, Ryu cells are maintained).

[0146] [Example 11] Barrier property of cell sheets of intestinal epithelial cell-like cells derived from intestinal structures Fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures prepared by the same method as in Example 3 were cultured on Transwell for 5 days until confluent, and then intestinal epithelial cell-like cells were 6.0×10 4 / Wells were further seeded to form a well, and co-cultured two-dimensionally for 7 days. For the culture, D-MEM medium supplemented with 15% FBS was used. After the culture, the obtained co-culture was used as a cell sheet, and its barrier property (endothelial cell resistance value) was measured using a chopstick-type electrode. After inserting the electrode into the cup, the resistance value at the time point 20 seconds after insertion was read and confirmed. The results are shown in Fig. 14. In Fig. 14, "Caco2" is a cell sheet of Caco-2 cells generally used as a drug discovery tool, and 6.0×10 4 / Wells were seeded to form a well and cultured for 11 days, and "Long05" represents a cell sheet of fibroblast-like cells derived from intestinal structures. Also, "RYU01", "RYU04", "RYU05" and "RYU07" refer to different intestinal epithelial cell-like cells prepared using different ES cells, and "RYU05 only" is a sample in which puromycin was used 2 days before the measurement of the barrier property (endothelial cell resistance value), and only the fibroblast-like cell layer was caused to undergo cell death to such an extent that the sheet did not peel off.

[0147] From the results shown in Fig. 14, it was shown that the cell sheets of "RYU05 only", "RYU01", "RYU04", "RYU05" and "RYU07" obtained by co-culturing fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures have a barrier property (endothelial cell resistance value) comparable to or higher than that of the cell sheet of Caco-2 cells generally used as a drug discovery tool.

[0148] [Example 12] Shape and Transporter Expression of Co-Culture of Fibroblast-Like Cells and Intestinal Epithelial Cell-Like Cells Derived from Intestinal Structures 1 Fibroblast-like cells and intestinal epithelial cell-like cells derived from an intestinal structure prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage 12 and fibroblast-like cells at passage 7 were co-cultured two-dimensionally for 7 days. For the culture, ESTEM-HE medium was used. After the culture was completed, the shape of the obtained co-culture was observed with a microscope, and the co-culture was further embedded in an IP gel. The co-culture embedded and fixed in the IP gel was sliced to obtain a section of the co-culture, and the obtained section was immunostained with antibodies against PEPT1, BCRP, and SLC10A2, which are transporters of intestinal epithelial cells, to confirm the expression of each. The results are shown in Fig. 15. In each photograph of Fig. 15, the scale bar indicates 50 μm in all cases.

[0149] From the photographs shown in Fig. 15, it was confirmed that the co-culture of fibroblast-like cells and intestinal epithelial cell-like cells derived from the structure takes a shape similar to that of the intestinal epithelium, and that the transporters are localized on the luminal side (i.e., the Apical side) of the living intestinal epithelium, similar to that in the living intestinal epithelium. These photographs suggest that the co-culture of fibroblast-like cells and intestinal epithelial cell-like cells derived from the intestinal structure has a functional similarity to the mucosal structure of the intestine.

[0150] [Example 13] Expression of Transporters and Metabolic Enzymes in Fibroblast-Like Cells and Intestinal Epithelial Cell-Like Cells Derived from an Intestinal Structure Fibroblast-like cells and intestinal epithelial cell-like cells derived from an intestinal structure prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage 12 and fibroblast-like cells at passage 7 were co-cultured two-dimensionally for 7 days. ESTEM-HE medium was used for the culture. For fibroblast-like cells and intestinal epithelial cell-like cells at each passage, the expression of ABCB1, a transporter of intestinal epithelial cells, and metabolic enzymes CES2, CYP3A4, UGT1A8, and UGT1A6 was confirmed. The results are shown in Fig. 16. In Fig. 16, "RYU" represents the gene expression of intestinal epithelial cell-like cells derived from the intestinal structure, "LONG" represents the gene expression of fibroblast-like cells derived from the intestinal structure, and "human_SI" represents the gene expression based on total RNA of the human small intestine. Also, "5653" indicates that the cells were treated with puromycin.

[0151] From the results shown in Fig. 16, it was confirmed that in "RYU", which is an intestinal epithelial cell-like cell derived from the intestinal structure, the transporter ABCB1 and metabolic enzymes CES2, UGT1A8, and UGT1A6 were expressed. Also, since all of the RYU5653 treated with puromycin, an antibiotic having cytotoxicity in Fig. 16, survived and showed a high CYP3A4 transcription level, a high CYP responsiveness to puromycin was confirmed. From these facts, it was confirmed that "RYU", which is an intestinal epithelial cell-like cell derived from the intestinal structure, has high drug-metabolizing activity.

[0152] [Example 14] Confirmation of division of intestinal epithelial cell-like cells in co-cultures of fibroblast-like cells and intestinal epithelial cell-like cells Fibroblast-like cells and intestinal epithelial cell-like cells derived from an intestinal structure prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage 5 and fibroblast-like cells at passage 12 were co-cultured two-dimensionally for 7 days. ESTEM-HE medium was used for the culture. After the culture was completed, the co-culture was immunostained using an antibody against Ki-67, a marker for cell proliferation. The results are shown in Fig. 17. In each photograph of Fig. 17, the scale bar indicates 50 μm.

[0153] From the photograph of FIG. 17, it was shown that Ki-67, a marker of cell proliferation, was present non-locally. This result suggests that cell division occurs non-locally rather than locally from a single cell. From this result, at least two possibilities are suggested. The first possibility is that the result of FIG. 17 suggests the characteristics when target cells are proliferated by fibroblast-like cells derived from intestinal organoids (i.e., the feeder cells of the present disclosure). The second possibility is that FIG. 17 suggests that intestinal epithelial cell-like cells derived from intestinal organoids divide and proliferate. That is, it is usually thought that intestinal epithelial cells, which are only generated by differentiating from intestinal epithelial stem cells (Lgr5-positive stem cells) and do not divide and proliferate after differentiation, proliferate by the division of intestinal epithelial cells.

[0154] [Example 15] Confirmation of the mucosal formation ability of fibroblast-like cells and intestinal epithelial cells derived from intestinal organoids Fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal organoids prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage 7 and fibroblast-like cells at passage 12 were co-cultured two-dimensionally for 3 days. ESTEM-HE medium was used for the culture. After the culture was completed, the co-culture was embedded in IP gel and stained with Alcian blue. The results are shown in FIG. 18. In each photograph of FIGS. 18A and B, the scale bars indicate 50 μm and 200 μm, respectively.

[0155] The blue coloring shown in each photograph of Fig. 18 indicates the presence of mucin, suggesting the presence of goblet cells classified as intestinal epithelial cells. This demonstrates that the feeder cells of the present disclosure exhibit a feeder ability that contributes to the stable growth of intestinal epithelial cells over a long period (multiple passages), and indicates that intestinal epithelial-like cells grown and maintained in culture using the feeder cells of the present disclosure maintain intestinal endocrine function. From these results, it is suggested that by using the feeder cells of the present disclosure, it is possible to produce a large amount of mucin itself or cells that produce mucin, and thus it is also possible to produce a cell sheet having such a large amount of mucin. Since almost all mucus secreted by animals contains mucin and it is known that most mucous membranes in the animal body are covered with mucin, the feeder cells of the present disclosure are expected to be applied in the medical field, particularly in the medical field related to mucus and mucous membranes. Conventionally, Caco-2 cells have generally been used as a drug discovery tool, but since Caco-2 cells are generally considered not to have the ability to secrete mucin, it is also suggested that by using the feeder cells of the present disclosure, it is possible to provide an assay system closer to the living body compared to Caco-2 cells.

[0156] [Example 16] Confirmation of the feeder ability of fibroblast-like cells derived from intestinal structures 1 The feeder ability of fibroblast-like cells derived from intestinal structures prepared by the same method as in Example 3 was confirmed by comparison with conventional feeder cells derived from human embryonic stem cells. First, conventional feeder cells were prepared according to the following procedure.

[0157] Human embryonic stem cells (hES cells) EES2 were exposed to an ROCK inhibitor (Y-27632; 10 μM), then dissociated into single cells using 0.5 mM EDTA, and seeded at a concentration of 5×10 3 cells per well in a 96-well plate.

[0158] The seeded cells were cultured in EB medium (76% KnockOut DMEM, 20% 35 kGy irradiated Xeno-free KnockOut Serum Replacement (XF-KSR, Life Technologies), 2 mM GlutaMAX-I, 0.1 mM non-essential amino acids (NEAA), 50 U / ml penicillin - 50 μg / ml streptomycin (Pen-Strep), 50 μg / ml L-ascorbic acid 2-phosphate (Sigma-Aldrich)) for 4 days to form embryoid bodies.

[0159] The obtained embryoid bodies were transferred to a T25 flask coated with NMP collagen PS (Nippon Ham Co., Ltd.) and cultured in XF32 medium (85% KnockOut DMEM, 15% 35 kGy irradiated XF-KSR, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 μg / ml L-ascorbic acid 2-phosphate, 10 ng / ml heregulin-1β (Recombinant human NRG-beta 1 / HRG-beta 1 EGF domain; Fujifilm Wako Pure Chemical Corporation), 200 ng / ml recombinant human IGF-1 (LONGR3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Kaken Pharmaceutical Co., Ltd.)) for 60 - 70 days to obtain hES cell-derived cells. Since these cells had mesenchymal cell-like properties, they were also referred to as ES cell-derived mesenchymal cells.

[0160] The obtained hES cell-derived mesenchymal cells were maintained in α-MEM medium supplemented with 10% FBS (Gibco or HyClone) and 1% Pen-Strep to obtain feeder cells (hereinafter, also referred to as "conventional feeder cells").

[0161] Fibroblast-like cells derived from the intestinal structure prepared by the same method as in Example 3 (i.e., the feeder cells of the present disclosure) were examined for their feeder ability according to the following procedure. The feeder cells of the present disclosure were cultured in large quantities to obtain feeder cells at passage 7. In the same manner as in Example 3, intestinal epithelial cells were seeded on the obtained feeder cells and cultured, and passage was repeated. The PDL (Population doubling level: vertical axis) at each passage number of the intestinal epithelial cells after repeated passage is shown in FIG. 19 respectively.

[0162] From the results shown in FIG. 19, it was confirmed that the feeder cells of the present disclosure exhibit a stable growth ability of target cells (intestinal epithelial cells) at any time point from passage 1 to 16 (P1 - 16). That is, it was shown that the feeder cells of the present disclosure have a feeder ability that contributes to the stable growth of target cells over a long period (multiple passages).

[0163] [Example 17] Confirmation of the feeder ability of fibroblast-like cells derived from the intestinal structure 2 The feeder ability of fibroblast-like cells derived from the intestinal structure prepared by the same method as in Example 3 (i.e., the feeder cells of the present disclosure) was confirmed as a comparison with conventional feeder cells derived from human embryonic stem cells. Specifically, first, conventional feeder cells prepared by the same method as in Example 16 were prepared. Then, the feeder cells of the present disclosure and the conventional feeder cells were each seeded in a 24-well plate at a concentration of 5×10 4 cells per well, and cultured in α-MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. One day later, intestinal epithelial cell-like cells (passage 3) derived from the intestinal structure were seeded at 1.0×10 per well 5Seeding was performed at a concentration of

[0164] From the results shown in Fig. 20, when culturing intestinal epithelial cell-like cells using conventional feeder cells, very large and flat cells were observed in the center. Also, although the expression of Villin was confirmed, the expression of CDX2 was hardly confirmed. From these results, it was suggested that when using conventional feeder cells, the expression of CDX2, which is normally expressed in intestinal epithelial cells, is lost, and the characteristics of intestinal epithelial cell-like cells change in a short period (with a small number of passages). Although not shown in the results, when culturing intestinal epithelial cell-like cells using the feeder cells of the present disclosure, the expression of CDX2 and Villin was confirmed even with the same number of passages, and it was confirmed that the characteristics as intestinal epithelial cells were maintained.

[0165] [Example 18] Maintenance of chondrocytes by fibroblast-like cells derived from intestinal structures The feeder ability (maintenance of chondrocytes) of four different fibroblast-like cells (i.e., the feeder cells of the present disclosure) at passage 7 derived from intestinal structures prepared by the same method as in Example 3 was confirmed. Specifically, for each of the chondrocytes (YUB) collected from human children and the chondrocytes cultured together with the above four types of fibroblast-like cells, the expression of lubricin and COL2A1 was confirmed by qRT-PCR. The results are shown in Fig. 21. Also, in the table, "YUB" indicates chondrocytes collected from pediatric specimens, and "4R" to "7R" each mean chondrocytes cultured together with each fibroblast-like cell (note that "6R" in Fig. 21A did not yield data).

[0166] From the results of Fig. 21, it was confirmed that lubricin and COL2A1 were highly expressed in chondrocytes 4R - 7R cultured with the feeder cells of the present disclosure, as compared with YUB, which are chondrocytes collected from human children. Here, lubricin is a lubricant highly expressed in synovial cells known as feeder cells for chondrocytes, and COL2A1 is a lubricant highly expressed in chondrocytes. Also, chondrocytes are known to be cells that are prone to dedifferentiation. Therefore, the above results indicate that in YUB, chondrocytes underwent dedifferentiation and lost their original properties, and the expression of lubricin as well as COL2A1 was hardly observed. In contrast, in 4R - 7R, the feeder cells of the present disclosure exerted synoviocyte-like functions, resulting in high expression of lubricin, and furthermore, the original properties of chondrocytes were maintained and COL2A1 was highly expressed. From these results, it was suggested that the feeder cells of the present disclosure have feeder ability for chondrocytes.

[0167] [Example 19] Confirmation of Marker by Gene Expression Profile and Immunostaining of Feeder Cells of the Present Disclosure Cell markers of fibroblast-like cells (i.e., feeder cells of the present disclosure) derived from intestinal structures prepared by the same method as in Example 3 were confirmed by immunostaining. Specifically, cultured fibroblast-like cells were embedded in IP gel to prepare specimens, and protein expressions on the cell surfaces of PDGFRA and CD81 were stained using PDGF Receptor α (D1E1E) XP (registered trademark) Rabbit mAb manufactured by Cell Signaling Technology, Inc. and Anti-CD81, Mouse-Mono (M38) manufactured by EXBIO as primary antibodies, respectively. The results are shown in Fig. 22.

[0168] Also, the expressions of genes of Foxl1, CD34, and GREM1 obtained by comprehensive gene expression and normalized were confirmed. The results are shown in Fig. 23.

[0169] From the results shown in FIGS. 22 and 23, it was confirmed that in the feeder cells of the present disclosure, each of the genes PDGFRA, CD81, Foxl1, and GREM1 was expressed, while CD34 was not expressed.

[0170] [Example 20] Preparation of cell sheet A cell sheet was prepared using the feeder cells of the present disclosure. Specifically, the feeder cells of the present disclosure were seeded and cultured until they reached a confluent state. Next, intestinal epithelial cells were seeded in a manner of being stacked on the confluent feeder cells and cultured until the intestinal epithelial cells became confluent and formed a sheet structure by cell-cell binding. Next, the laminate of the layer of the feeder cells of the present disclosure and the layer of intestinal epithelial cells was detached from the culture plane to obtain a cell sheet containing the layer of feeder cells and the layer of intestinal epithelial cells.

[0171] A photograph of the obtained cell sheet is shown in FIG. 24. From the results shown in FIG. 24, it was confirmed that the cell sheet of the present disclosure has a diameter of 1 cm or more. Industrial applicability

[0172] According to the present disclosure, there are provided feeder cells for two-dimensionally culturing, maintaining, or growing cells that have conventionally been considered difficult to maintain and grow by two-dimensional culturing, and a method for producing the same.

Claims

1. 1. A method for producing feeder cells for maintaining or growing cells, comprising: (A) providing an intestinal structure produced from pluripotent stem cells and comprising endodermal-derived cells and mesodermal-derived cells; (B) culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure; and (C) isolating the fibroblast-like cells to obtain feeder cells. A method comprising:

2. The intestinal structure comprises: (a) preparing a cell culture substrate comprising a substrate, a plurality of isolated cell adhesive regions formed on the substrate, and a cell non-adhesive region surrounding each of the cell adhesive regions; (b) seeding pluripotent stem cells onto the cell culture substrate; and (c) culturing the seeded pluripotent stem cells in a medium. The method comprises the steps of: The method of claim 1.

3. The method of claim 1 or 2, further comprising the step of (D) expanding the isolated fibroblast-like cells.

4. The method according to claim 1 or 2, wherein in the step (B), the intestinal structure is cultured in a culture vessel, and at least a portion of the intestinal structure is in contact with the culture vessel.

5. The method according to claim 2 , wherein the culture period in step (c) is 60 days or longer.

6. A feeder cell produced by the method according to claim 1 or 2.

7. Fibroblast-like cells that are positive for PDGFRA and CD81 and express Foxl1 and GREM1.

8. The fibroblast-like cell of claim 7, which is positive for PDGFRA and CD81, negative for CD34, and expresses Foxl1 and GREM1.

9. 8. The fibroblast-like cell of claim 7, derived from a culture of intestinal structures derived from or produced from pluripotent stem cells, the intestinal structures comprising endoderm-derived and mesoderm-derived cells.

10. The intestinal structure comprises: (a) preparing a cell culture substrate comprising a substrate, a plurality of isolated cell adhesive regions formed on the substrate, and a cell non-adhesive region surrounding each of the cell adhesive regions; (b) seeding pluripotent stem cells onto the cell culture substrate; and (c) culturing the seeded pluripotent stem cells in a medium. The method comprises the steps of: The fibroblast-like cell of claim 9.

11. The fibroblast-like cell of claim 9 , wherein the pluripotent stem cell is of human origin.

12. The feeder cell according to claim 6 or the fibroblast-like cell according to claim 7, which is a feeder cell for maintaining or growing at least one type of cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

13. The feeder cell according to claim 6 or the fibroblast-like cell according to claim 7, which is a feeder cell for maintaining or growing the cell by plate culture.

14. The feeder cell of claim 6 or the fibroblast-like cell of claim 7, wherein the cell is a feeder cell capable of being passaged three or more times.

15. A cell sheet comprising the feeder cells according to claim 6 or the fibroblast-like cells according to claim 7.

16. A co-culture comprising at least one type of cell selected from the group consisting of small intestinal epithelial cells and hepatic cells, and the feeder cell according to claim 6 or the fibroblast-like cell according to claim 7.

17. The co-culture of claim 16, wherein the cells have been passaged three or more times.

18. A cell sheet comprising the co-culture described in claim 16.

19. The cell sheet according to claim 18, which is circular or approximately circular with a diameter of 1 cm or more.

20. The cell sheet according to claim 18, comprising a layer of at least one type of cell selected from the group consisting of small intestinal epithelial cells and hepatic cells.

21. The cell sheet of claim 20, further comprising a layer of feeder cells.

22. A transplant material comprising the cell sheet according to claim 15 or 18.

23. 8. A method for maintaining or expanding cells, comprising culturing said cells in the presence of feeder cells according to claim 6 or fibroblast-like cells according to claim 7.

24. The method of claim 23, wherein the cell culture is a plate culture.

25. The method according to claim 23 or 24, wherein the cells are at least one type of cells selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

26. The method of claim 23 or 24, wherein the cells and the feeder cells are derived from the same individual.

27. The method of claim 23 or 24, wherein the cells and the feeder cells are derived from different individuals.

28. A cell expressing at least one gene selected from the group consisting of DCN, ACTA2 and NOG, and expressing the genes ADAMTS19, ANO1, BLID, LOC100507053, LHX8, SFRP4, AHRR, SLC14A1, FMO3, PZP, ABCG4, EYA2, TMEM92-AS1, ANO10, EGFL6, SNCAIP, LGSN, PCDHB15 and KRTAP1-5.

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