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

Abstract

The present invention addresses the problem of providing: a feeder cell for maintaining or proliferating a cell, which is considered to be difficult to maintain and proliferate by two-dimensional culture, by two-dimensional culture; and a method for producing the feeder cell. A feeder cell is produced by proliferating and isolating fibroblast-like cells derived from an intestinal structure comprising endoderm-derived cells and mesoderm-derived cells, said intestinal structure being produced from pluripotent stem cells.

Description

Technical Field

[0001] The present disclosure relates to feeder cells for maintaining or growing cells, a cell sheet containing the feeder cells, and methods for producing the same, and a method for maintaining or growing cells using the feeder cells. Background Art

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

[0003] In these fields, it is required to differentiate pluripotent stem cells to proliferate cells with specific properties, or to organize multiple cells with specific properties to form tissues and organs. In the past, such technology has been actively studied, for example, a technology has been proposed to differentiate intestinal epithelial cells from pluripotent stem cells, and then three-dimensionally culture the differentiated intestinal epithelial cells to maintain and proliferate them to form an intestinal structure (for example, Patent Document 1).

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

[0005] In order to induce differentiation of pluripotent stem cells into cells with specific properties or to maintain and promote proliferation of cells after differentiation induction, feeder cells that produce and supply specific factors (such as differentiation inducing factors) are sometimes used (for example, Patent Document 2).

[0006] However, there is no known technique for maintaining or proliferating intestinal epithelial cells even when they are cultured in two dimensions by using feeder cells. Therefore, it is desirable to find a practical method for maintaining or proliferating intestinal epithelial cells and other cells that have been considered difficult to maintain and proliferate in two-dimensional culture.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application Publication No. 2017-184749

[0010] Patent Document 2: Japanese Patent Application Publication No. 2022-7610 Summary of the invention

[0011] Problems to be solved by the invention

[0012] Under such circumstances, it is desired to find a practical method for maintaining or growing cells such as intestinal epithelial cells by two-dimensional culture, which has been considered difficult to maintain and grow by two-dimensional culture.

[0013] Therefore, the present disclosure provides a feeder cell for maintaining or proliferating cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate in two-dimensional culture by two-dimensional culture, and a method for producing the feeder cell. In addition, the present disclosure provides a cell sheet comprising the feeder cell.

[0014] Means for solving problems

[0015] The embodiments of the present disclosure relate to the following [1] to

[28] .

[0016] [1] A method for producing feeder cells for maintaining or growing cells, the method comprising:

[0017] (A) a step of preparing an intestinal structure composed of endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem cells;

[0018] (B) a step of culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure; and

[0019] (C) A step of isolating the fibroblast-like cells to obtain feeder cells.

[0020] [2] The method according to [1], wherein the intestinal structure is produced by a method comprising the following steps:

[0021] (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and a cell non-adhesion region surrounding each of the cell adhesion regions;

[0022] (b) a step of seeding the pluripotent stem cells on the cell culture substrate; and

[0023] (c) a step of culturing the seeded pluripotent stem cells in a culture medium.

[0024] [3] The method according to [1] or [2], further comprising (D) a step of proliferating the isolated fibroblast-like cells.

[0025] [4] The method according to [1] or [2], wherein in the step (B), the intestinal structure is cultured in a culture container, and at least a portion of the intestinal structure is in contact with the culture container.

[0026] [5] The method according to [2], wherein the culturing time in the step (c) is 60 days or longer.

[0027] [6] A feeder cell produced by the method described in [1] or [2].

[0028] [7] A fibroblast-like cell, which is positive for PDGFRA and CD81 and expresses Foxl1 and GREM1.

[0029] [8] The fibroblast-like cells according to [7], wherein the cells are positive for PDGFRA and CD81, negative for CD34, and express Foxl1 and GREM1.

[0030] [9] The fibroblast-like cells described in [7], which are derived from pluripotent stem cells or from a culture of an intestinal structure, wherein the intestinal structure is produced from pluripotent stem cells and contains endoderm-derived cells and mesoderm-derived cells.

[0031]

[10] The fibroblast-like cell according to [9], wherein the intestinal structure is produced by a method comprising the following steps:

[0032] (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and a cell non-adhesion region surrounding each of the cell adhesion regions;

[0033] (b) a step of seeding the pluripotent stem cells on the cell culture substrate; and

[0034] (c) a step of culturing the seeded pluripotent stem cells in a culture medium.

[0035]

[11] The fibroblast-like cells according to [9], wherein the pluripotent stem cells are derived from humans.

[0036]

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

[0037]

[13] The feeder cell according to [6] or the fibroblast-like cell according to [7], which is a feeder cell for maintaining or growing the above cells by planar culture.

[0038]

[14] The feeder cell according to [6] or the fibroblast-like cell according to [7], which is a feeder cell capable of passage the cell three or more times.

[0039]

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

[0040]

[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].

[0041]

[17] The co-culture according to

[16] , wherein the cells have been passaged three or more times.

[0042]

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

[16] .

[0043]

[19] The cell sheet according to

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

[0044]

[20] The cell sheet according to

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

[0045]

[21] The cell sheet according to

[20] , further comprising a layer of the above-mentioned feeder cells.

[0046]

[22] A transplant material comprising the cell sheet described in

[15] or

[18] .

[0047]

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

[0048]

[24] The method as described in

[23] , wherein the cell culture is planar culture.

[0049]

[25] The method of

[23] or

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

[0050]

[26] The method according to

[23] or

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

[0051]

[27] The method of

[23] or

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

[0052]

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

[0053] Effects of the Invention

[0054] According to the present disclosure, a feeder cell and a method for making such a feeder cell can be provided, and the feeder cell is used to maintain or proliferate cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate through two-dimensional culture by two-dimensional culture. In addition, according to the present disclosure, a cell sheet containing the above-mentioned feeder cells can be provided. Moreover, the above-mentioned feeder cells have good feeding ability even if the number of passages is repeated more than 10 times, so they can be widely popularized even if immortalization does not occur. The culture system using the feeder cells can also be expected to function as a standard model of a culture system closer to the human body compared to the commonly used feeder cells-mouse embryonic fibroblasts (MEF) and immortalized feeder cells. In addition, the feeder cells have adhesion to general culture surfaces, show expression of CD90, and exist in the positional relationship where mesenchymal stem cells originally exist in the organelles, so it can be easily inferred that it is a kind of mesenchymal stem cells, and the therapeutic effect brought by the transplantation of the feeder cells can also be expected. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 These are photographs of intestinal structures produced by inducing differentiation of pluripotent stem cells using immunostaining using antibodies that serve as intestinal differentiation markers (anti-villin antibody and anti-5TH antibody).

[0056] Figure 2 The photographs show immunostaining of co-cultures of fibroblast-like cells and intestinal epithelial-like cells from intestinal structures using antibodies against intestinal epithelial cell markers (CDX2 and villin).

[0057] Figure 3 The photographs show immunostaining of a culture of intestinal epithelial-like cells from intestinal structures using an antibody against a marker of intestinal epithelial cells (CDX2).

[0058] Figure 4 The photographs show immunostaining of a co-culture of fibroblast-like cells and intestinal epithelial-like cells from intestinal structures using an antibody against a marker of intestinal epithelial cells (CDX2).

[0059] Figure 5 The photographs show immunostaining of a co-culture of fibroblast-like cells and intestinal epithelial-like cells from intestinal structures using an antibody against a marker of intestinal epithelial cells (villin).

[0060] Figure 6 This is a microscopic photograph of a co-culture containing fibroblast-like cells from intestinal structures and human primary intestinal epithelial cells at passage number 2 and on day 5 of culture.

[0061] Figure 7 This is a microscopic photograph of a co-culture containing fibroblast-like cells derived from intestinal structures and human primary intestinal epithelial cells at passage number 4 and day 4 of culture.

[0062] Figure 8 This is a microscopic photograph of a co-culture of mouse embryonic fibroblasts (MEFs) and human primary intestinal epithelial cells at passage 2 and day 5 of culture.

[0063] Fig. 9 This is a microscopic photograph of a co-culture of mouse embryonic fibroblasts (MEFs) and human primary intestinal epithelial cells at passage 4 and day 4 of culture.

[0064] Fig.10 This is a microscopic photograph of a co-culture containing fibroblast-like cells derived from intestinal structures and hepatocytes on day 8 after the start of culture.

[0065] Fig.11 The results are from an analysis of gene expression in the intestine and site-specific cells that constitute the intestine in a co-culture of fibroblast-like cells and intestinal epithelial-like cells derived from intestinal structures.

[0066] Figure 12-1 The results are from an analysis of gene expression in the intestine and site-specific cells that constitute the intestine in a co-culture of fibroblast-like cells and intestinal epithelial-like cells derived from intestinal structures.

[0067] Figure 12-2 These are the results of analyzing gene expressions of various signaling proteins of the WNT family in fibroblast-like cells derived from intestinal structures.

[0068] Fig.13 The results of hierarchical clustering by comprehensive gene analysis of co-cultures of fibroblast-like cells and intestinal epithelial-like cells from intestinal structures.

[0069] Fig.14The results are the measurement results of the barrier properties (endothelial cell electrical resistance) of a cell sheet that is a co-culture of fibroblast-like cells and intestinal epithelial-like cells derived from intestinal structures.

[0070] Fig.15 These are photographs relating to the expression of transporters in intestinal epithelial cells in a co-culture of fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures.

[0071] Fig.16 The results are from the analysis of gene expression of transporters and metabolic enzymes of intestinal epithelial cells in co-cultures of fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures.

[0072] Fig.17 The photographs show immunostaining of a co-culture of fibroblast-like cells and intestinal epithelial-like cells from intestinal structures using an antibody against a cell proliferation marker (Ki-67).

[0073] Fig.18 This is a photograph showing a co-culture of fibroblast-like cells and intestinal epithelial cell-like cells derived from intestinal structures stained with Alcian blue.

[0074] Fig.19 It is a graph showing the feeding capacity of intestinal structure-derived fibroblast-like cells at each passage number.

[0075] Fig. 20 These are photographs of conventional co-cultures of feeder cells and intestinal epithelial-like cells immunostained with antibodies against intestinal epithelial cell markers (CDX2 and villin).

[0076] Fig.21 The graphs show the expression of lubricin and COL2A1 in fibroblast-like cells derived from intestinal structures and chondrocytes collected from human children, respectively.

[0077] Fig. 22 These are photographs showing immunostaining of fibroblast-like cells derived from intestinal structures using antibodies against PDGFRA and CD81.

[0078] Fig.23 These are the results of analyzing the expression of each gene of Foxl1, CD34, and GREM1 in fibroblast-like cells derived from intestinal structures.

[0079] Fig.24 This is a photograph of a cell sheet made using fibroblast-like cells derived from intestinal structures. DETAILED DESCRIPTION

[0080] The term "feeder cells" used in this specification refers to cells that are cultured (co-cultured) together with the target cells or cells before their differentiation, etc., in order to adjust the culture conditions that promote the maintenance and / or proliferation of the target cells, and are different from the target cells. In the present disclosure, the feeder cells can be inoculated simultaneously with the target cells, or they can be inoculated before the target cells are inoculated. In one embodiment, the feeder cells are inoculated before the target cells are inoculated to form a layer of feeder cells (i.e., feeder layer). The target cells and feeder cells can be cultured in a manner of contacting each other, or they can be cultured in a manner of not contacting each other. In one embodiment, the target cells and feeder cells are cultured in a manner in which at least a portion of the target cells are in contact with the feeder cells. It should be noted that the feeder cells can be inhibited from proliferating by known methods such as irradiation with radiation (e.g., gamma rays) and treatment with antibiotics (e.g., mitomycin C).

[0081] The term "pluripotent stem cell" used in this specification refers to a cell with so-called differentiation pluripotency. The terms "pluripotency" and "differentiation pluripotency" used in this specification refer to the ability of cells that can differentiate into all germ layers (i.e., ectoderm, mesoderm and endoderm) constituting an organism. As pluripotent stem cells, for example, embryonic stem cells (ES cells), ES cells (ntES cells) from somatic cells, induced pluripotent stem cells (iPS cells), stem cells from adult tissues or cord blood or umbilical cords, etc. can be cited.

[0082] The term "fibroblast-like cells" used in this specification refers not only to so-called fibroblasts, but also to cells having a flat or spindle-shaped shape differentiated from fibroblasts such as myofibroblasts. In addition, the source of fibroblast-like cells is not particularly limited. For example, fibroblast-like cells may be cells derived from the above-mentioned pluripotent stem cells or the intestinal structure described later. In addition, fibroblast-like cells may be cells that show a specific gene expression profile described later. It should be noted that the "fibroblast-like cells" in the present disclosure do not include the above-mentioned various naturally occurring cells, but are cells obtained by artificially manipulating naturally occurring cells or artificially obtained cells, that is, artificially obtained cells (artificial cells).

[0083] [Method for producing feeder cells]

[0084] According to one embodiment of the present disclosure, a method for producing feeder cells (hereinafter also referred to as "feeder cells of the present disclosure") for maintaining cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate in two-dimensional culture is provided. The feeder cells of the present disclosure can be produced by a method including the following steps (A) to (C) (hereinafter also referred to as "the production method of the present disclosure").

[0085] (A) a step of preparing an intestinal structure composed of endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem cells;

[0086] (B) a step of culturing the intestinal structure and causing fibroblast-like cells derived from the intestinal structure to proliferate; and

[0087] (C) A step of isolating fibroblast-like cells to obtain feeder cells.

[0088] Hereinafter, each step (A) to (C) will be described in detail.

[0089] <Step (A)>

[0090] In step (A), an intestinal structure comprising endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem cells is prepared. An intestinal structure is a structure comprising endoderm-derived cells and mesoderm-derived cells and having at least one function equivalent to or similar to that of the intestine.

[0091] The intestinal structure is not particularly limited as long as it has the above-mentioned cell composition and function, and can be obtained, for example, by inducing 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).

[0092] (Step (a))

[0093] Step (a) is a step of preparing a cell culture substrate comprising a substrate, a plurality of isolated cell adhesion regions (cell adhesive regions) formed on the substrate, and cell non-adhesive regions (cell non-adhesive regions) surrounding each of the cell adhesion regions.

[0094] " Cell adhesion " in this specification refers to the strength of adhered cells, i.e. the adhesion ease of cells. Therefore, " cell adhesion region " refers to the region where cell adhesion is higher, and " cell non-adhesion region " refers to the region where cell adhesion is lower. Specifically, cell adhesion refers to the degree of adhesion and stretching of cells caused by chemical properties, physical properties, etc. on substrate (substrate surface), easily causing the adhesion of cells in the cell adhesion region where cell adhesion is higher, and not easily causing the adhesion of cells in the cell adhesion region where cell adhesion is lower. Therefore, when inoculating cells on the substrate with the pattern of cell adhesion region and cell non-adhesion region as described above, cells are easily adhered in cell adhesion region, and cells are difficult to adhere in cell non-adhesion region, so the cells inoculated are arranged in a pattern on substrate.

[0095] Cell adhesion can be judged by using the cell adhesion stretching rate when culturing cells seeded on a substrate and arranged in a pattern as an indicator. The higher the cell adhesion stretching rate, the more efficiently the cells can be cultured. There is no particular limitation on the cell adhesion stretching rate of the cell adhesion area and the cell non-adhesion area as long as the effect of the present disclosure is exerted. For example, they can be set to be greater than 60% and less than 60%, respectively. The cell adhesion stretching rate of the cell adhesion area is preferably greater than 70%, more preferably greater than 80%, and further preferably greater than 90%. On the other hand, the cell adhesion stretching rate of the cell non-adhesion area is preferably less than 40%, more preferably less than 5%, and further preferably less than 2%. The cell adhesion stretching rate is defined as: the cell adhesion stretching rate is desired to be greater than 40% at a seeding density of 4,000 cm 2 More than 30,000 pieces / cm 2 Cells cultured within the range of are inoculated on the surface of the measurement object and kept in an incubator at a temperature of 37°C and a CO2 concentration of 5%. The proportion of cells that have adhered and spread after 14.5 hours of cultivation is ({(number of cells adhered to the surface of the measurement object) / (number of cells inoculated on the surface of the measurement object)}×100(%)).

[0096] The inoculation of cells on the measurement object is carried out in the following manner: the cells are suspended in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, the cell suspension is added to the measurement object, and then the measurement object to which the cells are added is slowly shaken to distribute the cells as evenly as possible. Furthermore, the cells that are not adhered to the measurement object are removed by replacing the culture medium immediately before the measurement, and then the cell adhesion extension rate is measured. In the measurement of the cell adhesion extension rate, the site other than the site where the cell density tends to become specific (for example, the center of a predetermined area where the density tends to increase, the periphery of a predetermined area where the density tends to decrease) is used as the measurement site.

[0097] In the present disclosure, the substrate used to make the intestinal structure is not particularly limited as long as the effect of the present disclosure is exerted, for example, the following substrate: the cell non-adhesion area is formed by immobilizing polyethylene glycol on the substrate, and the cell adhesion area is formed by oxidizing and / or decomposing at least a portion of the polyethylene glycol immobilized on the substrate. Such a substrate can be made, for example, as follows: a thin film of polyethylene glycol (PEG) is formed on the entire surface of the substrate, and then, the area where the cells are expected to adhere is subjected to oxidation and / or decomposition treatment to impart cell adhesion, thereby forming a cell adhesion area. The portion that is not subjected to any of the oxidation treatments and decomposition treatments becomes a cell non-adhesion area fixed with PEG.

[0098] As a substrate, as long as it is a substrate formed by a material on which a PEG film can be formed, there is no particular restriction. As the material of the substrate, inorganic materials such as metals, glass, ceramics, silicon, and organic materials represented by elastomers, plastics (such as polystyrene resins, polyester resins, polyethylene resins, polypropylene resins, ABS resins, nylons, acrylic resins, fluororesins, polycarbonate resins, polyurethane resins, methylpentene resins, phenolic resins, melamine resins, epoxy resins, vinyl chloride resins, etc.) can be cited. The shape of the substrate is not particularly limited, and flat shapes such as flat plates, flat films, films, porous films, and three-dimensional shapes such as cylinders, stamps, porous plates, and microchannels can be cited. In one embodiment, a film-like substrate is used. The thickness of the film-like substrate is not particularly limited, and is generally 0.1 to 1,000 μm, preferably 1 to 500 μm, and more preferably 10 to 200 μm.

[0099] Polyethylene glycol (PEG) contains at least one ethylene glycol chain (EG chain) composed of one or more ethylene glycol units (-(CH2)2-O-). The polyethylene glycol chain can be straight chain or branched chain. The ethylene glycol chain refers to, for example, the following formula:

[0100] -((CH2)2-O) m -

[0101] (m is an integer representing the degree of polymerization)

[0102] M is not particularly limited as long as the effects of the present disclosure are exhibited, but is preferably 1-13, more preferably 1-10.

[0103] PEG also includes ethylene glycol oligomers. In addition, PEG also includes PEG into which functional groups are introduced. As functional groups, for example, epoxy, carboxyl, N-hydroxysuccinimide, carbodiimide, amino, glutaraldehyde, (meth) acryloyl, etc. can be mentioned. The functional group is preferably introduced into the end of the PEG chain via a linker as required. As PEG into which functional groups are introduced, for example, PEG (meth) acrylate, PEG di(meth) acrylate, etc. can be mentioned.

[0104] The average thickness of the PEG film formed on the substrate is not particularly limited, preferably 0.8nm to 500μm, more preferably 0.8nm to 100μm, further preferably 1nm to 10μm, and particularly preferably 1.5nm to 1μm. By making the average thickness of the PEG film above 0.8nm, it is not easily affected by the area of ​​the substrate surface that is not covered by the PEG film during protein adsorption and cell adhesion. On the other hand, by making the average thickness below 500μm, it can be coated relatively easily. In addition, by making the thickness of the PEG film above a certain degree, the cell non-adhesion is reduced, and the cell adhesion can be suppressed from extending to the area outside the cell adhesion area. In addition, by making the thickness of the PEG film below a certain degree, the factors required for cell survival contained in the culture solution can also be spread to the cells close to the substrate in the cell adhesion area.

[0105] As a method for forming a PEG thin film on a substrate, there can be cited a method of directly adsorbing PEG on the surface of a substrate; a method of directly coating PEG on the surface of a substrate; a method of coating PEG on the surface of a substrate and then subjecting it to a crosslinking treatment; a method of forming a base layer on the surface of a substrate and then coating PEG in order to improve the adhesion between the substrate and the PEG thin film; a method of forming a polymerization starting point on the surface of a substrate and then polymerizing PEG; etc. In a preferred embodiment, the formation of the PEG thin film on a substrate is performed by forming a base layer on a substrate and then coating PEG.

[0106] The base layer on the substrate surface can be formed by the method described in Japanese Patent Application Laid-Open No. 2012-175983, for example. In a preferred embodiment, the base layer on the substrate surface can be formed by immobilizing the following silane coupling agent on the substrate surface, and the silane coupling agent has a functional group that can react with the hydroxyl group at the end of PEG or the functional group introduced to form a covalent bond, or a functional group that can be converted into such a functional group. As such a functional group, for example (meth) acryloyl, (1H- imidazole -1- base) carbonyl, succinimidyl oxycarbonyl, glycidyl, epoxy, aldehyde, amino, thiol, carboxyl, azido, cyano, active ester (1H- benzotriazole -1- base oxycarbonyl, pentafluorophenoxycarbonyl, p-nitrophenoxycarbonyl, etc.), halogenated carbonyl, isocyanate, maleimide, etc. Among these functional groups, preferred functional groups are (meth) acryloyl, glycidyl, epoxy.

[0107] For example, when a silane coupling agent having a methacryloyl group at the end (methacryloyl silane) is used, the water contact angle of the surface of the substrate to which the 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. It should be noted that 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.

[0108] The density and cell adhesion of the PEG immobilized on substrate can be evaluated simply with the water contact angle of substrate surface as an index. Specifically, the lower the water contact angle of substrate surface, the higher the density of the PEG present in substrate surface, and the result forms the low region of cell adhesion (i.e. cell non-adhesion region). That is, for example, when the water contact angle of the substrate surface after PEG is immobilized on the substrate surface is, for example, below 48 °, preferably below 40 °, more preferably below 30 °, it is believed that PEG is present in the surface of substrate with sufficient density, and cell adhesion is sufficiently low.

[0109] In the present disclosure, the oxidation treatment of polyethylene glycol used to form a cell adhesion region refers to a reaction in which an organic compound, i.e., PEG, reacts with oxygen to increase the oxygen content compared to before the reaction. In addition, in the present disclosure, the decomposition treatment of polyethylene glycol used to form a cell adhesion region refers to a reaction in which the bonds of the organic compound, i.e., PEG, are cut. Typical examples of "decomposition treatment" include decomposition caused by oxidation, decomposition caused by ultraviolet irradiation, etc., but are not limited to these. It should be noted that when the "decomposition treatment" is a decomposition accompanied by oxidation (i.e., oxidative decomposition), the "decomposition treatment" and the "oxidation treatment" refer to the same treatment.

[0110] Decomposition caused by ultraviolet irradiation refers to that PEG absorbs ultraviolet rays and decomposes through an excited state. It should be noted that when irradiating ultraviolet rays in a system where PEG and molecular species containing oxygen (such as oxygen, water, etc.) coexist, in addition to ultraviolet rays being absorbed by PEG and causing decomposition, the molecular species containing oxygen are sometimes activated and react with PEG. In this case, the latter reaction is classified as "oxidation". In addition, the reaction in which PEG is decomposed by oxidation caused by activated molecular species does not belong to "decomposition caused by ultraviolet irradiation", but is classified as "decomposition caused by oxidation". In this way, sometimes "oxidation" and "decomposition" are repeated as operations, and sometimes it is impossible to clearly distinguish between the two. In this manual, such a situation is also described as "oxidation and / or decomposition".

[0111] As the method of oxidation and / or decomposition, there can be cited a method of treating the PEG film with ultraviolet rays, a method of treating the film with a photocatalyst, a method of treating the film with an oxidant, etc. In the case of partially oxidizing and / or decomposing the PEG film, a mask or a stamp such as a photomask or a stencil mask can be used. In addition, oxidation and / or decomposition can also be carried out by direct drawing methods such as using a laser such as an ultraviolet laser.

[0112] In the case of ultraviolet irradiation treatment, it is preferred to use a lamp that radiates ultraviolet rays from the VUV region to the UV-C region, such as a mercury lamp that radiates ultraviolet rays with a wavelength of 185nm or 254nm or an excimer lamp that radiates ultraviolet rays with a wavelength of 172nm, as a light source. In the case of photocatalyst treatment, it is preferred to use a light source that radiates ultraviolet rays with a wavelength of less than 365nm, and it is more preferred to use a light source that radiates ultraviolet rays with a wavelength of less than 254nm. As a photocatalyst, it is preferred to use a titanium oxide photocatalyst, a titanium oxide photocatalyst activated by metal ions or metal colloids, etc. In addition, as an oxidant, organic acids and inorganic acids can be used without particular restrictions, but high-concentration acids are difficult to handle, so it is preferably diluted to a concentration of less than 10% for use. The optimal ultraviolet treatment time, photocatalyst treatment time, and oxidant 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 ability and concentration of the oxidant.

[0113] In one embodiment, the carbon amount of the cell adhesion region (including the basal layer in the presence of the basal layer) is preferably lower than the carbon amount of the cell non-adhesion region (including the basal layer in the presence of the basal layer). Specifically, the carbon amount of the cell adhesion region is preferably 20 to 99% relative to the carbon amount of the cell non-adhesion region. In addition, the value of the ratio (%) of carbon bonded to oxygen in the carbon of the cell adhesion region (including the basal layer in the presence of the basal layer) is preferably a value smaller than the value of the ratio (%) of carbon bonded to oxygen in the carbon of the cell non-adhesion region (including the basal layer in the presence of the basal layer). Specifically, the value of the ratio (%) of carbon bonded to oxygen in the carbon of the cell adhesion region is preferably 35 to 99% relative to the value of the ratio (%) of carbon bonded to oxygen in the carbon of the cell non-adhesion region. This is because, when the inoculated cells are arranged in a pattern on the substrate, as the amount of ultraviolet light exposure increases, the cell adhesion increases, but if the cell is highly adherent during recovery, the cell is difficult to peel off, and recovery becomes difficult.

[0114] In the present specification, the "carbon amount" in the cell adhesion region is defined as "the carbon amount obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectrometer", and the "ratio of carbon bonded to oxygen" is defined as "the ratio of carbon bonded to oxygen obtained from the analysis value of the C1s peak obtained using an X-ray photoelectron spectrometer".

[0115] In the substrate used in the present disclosure, the area of ​​each cell adhesion region is greater than 0.785 mm 2 , preferably 1.0 mm 2 More preferably 1.2 mm 2 More preferably, 1.5 mm 2 Above, most preferably 1.7 mm 2 Above, preferably 25mm 2 Less than, more preferably 15mm 2 Less than, more preferably 10 mm 2 Below, most preferably 5mm 2 By allowing pluripotent stem cells to adhere to a surface having a diameter greater than 0.785 mm 2 The cells can be cultured in a cell adhesion area of ​​25 mm without detaching, and the cells can be cultured in an adhered state, which promotes differentiation into intestinal structures. 2 Culturing cells in an adhesion region of the following area can effectively induce differentiation into intestinal structures.

[0116] The shape of each cell adhesion region is not particularly limited, and may be, for example, a polygon represented by a quadrilateral, a circle, an ellipse, etc. Among them, a circle is preferred, and a circle having a diameter greater than 1.0 mm, preferably greater than 1.2 mm, more preferably greater than 1.5 mm, preferably less than 6 mm, more preferably less than 4 mm, further preferably less than 3 mm, and particularly preferably less than 2 mm. In one substrate, the area and / or shape of multiple cell adhesion regions may be the same or different, but preferably the area and shape are the same.

[0117] In addition, in the substrate, each cell adhesion region is surrounded by a cell non-adhesion region, that is, isolated from each other, preferably separated from each other by more than 0.75 mm, more preferably separated from each other by more than 1.5 mm. 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 each radius) is preferably 0.75 mm or more, more preferably 1.5 mm or more. By separating each cell adhesion region by more than a certain distance, the cells in each cell adhesion region will not form intercellular bonds with the cells in other cell adhesion regions, and are evenly cultured at a certain interval, and a highly reproducible experimental system can be constructed.

[0118] The ratio of the cell adhesion area in the substrate is usually 5 to 80%, preferably 20 to 70%, and more preferably 40 to 60%. It should be noted that even when the substrate is placed in a culture dish, the ratio is the ratio of the cell adhesion area to the entire substrate (excluding the bottom of the culture dish). By making the amount of cells relative to the culture solution above a certain amount, cell death can be prevented, and by making it below a certain amount, the depletion of factors required for survival and the resulting damage to cells can be prevented.

[0119] In addition, each cell adhesion region is preferably regularly arranged at a certain interval, for example, in a grid pattern with the same pitch in the vertical and horizontal directions. By making the paracrine effect caused by the cell-produced substance in each cell adhesion region constant, the influence on differentiation can be made constant.

[0120] For example, a pattern having multiple circular cell adhesion areas can be formed as follows: using a photomask having multiple circular openings, a glass substrate having a PEG film formed thereon and the photomask are arranged relative to each other, ultraviolet rays are irradiated from the photomask side, and the areas in the PEG film corresponding to the openings of the photomask are oxidized.

[0121] In order to promote the adhesion of pluripotent stem cells to the cell adhesion area, the substrate used in the present disclosure is preferably pre-coated. Pre-coating can be carried out by coating substrates with extracellular matrix (e.g., collagen, fibronectin, proteoglycan, laminin, vitronectin, etc.), gelatin, lysine, peptides, gelatinous matrix containing them, serum, etc. By pre-coating the substrate, it is possible to promote the adhesion of pluripotent stem cells (e.g., ES cells, iPS cells, etc.) with low adhesion to the cell adhesion area, and it is possible to effectively carry out cell adhesion culture and differentiation induction.

[0122] Similarly, in order to promote the adhesion of pluripotent stem cells to the cell adhesion region, it is preferred to inoculate feeder cells before inoculating the pluripotent stem cells and culture them for about 24 hours, and then culture the pluripotent stem cells on the feeder cells. As feeder cells, any feeder cells commonly used in the art can be used without particular limitation, and examples thereof include fibroblasts. The feeder cells are 1.26×10 5 Pieces / cm 2 The density is preferably 6.3×10 4 Pieces / cm 2 The density is preferably 3.15×10 4 Pieces / cm 2 Inoculate at the above density.

[0123] The above-mentioned pre-coating treatment and feeder cell inoculation may be performed separately or in combination. It is preferred to perform either the pre-coating treatment or the feeder cell inoculation.

[0124] (Step (b))

[0125] Step (b) is a step of seeding pluripotent stem cells on the cell culture substrate having the cell adhesion region and the cell non-adhesion region prepared in step (a).

[0126] As pluripotent stem cells inoculated on a cell culture substrate, for example, embryonic stem cells (ES cells), ES cells (ntES cells) from somatic cells, induced pluripotent stem cells (iPS cells), stem cells from adult tissues or cord blood or umbilical cords, etc. can be cited. These pluripotent stem cells can be used alone or in combination of two or more. In a preferred embodiment, ES cells and iPS cells are used alone as pluripotent stem cells.

[0127] The ES cells used in the present disclosure are preferably ES cells of mammalian origin, for example, ES cells of rodents such as mice or primates such as humans can be used. In a preferred embodiment, as ES cells, ES cells of mouse or human origin are used. ES cells refer to stem cell lines made from the inner cell mass of a part of an embryo in the blastocyst stage belonging to the early stage of animal development, which can theoretically maintain the differentiation pluripotency of all tissues in vitro, and can be almost infinitely proliferated. As ES cells, for example, in order to easily confirm the degree of differentiation, cells into which a reporter gene has been introduced near the Pdx1 gene can be used. For example, 129 / Sv-derived ES cell lines with LacZ genes integrated in the Pdx1 site or ES cell SK7 lines with GFP reporter transgenes under the control of the Pdx1 promoter can be used. Alternatively, ES cell PH3 lines with mRFP1 reporter transgenes under the control of the Hnf3β endoderm-specific enhancer fragment and GFP reporter transgenes under the control of the Pdx1 promoter can also be used.

[0128] The iPS cells used in the present disclosure are cells with pluripotency obtained by initializing somatic cells. Regarding the preparation of induced pluripotent stem cells, a number of groups including the group of Professor Shinya Yamanaka of Kyoto University, the group of Rudolf Jaenisch of Massachusetts Institute of Technology, the group of James Thomson of the University of Wisconsin, and the group of Konrad Hochedlinger of Harvard University have achieved success. For example, the international publication WO2007 / 069666 bulletin records the nuclear initialization factor of somatic cells containing the gene products of Oct family genes, Klf family genes, and Myc family genes, and the nuclear initialization factor of somatic cells containing the gene products of Oct family genes, Klf family genes, Sox family genes, and Myc family genes, and also records the method of making induced pluripotent stem cells by nuclear initialization of somatic cells, including a step of contacting the above-mentioned nuclear initialization factor with somatic cells.

[0129] The type of somatic cells used herein is not particularly limited, and any somatic cells can be used. That is, in this specification, somatic cells include all cells other than the germ cells of the cells constituting the organism, and can be differentiated somatic cells or undifferentiated stem cells. The source of somatic cells can be any one of mammals, birds, fish, reptiles, and amphibians, and is not particularly limited, preferably mammals (e.g., rodents such as mice, primates such as humans), and particularly preferably mice or humans. In addition, when using human somatic cells, somatic cells of any one of a fetus, a newborn, or an adult can be used. As somatic cells, there can be mentioned, for example, fibroblasts (e.g., skin fibroblasts), epithelial cells (e.g., gastric epithelial cells, liver epithelial cells, alveolar epithelial cells), endothelial cells (e.g., blood vessels, lymphatic vessels), neural cells (e.g., neurons, glial cells), pancreatic cells, blood cells, bone marrow cells, muscle cells (e.g., skeletal muscle cells, smooth muscle cells, cardiomyocytes), hepatic parenchymal cells, hepatic non-parenchymal cells, adipocytes, osteoblasts, cells constituting periodontal tissue (e.g., periodontal ligament cells, cementoblasts, gingival fibroblasts, osteoblasts), cells constituting the kidney, eye, ear, etc.

[0130] iPS cells refer to stem cells that have the ability to self-replicate for a long time under predetermined culture conditions (e.g., conditions for culturing ES cells) and have the ability to differentiate into ectoderm, mesoderm, and endoderm under predetermined differentiation induction conditions. In addition, iPS cells in the present disclosure may be stem cells that have the ability to form teratomas when transplanted into experimental animals such as mice.

[0131] In order to make iPS cells from somatic cells, first, at least one resetting gene is introduced into the somatic cells. The resetting gene refers to a gene encoding a resetting factor that has the function of resetting somatic cells to become iPS cells. As specific examples of the combination of resetting genes, the following combinations can be cited, but are not limited to these.

[0132] (i) Oct gene, Klf gene, Sox gene, Myc gene

[0133] (ii) Oct gene, Sox gene, NANOG gene, LIN28 gene

[0134] (iii) Oct gene, Klf gene, Sox gene, Myc gene, hTERT gene, SV40 largeT gene (iv) Oct gene, Klf gene, Sox gene

[0135] The pluripotent stem cells before being inoculated on the cell culture substrate are maintained undifferentiated using a non-differentiation induction medium. Before and after inoculation on the surface of the cell culture substrate, the differentiation induction medium is switched to inoculation on the surface of the cell culture substrate, and the cells are directly proliferated in the cell adhesion area until they are confluent. By enzymatically treating the obtained cell aggregate, the target differentiation-induced cells can be obtained.

[0136] The non-differentiation induction 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, and examples thereof include: a medium containing a leukemia inhibitory factor known to have the property of maintaining the undifferentiated nature of mouse ES cells and mouse iPS cells; a medium containing a basic fibroblast growth factor (basic FGF) known to have the property of maintaining the undifferentiated nature of human iPS cells; etc. The differentiation induction medium is not particularly limited as long as it is a medium that induces differentiation of pluripotent stem cells, and examples thereof include a serum-containing medium, a serum-free medium containing a known component having the property of replacing serum, etc. 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.

[0137] 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, and 1-20mM ROCK inhibitor (e.g., Y27632) may be added. Liquid factors may be added to the differentiation induction medium, but according to the differentiation induction method of the intestinal structure disclosed in the present invention, the intestinal structure can be differentiated even without adding liquid factors. Therefore, in one embodiment, a differentiation induction medium that does not contain liquid factors is used to culture cells.

[0138] The method of seeding pluripotent stem cells on a cell culture substrate is not particularly limited, and the seeding can be performed by a method commonly used for seeding cells. In the present disclosure, pluripotent stem cells are, for example, less than 1.2×10 5 Pieces / cm 2 , preferably 3×10 4 Pieces / cm 2 Below, preferably 1.5×10 4 Pieces / cm 2 The above density was inoculated on the cell culture substrate.

[0139] (Step (c))

[0140] Step (c) is a step of culturing the pluripotent stem cells seeded on the cell culture substrate in step (b) in a culture medium to obtain an intestinal structure.

[0141] The pluripotent stem cells are usually cultured at 37° C. Preferably, the cells are cultured in an atmosphere of about 5% CO 2 concentration using a CO 2 cell culture apparatus or the like.

[0142] The culture medium used in this step (c) is not particularly limited as long as it is a culture medium for inducing differentiation of pluripotent stem cells, and can be appropriately selected according to the type of pluripotent stem cells. As culture medium, for example, MEM culture medium, BME culture medium, DMEM culture medium, DMEM-F12 culture medium, αMEM culture medium, IMDM culture medium, ES culture medium, DM-160 culture medium, Fisher culture medium, F12 culture medium, WE culture medium, RPMI1640 culture medium, etc. can be cited. In addition, in these culture mediums, serum or a substance having the property of replacing serum can also be added as required.

[0143] The culture time after the pluripotent stem cells are inoculated into the cell culture substrate is preferably 60 days or more, more preferably 80 days or more, further preferably 100 days or more, preferably 210 days or less, and more preferably 140 days or less. On the 60th day after the pluripotent stem cells were inoculated into the cell culture substrate, villin and 5TH, which are differentiation markers of the intestine, were detected, and a bag-like structure was confirmed, and peristaltic motion was 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.

[0144] As described above, through each step (a) to (c), an intestinal structure composed of endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem cells can be obtained. The obtained intestinal structure is subjected to the step (B) described below.

[0145] <Process (B)>

[0146] In step (B), the intestinal structure prepared in step (A) is cultured to proliferate fibroblast-like cells derived from the intestinal structure.

[0147] There is no particular limitation on the method of culturing the intestinal structure as long as the effects of the present disclosure are exerted. It can be a method of culturing cells by adhering to the culture container (so-called adhesion culture) or a method of culturing cells without adhering to the culture container (so-called suspension culture). In a preferred embodiment, the intestinal structure is cultured in a state where at least a portion of the intestinal structure is in contact with the culture container. The culture container and the intestinal structure can be in contact all the time or intermittently.

[0148] In one embodiment, the intestinal structure is suspended in a culture container, and the amount of culture medium added to the culture container is appropriately adjusted so that the culture container (e.g., bottom surface, inner wall, etc.) is in contact with at least a portion of the intestinal structure. For example, when a culture dish is used as the culture container, the culture medium is shallowly added to the culture dish, and the suspension culture is performed in a manner such that at least a portion of the intestinal structure is in contact with the culture dish.

[0149] The medium used in this step (B) is not particularly limited as long as it is a medium that induces differentiation and proliferation of fibroblasts, and examples thereof 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 a substance having the property of replacing serum may be added to these media as needed.

[0150] The intestinal structure is usually cultured at 37° C. It is preferably cultured in an atmosphere of about 5% CO 2 concentration using a CO 2 cell culture apparatus or the like.

[0151] The intestinal structure is preferably cultured until the fibroblast-like cells from the intestinal structure are confluent or substantially confluent. The culture time can be appropriately set according to the proliferation rate of the fibroblast-like cells, the shape and size of the culture container used, etc. The culture time of the intestinal structure can be set, for example, to 1 to 6 days, 6 to 12 days, 12 to 72 days, etc.

[0152] In this step (B), in addition to the above-mentioned fibroblast-like cells, cells with intestinal epithelial cell-like properties (hereinafter also referred to as "intestinal epithelial cell-like cells") sometimes proliferate. The intestinal epithelial cell-like cells from the intestinal structure obtained by this step (B) have different properties from the intestinal epithelial cells obtained by organoid culture using stem cells as the starting point in the past. For example, the inventors have revealed that in the intestinal epithelial cell-like cells from the intestinal structure obtained by this step (B), the cell division-related protein Ki-67, which is a proliferating cell marker, is locally present. Here, it is generally known that intestinal epithelial cells are generally differentiated only from Lgr5 (Leucine-rich repeat-containing G-protein-coupled receptor 5) positive stem cells known as intestinal epithelial stem cells, and the differentiated intestinal epithelial cells are not produced by proliferation. Therefore, as described above, the local presence of the proliferative cell marker Ki-67 in the intestinal epithelial cell-like cells from the intestinal structure obtained by this step (B) can be said to indicate that the intestinal epithelial cell-like cells divide to produce new intestinal epithelial cell-like cells. That is, from the perspective of the possibility of proliferation independent of the previous Lgr5-positive stem cells, it is believed that the intestinal epithelial cell-like cells from the intestinal structure obtained by this step (B) have specific properties. In addition, as the intestinal epithelial cell-like cells from the intestinal structure obtained by this step (B), various cells that bear the function of the small intestine, such as secretory cells confirmed by the expression of specific mucins and absorptive epithelial cells confirmed by the expression of specific transporters, can be obtained.

[0153] The intestinal structure-derived fibroblast-like cells (ie, feeder cells of the present disclosure) and intestinal epithelial cell-like cells obtained in the present step (B) may be used in combination with each other or may be used alone.

[0154] <Process (C)>

[0155] In step (C), the intestinal structure-derived fibroblast-like cells grown in step (B), that is, the feeder cells of the present disclosure, are isolated.

[0156] The separation of fibroblast-like cells can be carried out by the method commonly used when separating fibroblasts. That is, it can be carried out by known enzyme treatment, scraping with a scraper and other steps. These steps can be carried out alone or in combination of two or more.

[0157] 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.

[0158] In one embodiment, in step (C), in addition to the separation of the fibroblast-like cells from the intestinal structure described above, the separation of the intestinal epithelial cell-like cells from the intestinal structure is also performed. Specifically, the separation of the intestinal epithelial cell-like cells from the intestinal structure can be performed according to the following steps. First, the culture medium containing the intestinal structure after the fibroblast-like cells are separated as described above is made into an environment in which only the intestinal epithelial cell-like cells can survive. For example, by adding antibiotics such as puromycin and penicillin to the culture medium at 100 μg / ml, an environment in which only intestinal epithelial cell-like cells with the activity of metabolizing these antibiotics (drug metabolizing enzymes: CYP) can survive is formed. Then, dead cells are removed from the culture medium to separate the intestinal epithelial cell-like cells. It should be noted that in the presence of hepatocytes in the culture medium, the hepatocytes can survive by adding antibiotics as described above, but the hepatocytes can be easily distinguished under a microscope as a group of multinucleated cells, so the intestinal epithelial cell-like cells can be separated by removing the hepatocytes under a microscope. In addition, since hepatocytes are not stained in immunostaining using antibodies against CDX2, intestinal epithelial cell-like cells can also be separated by distinguishing and removing hepatocytes using such antibodies. Intestinal epithelial cell-like cells adhere to each other by strong intercellular adhesion, so when intestinal epithelial cell-like cells form a layer structure with other cells, intestinal epithelial cell-like cells can be separated by peeling them off from other cells using forceps or the like.

[0159] <Process (D)>

[0160] The method for producing feeder cells disclosed herein may further include, in addition to the above steps (A) to (C), a step (step (D)) of growing the fibroblast-like cells separated in the step (C).

[0161] The fibroblast-like cells separated in step (C) can be cultured by a method generally used for culturing fibroblasts.

[0162] The medium used in this step (D) is not particularly limited as long as it is a medium that allows fibroblasts to proliferate, and examples thereof 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 a substance having the property of replacing serum may be added to these media as needed.

[0163] The fibroblast-like cells are usually cultured at 37° C. Preferably, the cells are cultured in an atmosphere of about 5% CO 2 concentration using a CO 2 cell culture apparatus or the like.

[0164] The fibroblast-like cells are preferably cultured until the fibroblast-like cells are confluent or substantially confluent. The culture time can be appropriately set according to the proliferation rate of the fibroblast-like cells, the shape and size of the culture container used, etc. The culture time of the fibroblast-like cells can be set to, for example, 3 to 7 days, 7 to 12 days, etc.

[0165] [Feeder cells]

[0166] According to one embodiment of the present disclosure, a fibroblast-like cell (i.e., a feeder cell of the present disclosure) is provided, which is used to maintain cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate through two-dimensional culture. In one embodiment, the feeder cells of the present disclosure show a specific gene expression profile. In one embodiment, the feeder cells of the present disclosure are characterized in that they express each gene of platelet-derived growth factor receptor (PDGFRA), CD81, forkhead box protein (Forkhead box) L1 (Foxl1) and bone morphogenetic protein antagonist (Gremlin) -1 (GREM1). In addition, in another embodiment, the feeder cells of the present disclosure are characterized in that they express each gene of PDGFRA, CD81 and GREM1, and do not express the CD34 gene. In a preferred embodiment, the feeder cells of the present disclosure are characterized in that they express each gene of PDGFRA, CD81, Foxl1 and GREM1, and do not express the CD34 gene. According to these feeder cells of the present disclosure, cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate through two-dimensional culture can be maintained or proliferated by two-dimensional culture.

[0167] In the past, feeder cells for maintaining cells such as intestinal epithelial cells that are considered difficult to maintain and proliferate through two-dimensional culture have been studied in mice. However, feeder cells that can maintain cells such as intestinal epithelial cells alone (i.e., having complete feeding ability) have not been found so far, and the current situation is to achieve the maintenance of cells such as intestinal epithelial cells by combining a variety of feeder cells. In addition, as representative feeder cells discovered in the past, trophoblasts (Trophocytes), telocytes, etc. of mice can be cited. Trophocytes are known to express the genes of PDGFRA, CD81, and CD34, but not Foxl1. In addition, telocytes are known to express the genes of PDGFRA and Foxl1, but not the genes of CD81 and CD34. Therefore, cells expressing the genes of PDGFRA, CD81, Foxl1, and GREM1, and cells expressing the genes of PDGFRA, CD81, and GREM1 but not expressing the CD34 gene, as in the feeder cells disclosed in the present invention, have not been found at all, and it can be said that this is a surprising discovery that such cells have excellent feeding ability for cells such as intestinal epithelial cells, which was completely unknown before.

[0168] In one embodiment, the feeder cells of the present invention can be obtained by the above-mentioned steps (A) to (C) and the optional step (D). The feeder cells of the present invention can be used to maintain or proliferate cells that were previously considered difficult to maintain and proliferate by two-dimensional culture. In particular, compared with conventional feeder cells, the feeder cells of the present invention have the ability to feed the target cells in long-term culture (e.g., more than 10 passages).

[0169] The cells (hereinafter also referred to as "target cells") used as the object of the feeder cells of the present invention are not particularly limited, and are preferably cells that are considered difficult to maintain and proliferate through two-dimensional culture as described above. More specifically, it is preferred that the cells are prone to rapid dedifferentiation due to stress caused by passage and culture time during two-dimensional culture. As such target cells, for example, small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, chondrocytes, etc. can be cited. In a preferred embodiment, the target cells are small intestinal epithelial cells and hepatocytes. Therefore, in one embodiment, the feeder cells of the present invention are feeder cells for maintaining or proliferating the above-mentioned cells through two-dimensional culture (planar culture). That is, even when the target cells are passaged multiple times (for example, more than 3 times) through two-dimensional culture, as long as the target cells maintain their original properties, the feeder cells of the present invention can inhibit the loss of their properties and proliferate them. It should be noted that in the present disclosure, feeder cells and target cells may be from the same individual (feeder cells are derived from pluripotent stem cells from a specific individual, and target cells are derived from pluripotent stem cells or natural cells from the same individual), or from different individuals (feeder cells are derived from pluripotent stem cells from a specific individual, and target cells are derived from pluripotent stem cells or natural cells from other individuals of the same species). It should be noted that "natural cells" refer to cells that exist in an individual and are not artificially manipulated (i.e., "natural"), and specifically, small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, chondrocytes, etc. collected from humans can be cited.

[0170] In one embodiment, the target cells are cells derived from pluripotent stem cells, preferably small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes derived from pluripotent stem cells, and more preferably small intestinal epithelial cells and hepatocytes derived from pluripotent stem cells.

[0171] In a preferred embodiment, the target cell is an intestinal epithelial cell-like cell collected from the above-mentioned intestinal structure, and is particularly preferably an intestinal epithelial cell-like cell collected from the same intestinal structure as the intestinal structure prepared in step (A) to obtain the feeder cells of the present invention. In this way, by making the feeder cells and intestinal epithelial cell-like cells of the present invention all come from the same intestinal structure made by the same pluripotent stem cell, there are advantages as described below. As the first advantage, since the feeder cells and intestinal epithelial cell-like cells are derived from the same pluripotent stem cell, their respective safety can be easily confirmed. Specifically, before the feeder cells and intestinal epithelial cell-like cells are differentiated and induced, the safety of the feeder cells and intestinal epithelial cell-like cells after differentiation and induction can be confirmed only by confirming the safety of the pluripotent stem cells that are their source. In addition, since the feeder cells and intestinal epithelial cell-like cells are derived from the same pluripotent stem cell, no components of heterologous origin will be introduced after differentiation and induction. Therefore, when using one or both of these differentiated or induced cells, there is no problem of mixing in components of a xenogeneic origin, and in particular, in medical applications, so-called "xeno-free" cells can be used directly.

[0172] In one embodiment, the intestinal epithelial cell-like cells collected from the above-mentioned intestinal structure express at least one intestinal epithelial cell marker 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 above-mentioned intestinal structure express at least one intestinal epithelial cell marker selected from the group consisting of CDX2 and villin.

[0173] When the target cells are cultured using the feeder cells of the present disclosure, the number of passages of the feeder cells used is not particularly limited, and may be, for example, 2 or more, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, etc. On the other hand, the number of passages of the feeder cells used may be, for example, 20 or less, 15 or less, 12 or less, etc.

[0174] The feeder cells disclosed herein may be cultured alone to proliferate only the feeder cells. Alternatively, the feeder cells disclosed herein may be co-cultured with specific target cells to proliferate both the feeder cells and the target cells.

[0175] By culturing the feeder cells of the present invention alone, a culture (cell colony) containing the feeder cells of the present invention or a culture composed of the feeder cells of the present invention can be obtained. Specifically, by culturing the feeder cells of the present invention alone in two dimensions, a culture having a two-dimensional shape (e.g., sheet-like) containing the feeder cells of the present invention or a culture having a two-dimensional shape (e.g., sheet-like) composed of the feeder cells of the present invention can be obtained. It should be noted that in this specification, with respect to "culture" and "co-culture", "comprising" specific cells means that cells other than specific cells may be included in the culture, and "consisting of" specific cells means that cells other than specific cells are not intentionally included in the culture.

[0176] In addition, as described above, the feeder cells of the present invention can maintain or proliferate the target cells, especially the cells that are considered difficult to maintain and proliferate through two-dimensional culture. Therefore, by using the feeder cells of the present invention to culture the target cells (that is, the feeder cells of the present invention and the target cells are co-cultured), it is possible to obtain 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 invention and the target cells. In particular, by using the feeder cells of the present invention, it is possible to maintain or proliferate cells that were previously considered difficult to maintain and proliferate through two-dimensional culture in a two-dimensional manner, so even such cells can be obtained in the form of a co-culture having a two-dimensional shape (e.g., sheet-like). For example, it can be obtained in the form of a co-culture of a layer containing feeder cells and a layer of target cells. In addition, by separating and removing a part or all of the feeder cells of the present invention from such a co-culture, it is possible to obtain a culture containing the target cells, or a culture composed of the target cells. Specifically, the feeder cells of the present disclosure are made into a sheet, and the target cells that are considered difficult to maintain and proliferate by two-dimensional culture are co-cultured on the sheet to maintain or proliferate them, thereby obtaining a co-culture of the target cells in a two-dimensional shape (e.g., sheet shape), and then a part or all of the feeder cells of the present disclosure are separated and removed from the obtained co-culture, thereby obtaining a culture having a two-dimensional shape containing the target cells, or a culture having a two-dimensional shape composed of the target cells. On the other hand, a part or all of the target cells can also be separated and removed from the co-culture obtained as described above, thereby obtaining 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.

[0177] In one embodiment, the target cells are intestinal epithelial cells (eg, small intestinal epithelial cells, large intestinal epithelial cells, etc.), hepatocytes, chondrocytes, etc., which are considered difficult to maintain and proliferate in two-dimensional culture, preferably intestinal epithelial cells.

[0178] The separation of the feeder cells of the present disclosure contained in the co-culture and the target cells can generally be performed using a known method used in the field of cell culture. In one embodiment, when the feeder cells of the present disclosure and intestinal epithelial cell-like cells are contained in the co-culture, the intestinal epithelial cell-like cells are separated from the co-culture using the method disclosed in the pamphlet of International Publication No. 2019 / 131938.

[0179] In one embodiment, such a co-culture of feeder cells and target cells disclosed herein, a co-culture containing target cells, and a co-culture containing feeder cells can all be used as so-called cell sheets. A cell sheet is a thin film made by culturing cells into a sheet shape, which has the advantages of being able to transplant biological tissues without using sutures and being able to transplant in a short time. Such characteristics are effectively utilized and used for the treatment of various diseases and tissue regeneration. Therefore, in one embodiment, a cell sheet is used for the treatment of diseases and for transplantation materials for tissue regeneration.

[0180] As described above, the feeder cells disclosed in the present invention can maintain or proliferate the target cells through two-dimensional culture, so it is easy to make co-cultures such as cell sheets with a two-dimensional shape. In addition, by appropriately setting the size, shape, culture time, etc. of the culture container in which the feeder cells disclosed in the present invention and the target cells are co-cultured, it is possible to make cell sheets with a desired size and two-dimensional shape. In the past, it was difficult to maintain and proliferate cells such as intestinal epithelial cells through two-dimensional culture, and it was not easy to make co-cultures such as cell sheets mainly containing these cells with a two-dimensional shape. Therefore, for example, in the case of making large cell sheets, multiple cell sheets with smaller two-dimensional shapes that can be made can only be used in combination. On the other hand, by using the feeder cells disclosed in the present invention, even such cells can be maintained or proliferated through two-dimensional culture, and their size and shape can also be appropriately set. Therefore, as described above, large cell sheets can be made without combining multiple cell sheets. For example, by using the feeder cells disclosed in the present invention, it is possible to make a giant cell sheet such as a circle, an ellipse, or a polygon of the same size (such as a quadrilateral, etc.) with a diameter of more than 1 cm, more than 2 cm, more than 3 cm, more than 5 cm, or more than 10 cm into one cell sheet without combining multiple cell sheets. In this way, for cells that were previously difficult to maintain and proliferate through two-dimensional culture, it is possible to make a large cell sheet without combining multiple cell sheets, which can be said to be one of the excellent effects brought about by the feeder cells disclosed in the present invention. For example, in Crohn's disease, the size (diameter) of the ulcer is used as an indicator to judge the condition, and it is judged to be difficult to cure when the diameter is more than 2 cm. Therefore, it can be said that by efficiently making such a giant cell sheet with one cell sheet, the treatment of the cell sheet can make a great contribution to the effective treatment of Crohn's disease, etc.

[0181] In addition, the following method is known in the past: a precursor cell in the previous stage of differentiation into a specific cell is made from pluripotent stem cells, etc., and after being proliferated by a proliferation medium through two-dimensional culture, differentiation is promoted using a differentiation medium to obtain a structure composed of functional cells. Here, in the case where the target cell is a cell that is prone to rapid dedifferentiation due to stress caused by passage or culture time during two-dimensional culture, dedifferentiation occurs during the differentiation promotion stage using a differentiation medium, and the properties of the target cell cannot usually be maintained for a long time. That is, a structure that maintains the properties of the target cell for a long time cannot be obtained. However, by using the feeder cells disclosed in the present invention, dedifferentiation in this differentiation promotion stage can be suppressed, and the properties of the target cell can be maintained for a long time. Therefore, the feeder cells disclosed in the present invention can also be appropriately used in such uses as structures (such as transplant materials, drug development tools, etc.) that need to maintain the properties of the target cell for a long time.

[0182] In addition, the feeder cells disclosed in the present invention can be produced in a manner that does not contain xenogeneic components (so-called xeno-free conditions). Therefore, when used as transplant materials such as cell sheets as described above, the risk caused by xenogeneic components during transplantation can be eliminated. In addition, as shown in the examples, the feeder cells disclosed in the present invention can be used as a scaffold to proliferate primary intestinal epithelial cells collected from humans, so it is speculated that the single transplantation of the feeder cells disclosed in the present invention can contribute to rapid autologous tissue regeneration.

[0183] In addition, the feeder cells of the present disclosure are believed to have various functions in addition to the above-mentioned functions as feeder cells. Therefore, the use of the feeder cells of the present disclosure is not limited to such uses as feeder cells, and can also be used for uses where functions different from the functions as feeder cells are expected.

[0184] For example, according to one embodiment of the present invention, a pharmaceutical composition is provided, which comprises feeder cells (fibroblast-like cells) disclosed herein, or feeder cells (fibroblast-like cells) and various cells such as small intestinal epithelial cells, hepatocytes, etc. co-culture. Here, the pharmaceutical composition refers to a composition obtained by preparing feeder cells (fibroblast-like cells) or co-cultures disclosed herein into oral preparations or non-oral preparations according to conventional methods. In formulation, additives that are permitted by formulation can be used in combination. As additives that are permitted by formulation, for example, excipients, stabilizers, preservatives, wetting agents, emulsifiers, lubricants, sweeteners, colorants, spices, buffers, antioxidants, pH regulators, etc. can be cited. In the case where the pharmaceutical composition is an oral preparation, it can be in the form of solid preparations such as tablets, powders, fine granules, granules, capsules, pills, sustained-release agents, solutions, suspensions, emulsions, and other liquid preparations. When the pharmaceutical composition is a parenteral preparation, it can be in the form of an injection, a suppository, a sheet-like patch, a suspension for application, a spray, or the like.

[0185] The dosage (application) of the pharmaceutical composition is not particularly limited as long as the effects of the present disclosure are exerted, and can be appropriately adjusted according to the age, health status, weight, etc. of the subject of administration (application). For example, when the feeder cells of the present disclosure are used as a sheet-shaped patch for treating an ulcer of the mucous membrane of the subject, the patch is applied in a manner that the ulcer portion and its surrounding portion are completely covered. In the case of a large ulcer, a plurality of patches are combined and applied in a manner that the patch completely covers the ulcer portion and its surrounding portion.

[0186] In order to better exert its effect, feeder cells (fibroblast-like cells) and co-cultures are preferably taken up (administered) continuously for a long period of time, and the intake (administration) period can be set to, for example, 1 to 6 weeks, 1 to 12 weeks, 2 to 10 weeks, 4 to 10 weeks, 4 to 12 weeks, etc. In this specification, "continuously" means that a prescribed amount of the pharmaceutical composition is taken up (administered) continuously every day.

[0187] According to another aspect of the present disclosure, a method for treating a disease in a subject is provided, comprising causing the subject to ingest (administer) an effective amount of feeder cells (fibroblast-like cells) or co-culture. The above method may be a non-therapeutic method or a therapeutic method.

[0188] In the method for treating a disease, the dosage of feeder cells (fibroblast-like cells) and co-culture and the time of ingestion (administration) are not particularly limited as long as the effects of the present disclosure are exerted, and can be appropriately adjusted according to the type of disease, the age, health condition, weight, etc. of the ingestion (administration) subject.

[0189] According to another aspect of the present disclosure, a method for treating a disease in a subject is provided, comprising applying the cell sheet to the subject. The method may be a non-therapeutic method or a therapeutic method.

[0190] 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 exerted, and can be appropriately adjusted according to the type of disease, the type of organ or tissue to be applied, and the shape and size of the part to be applied.

[0191] According to another aspect of the present disclosure, there is provided use of the feeder cells (fibroblast-like cells) and co-cultures of the present disclosure for producing a composition for treating a disease.

[0192] In each of the above aspects, diseases that can be treated using the feeder cells (fibroblast-like cells), cocultures, and cell sheets disclosed herein include diseases accompanied by digestive tract ulcers such as Crohn's disease and ulcerative colitis, and complications associated with digestive tract surgery such as postoperative colon cancer surgery.

[0193] In each of the above-mentioned embodiments, the cell sheet comprising the feeder cells (fibroblast-like cells) disclosed herein, the cell sheet comprising the feeder cells (fibroblast-like cells) and the co-culture, and the cell sheet comprising the co-culture can be used for transplantation into the body of a human or non-human animal, in particular, for covering inflamed areas, wound areas, lesions, sutured areas after surgery, abdominal cavities, defective intestinal tissues, and the like in organs and the like.

[0194] 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 the ability to secrete mucus, specifically, the ability to secrete mucin, the cell sheet of the present disclosure has a high protective effect on the application site (e.g., the healing site in ulcers, etc.), and is therefore preferred. The cell sheet of the present disclosure is preferably applied to an inflamed site, a wound site, a lesion site, a suture site after surgery, the abdominal cavity, etc., to heal the site, thereby protecting the application site.

[0195] The cell sheet of the present disclosure can preferably be prevented from leaking out of or into the healing site, or from invading the healing site by healing with the inflamed site, wound site, lesion site, sutured site after surgery, or abdominal cavity.

[0196] The cell sheet disclosed in the present invention can preferably provide cells or cell scaffolds required for the healing of the healing part by healing with the inflamed part, the wound part, the lesion part, the sutured part after surgery, or the abdominal cavity.

[0197] The cell sheet disclosed in the present invention can preferably prevent the adhesion of the healing part to the ectopic site by healing with the inflamed part, wound part, lesion part, sutured part after surgery or abdominal cavity.

[0198] The cell sheet disclosed herein can preferably suppress immune responses, such as suppressing the infiltration of blood cell lineage cells associated with autoimmune responses into digestive tract tissues, by healing with inflamed areas, wound areas, lesions, sutured areas after surgery, or the abdominal cavity.

[0199] The cell sheet disclosed herein can alleviate the hyperinflammation of the healing part by preferably healing with the inflamed part, the wound part, the lesion part, the suture part after surgery or the abdominal cavity. The cell sheet disclosed herein can prevent postoperative complications by preferably healing with the suture part after surgery. The cell sheet disclosed herein can replace the intestinal function by preferably transplanting into the defective intestinal tissue.

[0200] The cell sheet disclosed herein can be held by forceps or the like in any surgery such as endoscopic surgery, laparotomy, and laparoscopic surgery, and attached to the desired transplant site. In the case of transplantation to a site that is difficult to transplant by laparoscopic surgery, the cell sheet disclosed herein can be transplanted in a state of being integrated with a support, or can be transplanted by holding it with a carrier such as a thread. The cell sheet disclosed herein can be fixed to a biocompatible support and transplanted together with the support.

[0201] When the cell sheet of the present disclosure is transplanted into a living body, a biocompatible adhesion-assisting substance such as fibrin may be applied to the cell sheet of the present disclosure and / or the transplant site. In addition, the cell sheet of the present disclosure may be sutured to the transplant site.

[0202] When attaching the cell sheet of the present invention to the transplant site, for example, the cell sheet of the present invention can be attached to the transplant site by being unfolded with forceps, or by being placed on the transplant site and then unfolded. In addition, the cell sheet of the present invention can be attached to the transplant site by pressing it with a clamp having a balloon structure.

[0203] Other aspects of the present disclosure relate to the following [1] to

[28] .

[0204] [1] A method for producing feeder cells for maintaining or growing cells, comprising:

[0205] (A) a step of preparing an intestinal structure composed of endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem cells;

[0206] (B) a step of culturing the intestinal structure to proliferate fibroblast-like cells derived from the intestinal structure; and

[0207] (C) A step of isolating the fibroblast-like cells to obtain feeder cells.

[0208] [2] The method according to [1], wherein the intestinal structure is produced by a method comprising the following steps:

[0209] (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and a cell non-adhesion region surrounding each of the cell adhesion regions;

[0210] (b) a step of seeding the pluripotent stem cells on the cell culture substrate; and

[0211] (c) a step of culturing the seeded pluripotent stem cells in a culture medium.

[0212] [3] The method according to [1] or [2], further comprising (D) a step of proliferating the isolated fibroblast-like cells.

[0213] [4] The method according to any one of [1] to [3], wherein in the step (B), the intestinal structure is cultured in a culture container, and at least a portion of the intestinal structure is in contact with the culture container.

[0214] [5] The method according to [2], wherein the culturing time in the step (c) is 60 days or longer.

[0215] [6] A feeder cell produced by the method according to any one of [1] to [5].

[0216] [7] A fibroblast-like cell, which is positive for PDGFRA and CD81 and expresses Foxl1 and GREM1.

[0217] [8] The fibroblast-like cells according to [7], wherein the cells are positive for PDGFRA and CD81, negative for CD34, and express Foxl1 and GREM1.

[0218] [9] The fibroblast-like cells described in [7] or [8], which are derived from pluripotent stem cells or from a culture of an intestinal structure, wherein the intestinal structure is produced from pluripotent stem cells and contains endoderm-derived cells and mesoderm-derived cells.

[0219]

[10] The fibroblast-like cell according to [9], wherein the intestinal structure is produced by a method comprising the following steps:

[0220] (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and a plurality of isolated cell adhesion regions formed on the substrate and a cell non-adhesion region surrounding each of the cell adhesion regions;

[0221] (b) a step of seeding the pluripotent stem cells on the cell culture substrate; and

[0222] (c) a step of culturing the seeded pluripotent stem cells in a culture medium.

[0223]

[11] The fibroblast-like cells according to [9] or

[10] , wherein the pluripotent stem cells are derived from humans.

[0224]

[12] The feeder cell described in [6] or the fibroblast-like cell described in any one of [7] to

[11] , which is a feeder cell for maintaining or growing at least one cell selected from the group consisting of small intestinal epithelial cells, large intestinal epithelial cells, hepatocytes, and chondrocytes.

[0225]

[13] The feeder cell according to [6] or the fibroblast-like cell according to any one of [7] to

[12] , which is a feeder cell for maintaining or growing the cell in a planar culture.

[0226]

[14] The feeder cell according to [6] or the fibroblast-like cell according to any one of [7] to

[13] , which is a feeder cell capable of passage the cell three or more times.

[0227]

[15] A cell sheet comprising the feeder cell according to [6] or the fibroblast-like cell according to any one of [7] to

[14] .

[0228]

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

[14] .

[0229]

[17] The co-culture according to

[16] , wherein the cells have been passaged three or more times.

[0230]

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

[16] or

[17] .

[0231]

[19] The cell sheet according to

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

[0232]

[20] The cell sheet according to

[18] or

[19] , comprising a layer of at least one cell type selected from the group consisting of small intestinal epithelial cells and hepatocytes.

[0233]

[21] The cell sheet according to any one of

[18] to

[20] , further comprising a layer of the feeder cells.

[0234]

[22] A transplant material comprising the cell sheet according to any one of

[15] and

[18] to

[21] .

[0235]

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

[14] .

[0236]

[24] The method as described in

[23] , wherein the cell culture is planar culture.

[0237]

[25] The method of

[23] or

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

[0238]

[26] The method according to any one of

[23] to

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

[0239]

[27] The method according to any one of

[23] to

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

[0240]

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

[0241] Example

[0242] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to these examples.

[0243] [Example 1] Preparation of cell culture substrate

[0244] Mix 39.0g of toluene and 13.5g of methacryl silane TSL8370 (manufactured by GE Toshiba Silicone), and add 450μl of triethylamine under stirring. After stirring for several minutes at room temperature, transfer all of it to a glass dish. Immerse a 5cm square glass substrate after UV cleaning in it and leave it at room temperature for 16 hours. Then, wash the glass substrate with ethanol and water and dry it by blowing nitrogen. Thus, a film containing methacryl is formed on the surface of the glass substrate.

[0245] Dissolve 0.1 g of polymerization initiator 2,2'-dimethoxy-2-phenylacetophenone (DMPA, Aldrich) in 10 g of polyethylene glycol diacrylate (PEGdA, Aldrich) at room temperature. Spin coat it on the above-mentioned methacrylated substrate at 1500 rpm for 5 seconds. Then, quickly irradiate the entire surface of the substrate with ultraviolet light for 3 seconds under a nitrogen atmosphere. Then, post-bake at 160°C for 10 minutes. After immersing the PEGdA substrate in water overnight, wash it with water and then dry it. It should be noted that the average dry film thickness is 0.33 μm.

[0246] The photomask used was a 5-inch photomask having a pattern in which a plurality of circular openings with a diameter of 1.5 mm were formed. The spaces between the openings of the photomask, that is, the shortest distance between the openings, were all 0.35 mm.

[0247] The mask was gently placed on the PEGdA surface of the PEGdA-treated substrate and irradiated from the back side of the mask with a xenon excimer lamp (172 nm, 10 mW / cm 2 ) as the light source for 1 minute. Thus, the area on the surface of the PEGdA film corresponding to the opening of the photomask was oxidized. Next, the substrate was cut into 2.5 cm squares and used as a cell culture substrate.

[0248] The cell adhesion regions in the obtained cell culture substrate were circular in shape with a diameter of 1.5 mm, and the spaces between the cell adhesion regions, that is, the shortest distances between the cell adhesion regions were all 0.35 mm.

[0249] [Example 2] Preparation of intestinal structure

[0250] The surface of the cell culture substrate prepared in Example 1 was inoculated with about 1×10 7 Pieces / cm 2 Pluripotent stem cells (ES cell line ─ SEES-2 cells established by the National Center for Developmental Medicine, National Research and Development Corporation of Japan) were cultured in an incubator at 37°C and 5% CO2 concentration for 60 days for differentiation induction. XF32 medium was used for the culture of pluripotent stem cells. After 60 days of differentiation induction, the culture was gelled using a cell suspension gelling agent (iPGell, manufactured by Genostaff Co., Ltd.), and the gel-embedded culture was cut to make slices of the culture. The composition of the XF32 medium used in the culture of pluripotent stem cells is shown in Table 1 below.

[0251] [Table 1]

[0252]

[0253] 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 area, confirming the formation of a bag-like structure (i.e., intestinal structure) ( Figure 1 ). It was confirmed that the bag-like structure was peristaltic, and thus the presence of cells from the endoderm, mesoderm, and ectoderm required for peristaltic movement was confirmed. In addition, observations using a stereomicroscope also revealed peristaltic-like movements in the bag-like structure, and the presence of Cajal cells of the ectoderm, intestinal epithelial cells of the endoderm, and smooth muscles of the mesoderm could be confirmed.

[0254] [Example 3] Maintenance and proliferation of fibroblast-like cells and intestinal epithelial cells from intestinal structures 1

[0255] The intestinal structure prepared in Example 2 was transferred to another culture dish with adhesion, and cultured for 17 days in an incubator at 37°C and a CO2 concentration of 5%, so that the fibroblast-like cells and intestinal epithelial-like cells from the intestinal structure proliferated. XF32 culture medium was used for the culture of the intestinal structure. It should be noted that when culturing the intestinal structure, the amount of the culture medium was adjusted so that at least a portion of it was always in contact with the bottom surface of the culture container.

[0256] Using D-MEM supplemented with 10% FBS, only the obtained fibroblast-like cells were expanded and cultured by replacing the medium twice a week and subculturing every week, and a large amount of stock solution with a subculture number of 7 was obtained. It should be noted that the obtained stock solution was divided into two parts, which were stored in a freezer at -80°C or in the gas phase of a tank filled with liquid nitrogen, and the stock solution stored in the tank filled with liquid nitrogen was transferred to a freezer at -80°C before use. The fibroblast-like cells and intestinal epithelial cell-like cells were cultured together for 5 days. The culture was carried out according to the following steps: the fibroblast-like cells were pre-cultured with D-MEM supplemented with 10% FBS for about 1 week, and then the intestinal epithelial cell-like cells were inoculated on the fibroblast-like cells. In the culture after inoculating the intestinal epithelial cell-like cells, ESTEM-HE medium (GlycoTechnica) containing R-spondin 1 and Wnt3a was used. Antibodies against intestinal epithelial cell marker CDX2 and fibroblast marker vimentin were used to immunostain the intestinal epithelial-like cells after 21 days of culture (passage 3) and the fibroblast-like cells at passage 7 that were replaced at each passage and cultured for 1 week. The results showed that CDX2 and vimentin were detected. Figure 2 ). It should be noted that Figure 2 In the figure, the lower left photo shows the immunostaining photo of CDX2, the upper right photo shows the staining photo based on DAPI, the lower right photo shows the immunostaining photo using the antibody against vimentin, and the upper left photo shows the overlap (merge) of the above three photos. Figure 2 In each of the photos, the scale bar represents 100 μm. Figure 2 The photo shows that in the case of co-culture of fibroblast-like cells and intestinal epithelial-like cells, intestinal epithelial-like cells can maintain and proliferate. It should be noted that vimentin is a marker of the mesoderm, and intestinal epithelial cells expressing CDX2 and villin are considered to be cells derived from the endoderm, so it can be said that Figure 1 and Figure 2 The presence of cells derived from the endoderm and mesoderm in the intestinal structure was demonstrated.

[0257] On the other hand, the obtained intestinal epithelial cell-like cells were cultured for 14 days in the second passage. The above-mentioned ESTEM-HE medium was used for the culture. The culture after 14 days of culture (second passage) was immunostained using an antibody against the intestinal epithelial cell marker CDX2. As a result, CDX2 was detected only in a very limited number of cells ( Figure 3 ). It should be noted that Figure 3 In the figure, the lower left photo shows CDX2 immunostaining, the upper right photo shows DAPI-based staining, and the upper left photo shows the merge of the two photos. Figure 3In each of the photos, the scale bar represents 100 μm. Figure 3 The photographs show that when the intestinal epithelial-like cells are cultured alone, the intestinal epithelial-like cells cannot be maintained and the cells with this property cannot proliferate.

[0258] [Example 4] Maintenance and proliferation of fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures 2

[0259] Fibroblast-like cells and intestinal epithelial-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial-like cells at passage number 12 and fibroblast-like cells at passage number 7 were co-cultured in two dimensions for 7 days. ESTEM-HE medium was used for the culture. The co-culture was embedded in IP gel. The co-culture embedded and fixed by IP gel was sliced ​​to obtain a slice of the co-culture, and the obtained slice was immunostained with an antibody against intestinal epithelial cell marker CDX2, and the result showed that CDX2 ( Figure 4 ). It should be noted that Figure 4 In the photographs, the scale bar indicates 50 μm.

[0260] [Example 5] Cell polarity of fibroblast-like cells and intestinal epithelial cells from intestinal structures

[0261] Fibroblast-like cells and intestinal epithelial-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial-like cells at passage number 12 and fibroblast-like cells at passage number 7 were co-cultured in two dimensions for 7 days. ESTEM-HE medium was used for the culture. The co-culture was embedded in IP gel. The co-culture embedded and fixed by IP gel was sliced ​​to obtain a slice of the co-culture, and the obtained slice was immunostained with an antibody against villin, a marker of intestinal epithelial cells, and villin ( Figure 5 ). It was further confirmed that villin is locally present on the lumen side (i.e., the apical side) of the intestinal epithelium of the organism ( Figure 5 ). This is the same as the local presence of villin in the intestinal epithelium of an organism. Figure 5 In the figure, the upper left photo shows the immunostaining of villin, the upper right photo shows the staining based on DAPI, and the lower left photo shows the overlap (merge) of the above two photos. Figure 5 In each of the photographs, the scale bar indicates 50 μm.

[0262] [Example 6] Maintenance of intestinal epithelial cells by fibroblast-like cells from intestinal structures

[0263] Fibroblast-like cells of passage number 7 from intestinal structures prepared by the same method as in Example 3 and primary intestinal epithelial cells collected from humans were co-cultured. The culture was carried out according to the following steps: fibroblast-like cells were pre-cultured for about 1 week using D-MEM supplemented with 10% FBS, and then intestinal epithelial cells were inoculated on the fibroblast-like cells. In the culture after inoculation of intestinal epithelial cells, ESTEM-HE medium was used. Microscopic photographs of cells at each time point of passage number 2 (5th day of culture) and passage number 4 (4th day of culture) are shown in Figure 6 and Figure 7 On the other hand, as feeder cells, mouse embryonic fibroblasts (MEFs), which are commonly used feeder cells, were used instead of fibroblast-like cells from intestinal structures, and primary intestinal epithelial cells collected from humans were co-cultured under the same conditions as above. Microscopic photographs of cells at each time point of passage number 2 (5th day of culture) and passage number 4 (4th day of culture) are shown in Figure 8 and Fig. 9 It should be noted that Figure 6 to Figure 9 In each of the photographs, the scale bar represents 100 μm.

[0264] Depend on Figure 6 and 7 The comparison showed that primary intestinal epithelial cells collected from humans can be maintained and proliferated by fibroblast-like cells derived from intestinal structures. In particular, primary intestinal epithelial cells that are not derived from intestinal structures can be maintained and proliferated.

[0265] On the other hand, by Figure 8 and 9 Comparisons showed that primary intestinal epithelial cells collected from humans could not be maintained and proliferated by feeder cells other than fibroblast-like cells from intestinal structures. Fig. 9 In the 3D images, the cells clearly changed to a flattened shape, which is typical of dedifferentiation.

[0266] [Example 7] Maintenance of hepatocytes by fibroblast-like cells from intestinal structures

[0267] Fibroblast-like cells from intestinal structures at passage number 7 prepared in the same manner as in Example 3 were co-cultured with hepatocytes. The culture was carried out as follows: the fibroblast-like cells were pre-cultured for about 1 week in D-MEM supplemented with 10% FBS, and then the hepatocytes were seeded on the fibroblast-like cells. In the culture after seeding the hepatocytes, ESTEM-HE medium was used. The microscopic photograph of the cells on the 8th day after the start of the culture is shown in FIG. Fig.10 shown.

[0268] exist Fig.10 Polygonal cells (arrows) characteristic of stem cells were observed in the center of the photograph, indicating that hepatocytes can be maintained and proliferated by fibroblast-like cells derived from intestinal structures.

[0269] In addition, the culture of hepatocytes usually requires appropriate coating of the culture container and feeder cells such as mouse embryonic fibroblasts (MEF), but the above results show that by using the feeder cells of the present disclosure, hepatocytes can be cultured without these. This result suggests that the feeder cells of the present disclosure are excellent as a scaffold for culture, and also suggests the use of isolating only the extracellular matrix (ECM) produced by the feeder cells of the present disclosure by methods such as decellularization treatment for use.

[0270] [Example 8] Genetic analysis of fibroblast-like cells from intestinal structures 1

[0271] The gene expression of the intestinal fibroblast-like cells and intestinal epithelial cell-like cells produced by the same method as in Example 3 was analyzed for the intestinal fibroblast-like cells and intestinal epithelial cell-like cells constituting the intestinal fibroblast-like cells. The gene expression of various fibroblasts in the organism was also analyzed. The results are shown in Fig.11 . Fig.11 In the table, "RYU" and "LONG" respectively represent the gene expression of intestinal epithelial-like cells and fibroblast-like cells derived from intestinal structures prepared by the same method as in Example 3. In addition, "human_SI" represents the gene expression based on total RNA of human small intestine, and DuSMFs, DuSPFs, ILSMFs and ILSPFs represent the gene expression of fibroblasts near the mucosa of the duodenum, subperitoneum of the duodenum, submucosa of the ileum and subperitoneum of the ileum, and fibroblasts near the serosal membrane, respectively.

[0272] Depend on Fig.11 The results shown show that the fibroblast-like cells from the intestinal structure express all the genes expressed by the intestinal tract shown in the figure and the site-specific cells constituting the intestinal tract. It is also shown that the fibroblast-like cells from the intestinal structure have a gene expression pattern that is very different from the various fibroblasts in the organism in terms of the genes expressed by the site-specific cells constituting the intestinal tract. According to these results, it is suggested that the fibroblast-like cells from the intestinal structure are different from the various fibroblasts in the organism and can contribute to the maintenance and / or proliferation of cells such as the intestinal epithelial cells constituting the intestinal tract.

[0273] [Example 9] Genetic analysis of fibroblast-like cells from intestinal structures 2

[0274] The gene expression of the intestinal fibroblast-like cells and intestinal epithelial cell-like cells produced by the same method as in Example 3 was analyzed for the intestinal and site-specific cells constituting the intestinal tract. Furthermore, the gene expression based on the total RNA of the human small intestine was analyzed. The results are shown in Figure 12-1 . Figure 12-1 In the above, "RYU01", "RYU02" and "RYU03" all represent the gene expression of intestinal epithelial-like cells from intestinal structures prepared by the same method as in Example 3. In addition, "LONG01" represents the gene expression of fibroblast-like cells from intestinal structures prepared by the same method as in Example 3. In addition, "human_SI" represents the gene expression based on total RNA of human small intestine.

[0275] Depend on Figure 12-1 The results shown show that the fibroblast-like cells from the intestinal structure express the genes NOG, HGF, WNT5A, and GREM1 expressed by the intestinal and site-specific cells constituting the intestinal tract shown in the figure. Here, NOG, WNT5A, and GREM1 are known to act as factors that maintain intestinal epithelial cells (refer to Hans Clevers et al., Gastroenterology, 2012; 143: 1518-1529, "Redundant Sources of Wnt Regulate Intestinal Stem Cells and Promote Formation of Paneth"). In addition, HGF is known to contribute to the maintenance of the homeostasis of colon epithelial cells. Therefore, these results suggest that fibroblast-like cells from the intestinal structure can contribute to the maintenance and / or proliferation of cells such as intestinal epithelial cell-like cells that constitute the intestine.

[0276] In addition, the intestinal epithelial cell-like cells derived from the intestinal structure and the fibroblast-like cells prepared by the same method as in Example 3 were analyzed for the gene expression of the WNT family, which are various signal transduction proteins. The results are shown in Figure 12-2 . Figure 12-2 In the figure, "LONG01" indicates the gene expression of fibroblast-like cells derived from intestinal structures prepared by the same method as in Example 3. In addition, "human_SI" indicates the gene expression based on total RNA of human small intestine.

[0277] Depend on Figure 12-2 The results shown show that the expression pattern of WNT family genes in fibroblast-like cells from intestinal structures is different from that in human small intestine. This result suggests that fibroblast-like cells from intestinal structures are different from human small intestine, that is, natural fibroblasts.

[0278] [Example 10] Hierarchical clustering by comprehensive gene analysis of fibroblast-like cells from intestinal structures

[0279] Hierarchical clustering based on comprehensive gene expression analysis was performed on fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures prepared by the same method as in Example 3. Furthermore, hierarchical clustering based on comprehensive gene expression analysis was also performed on primary intestinal epithelial cells collected from humans. The results are shown in Fig.13 It should be noted that Fig.13 In the present invention, “Ryu cells” and “Long cells” refer to intestinal epithelial-like cells and fibroblast-like cells derived from intestinal structures, respectively.

[0280] according to Fig.13 The results shown showed that there was no correlation between the number of passages and the gene expression pattern of the intestinal epithelial cells Ryu cells co-cultured with the fibroblast-like cells Long cells from the intestinal structure in comparison with the human primary intestinal epithelial cells. Based on this result, it is strongly suggested that in the intestinal epithelial cell-like cells Ryu cells co-cultured with the fibroblast-like cells Long cells from the intestinal structure, dedifferentiation due to the number of passages did not occur (i.e., Ryu cells were maintained).

[0281] [Example 11] Barrier properties of cell sheets of intestinal epithelial-like cells derived from intestinal structures

[0282] The intestinal structure-derived fibroblast-like cells and intestinal epithelial cell-like cells prepared by the same method as in Example 3 were cultured on Transwell for 5 days. After reaching confluence, 6.0×10 4 Intestinal epithelial cells were further inoculated into each well and co-cultured in two dimensions for 7 days. D-MEM medium supplemented with 15% FBS was used for the culture. After the culture was completed, the co-cultured product was used as a cell sheet, and its barrier property (endothelial cell resistance value) was confirmed as follows: using a chopstick-shaped electrode, the resistance value was read 20 seconds after the electrode was inserted into the cup. The results are shown in Fig.14 It should be noted that Fig.14 In the figure, “Caco2” refers to a cell sheet of Caco-2 cells, which is commonly used as a drug development tool. The cells were plated on a Transwell plate at 6.0×10 4 / well inoculated and cultured for 11 days; "Long05" indicates a cell sheet of fibroblast-like cells from intestinal structures. In addition, "RYU01", "RYU04", "RYU05" and "RYU07" refer to different intestinal epithelial-like cells made using different ES cells, respectively, and "RYU05 only" is a sample in which puromycin was used 2 days before measuring the barrier property (endothelial cell resistance value) to cause cell death only in the fibroblast-like cell layer to the extent that the sheet did not peel off.

[0283] Depend on Fig.14 The results shown indicate that the cell sheets of "RYU05 only", "RYU01", "RYU04", "RYU05" and "RYU07", which were obtained by co-culturing fibroblast-like cells and intestinal epithelial-like cells from intestinal structures, have barrier properties (endothelial cell resistance value) that are equal to or higher than those of cell sheets of Caco-2 cells, which are commonly used as drug development tools.

[0284] [Example 12] Shape and transporter expression of co-cultured fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures 1

[0285] Fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage number 12 and fibroblast-like cells at passage number 7 were co-cultured in two dimensions for 7 days. ESTEM-HE medium was used for the culture. After the culture was completed, the shape of the co-culture was observed under a microscope, and the co-culture was then embedded in IP gel. The co-culture embedded and fixed by IP gel was sliced ​​to obtain a slice of the co-culture, and the obtained slice was immunostained using antibodies against the intestinal epithelial cell transporters PEPT1, BCRP and SLC10A2 to confirm their respective expressions. The results are shown in Fig.15 It should be noted that Fig.15 In each of the photographs, the scale bar indicates 50 μm.

[0286] Depend on Fig.15 The photos shown confirm that the co-culture of fibroblast-like cells and intestinal epithelial-like cells from the structure has a shape similar to the intestinal epithelium, and that the transporter is localized on the luminal side (i.e., the apical side) in the intestinal epithelium of the organism, as in the intestinal epithelium of the organism. These photos suggest that the co-culture of fibroblast-like cells and intestinal epithelial-like cells from the intestinal structure has functional similarities with the mucosal structure of the intestine.

[0287] [Example 13] Expression of transporters and metabolic enzymes in fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures

[0288] Fibroblast-like cells and intestinal epithelial cell-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial cell-like cells at passage number 12 and fibroblast-like cells at passage number 7 were co-cultured in a two-dimensional manner for 7 days. ESTEM-HE medium was used for culture. For fibroblast-like cells and intestinal epithelial cell-like cells at each passage number, the expression of intestinal epithelial cell transporter ABCB1 and metabolic enzymes CES2, CYP3A4, UGT1A8, and UGT1A6 was confirmed. The results are shown in Fig.16 It should be noted that Fig.16 In the table, "RYU" indicates gene expression of intestinal epithelial-like cells from intestinal structures, "LONG" indicates gene expression of fibroblast-like cells from intestinal structures, and "human_SI" indicates gene expression based on total RNA of human small intestine. In addition, "5653" indicates that the cells were treated with puromycin.

[0289] Depend on Fig.16 The results shown confirmed that the transporter ABCB1 and metabolic enzymes CES2, UGT1A8, and UGT1A6 were expressed in the intestinal epithelial cell-like cells "RYU" derived from the intestinal structure. Fig.16 In the experiment, RYU5653 cells treated with puromycin, a cytotoxic antibiotic, survived and showed high CYP3A4 transcription, thus confirming high CYP responsiveness to puromycin. Thus, it was confirmed that the intestinal epithelial cell-like cells "RYU" derived from the intestinal structure have high drug metabolism activity.

[0290] [Example 14] Confirmation of division of intestinal epithelial cell-like cells in co-culture of fibroblast-like cells and intestinal epithelial cell-like cells

[0291] Fibroblast-like cells and intestinal epithelial-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial-like cells at passage number 5 and fibroblast-like cells at passage number 12 were co-cultured in a two-dimensional manner for 7 days. ESTEM-HE medium was used for culture. After the culture was completed, the co-culture was immunostained using an antibody against the cell proliferation marker Ki-67. The results are shown in Fig.17 It should be noted that Fig.17 In each photograph, the scale bar indicates 50 μm.

[0292] Depend on Fig.17The photos show that the cell proliferation marker Ki-67 exists non-locally. This result suggests that cell division is not a local division produced by a single cell, but is produced non-locally. This result suggests at least two possibilities. The first possibility is that Fig.17 The results suggest the characteristics of the case where the target cells are proliferated by fibroblast-like cells from intestinal structures (i.e., feeder cells of the present disclosure). The second possibility is that Fig.17 This suggests that intestinal epithelial cell-like cells derived from intestinal structures divide and proliferate. In other words, it suggests that intestinal epithelial cells, which are usually generated only from intestinal epithelial stem cells (Lgr5-positive stem cells) and do not divide and proliferate after differentiation, proliferate through the division of intestinal epithelial cells.

[0293] [Example 15] Confirmation of the mucosal formation ability of fibroblast-like cells and intestinal epithelial cells derived from intestinal structures

[0294] Fibroblast-like cells and intestinal epithelial-like cells from intestinal structures prepared by the same method as in Example 3 were co-cultured. Specifically, intestinal epithelial-like cells at passage number 7 and fibroblast-like cells at passage number 12 were co-cultured in a two-dimensional manner for 3 days. ESTEM-HE medium was used for 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 It should be noted that Fig.18 In each of the photographs of A and B, the scale bars represent 50 μm and 200 μm, respectively.

[0295] Fig.18 The blue coloration shown in each photo indicates the presence of mucin, suggesting the presence of goblet cells that are distinguished as intestinal epithelial cells. This shows that the feeder cells disclosed in the present invention exert a feeding ability that contributes to the long-term (multiple passages) stable proliferation of intestinal epithelial cells, and the intestinal epithelial-like cells that proliferate and maintain culture using the feeder cells disclosed in the present invention maintain the enteroendocrine ability. This result suggests that by using the feeder cells disclosed in the present invention, it is possible to produce a large amount of mucin itself or cells that produce mucin, and then it is also possible to produce a large amount of cell sheets having such mucin. It is known that almost all mucus secreted by animals contains mucin, and most of the mucosa in the animal body is covered with mucin, so the feeder cells disclosed in the present invention are expected to be used in the medical field, especially in the medical field related to mucus and mucosa. In addition, in the past, Caco-2 cells were generally used as drug development tools, but Caco-2 cells generally do not have the ability to secrete mucin, so it is also suggested that by using the feeder cells disclosed in the present invention, a detection system that is closer to the organism than Caco-2 cells can be provided.

[0296] [Example 16] Confirmation of the feeding ability of fibroblast-like cells from intestinal structures 1

[0297] The feeder ability of the intestinal structure-derived fibroblast-like cells prepared by the same method as in Example 3 was confirmed by comparing with conventional feeder cells derived from human embryonic stem cells. First, conventional feeder cells were prepared according to the following procedure.

[0298] EES2 human embryonic stem cells (hES cells) were exposed to a ROC inhibitor (Y-27632; 10 μM) and then dissociated into single cells using 0.5 mM EDTA. 5×10 cells were plated per well. 3 The cells were seeded in 96-well plates at different concentrations.

[0299] 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.

[0300] The obtained embryoid bodies were transferred to a T25 flask coated with NMP collagen PS (Nipponham Co., Ltd.), and cultured for 60 to 70 days 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-β1 / HRG-β1 EGF domain; Fujifilm Wako Pure Chemical Industries, Ltd.), 200 ng / ml recombinant human IGF-1 (LONGR3-IGF-1; Sigma-Aldrich), and 20 ng / ml human bFGF (Kakyo Pharmaceutical Co., Ltd.)) to obtain hES cell-derived cells. These cells have mesenchymal cell-like properties and are therefore also called ES cell-derived mesenchymal cells.

[0301] The obtained hES cell-derived mesenchymal cells were maintained in an 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”).

[0302] The feeding ability of the fibroblast-like cells from the intestinal structure (i.e., the feeder cells of the present disclosure) prepared by the same method as in Example 3 was confirmed by the following steps. The feeder cells of the present disclosure were cultured in large quantities to obtain feeder cells of passage number 7. Intestinal epithelial cells were inoculated on the obtained feeder cells and cultured and passaged repeatedly in the same manner as in Example 3. The PDL (Population doubling level: vertical axis) of the repeatedly passaged intestinal epithelial cells at each passage number is shown in Fig.19 .

[0303] Depend on Fig.19 The results shown confirmed that the feeder cells of the present disclosure exhibited a stable proliferation ability of the target cells (intestinal epithelial cells) at any time point of passage numbers 1 to 16 (P1 to 16). In other words, it was shown that the feeder cells of the present disclosure exhibited a feeding ability that contributed to the stable proliferation of the target cells for a long period of time (multiple passages).

[0304] [Example 17] Confirmation of the feeding ability of fibroblast-like cells from intestinal structures 2

[0305] The feeding ability of the fibroblast-like cells from the intestinal structure produced by the same method as in Example 3 (i.e., the feeder cells of the present disclosure) was compared with the conventional feeder cells from human embryonic stem cells to confirm. Specifically, first, conventional feeder cells produced by the same method as in Example 16 were prepared. Then, the feeder cells of the present disclosure and the conventional feeder cells were respectively cultured at 5×10 4 The cells were seeded in a 24-well plate and cultured in α-MEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. After one day, 1.0 × 10 cells were added to each well. 5 Intestinal epithelial cell-like cells from intestinal structures were inoculated at a concentration of 1.5 μg / mL (passage 3). ESTEM-HE medium was used for culture. Antibodies against intestinal epithelial cell markers CDX2 and villin were used to immunostain the culture after 7 days of culture (1 passage). The results of culturing intestinal epithelial cell-like cells using conventional feeder cells are shown in Fig. 20 It should be noted that Fig. 20 In the figure, the upper left photograph shows immunostaining of CDX2, the upper right photograph shows immunostaining using villin, the lower left photograph shows immunostaining using DAPI, and the lower right photograph shows a microscope photograph of cultured intestinal epithelial-like cells.

[0306] according to Fig. 20The results shown, when using the previous feeder cells to culture intestinal epithelial cell-like cells, observed very large and flat cells in the center. In addition, although the expression of villin was confirmed, the expression of CDX2 was hardly confirmed. This result suggests that when using the previous feeder cells, the expression of CDX2 usually expressed in intestinal epithelial cells disappears, and the properties of intestinal epithelial cell-like cells change in a short time (very few passages). It should be noted that, although the results are not shown, when using the feeder cells disclosed in the present invention to culture intestinal epithelial cell-like cells, even the same number of passages is confirmed to express CDX2 and villin, and it is confirmed that the properties as intestinal epithelial cells are maintained.

[0307] [Example 18] Maintenance of chondrocytes by fibroblast-like cells from intestinal structures

[0308] The feeding capacity (maintenance of chondrocytes) of four different fibroblast-like cells (i.e., feeder cells disclosed in the present invention) at passage number 7 from intestinal structures prepared by the same method as in Example 3 was confirmed. Specifically, the expression of lubricin and COL2A1 was confirmed by qRT-PCR for chondrocytes collected from human children (YUB) and chondrocytes cultured with the above four fibroblast-like cells. The results are shown in Fig.21 In addition, in the table, "YUB" represents chondrocytes collected from children's samples, and "4R" to "7R" represent chondrocytes cultured together with fibroblast-like cells (it should be noted that Fig.21 (No data was obtained for "6R" in A).

[0309] Depend on Fig.21 The results confirmed that lubricin and COL2A1 were highly expressed in chondrocytes 4R to 7R cultured with the feeder cells of the present invention, compared with chondrocytes YUB collected from human children. Here, lubricin is a lubricating substance highly expressed in synovial cells, which are feeder cells known as chondrocytes, and COL2A1 is a lubricating substance highly expressed in chondrocytes. In addition, chondrocytes are known to be cells that are prone to dedifferentiation. Therefore, the above results can be considered to mean that in YUB, chondrocytes dedifferentiate and lose their original properties, not to mention lubricin, and even COL2A1 is almost no longer confirmed to be expressed; in contrast, in 4R to 7R, the feeder cells of the present invention exert synovial cell-like functions, lubricin is highly expressed, and the original properties of chondrocytes are maintained, and COL2A1 is highly expressed. These results suggest that the feeder cells of the present invention have the ability to feed chondrocytes.

[0310] [Example 19] Gene expression profile of feeder cells disclosed herein and marker confirmation based on immunostaining

[0311] The cell markers of the fibroblast-like cells (i.e., the feeder cells disclosed in the present invention) from the intestinal structure prepared by the same method as in Example 3 were confirmed by immunostaining. Specifically, the cultured fibroblast-like cells were embedded in IP gel to prepare a specimen, and the PDGF receptor α (D1E1E) XP (registered trademark) rabbit mAb manufactured by Cell Signaling Technology, Inc. and Anti-CD81, Mouse-Mono (M38) manufactured by EXBIO were used as primary antibodies to stain the protein expression of PDGFRA and CD81 on the cell surface. The results are shown in Fig. 22 .

[0312] In addition, the expression of each gene of Foxl1, CD34 and GREM1 was confirmed by normalization of the comprehensive gene expression. Fig.23 .

[0313] Depend on Fig. 22 and 23 The results shown confirmed that, in the feeder cells of the present disclosure, each of the genes PDGFRA, CD81, Fox11, and GREM1 was expressed, whereas CD34 was not expressed.

[0314] [Example 20] Preparation of cell sheets

[0315] A cell sheet is prepared using the feeder cells disclosed herein. Specifically, the feeder cells disclosed herein are inoculated and cultured until a confluent state is reached. Next, intestinal epithelial cells are inoculated in a stacked form on the feeder cells that have reached a confluent state, and cultured until the intestinal epithelial cells reach confluence and form a sheet structure through cell-to-cell bonding. Next, the stack of the layer of feeder cells disclosed herein and the layer of intestinal epithelial cells is peeled off from the culture plane to obtain a cell sheet comprising a layer of feeder cells and a layer of intestinal epithelial cells.

[0316] The photograph of the obtained cell sheet is shown in Fig.24 .Depend on Fig.24 The results shown confirmed that the diameter of the cell sheet of the present disclosure was 1 cm or more.

[0317] Industrial Applicability

[0318] According to the present disclosure, a feeder cell and a method for producing the feeder cell are provided, wherein the feeder cell is used to maintain or proliferate cells that have been considered difficult to maintain and proliferate in two-dimensional culture by two-dimensional culture.

Claims

1. A method for producing feeder cells for maintaining or proliferating cells, comprising: (A) a step of preparing an intestinal structure composed of endoderm-derived cells and mesoderm-derived cells produced from pluripotent stem 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.

2. The method of claim 1, wherein: The intestinal structure is prepared by a method comprising the following steps: (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and 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 culture medium.

3. The method according to claim 1 or 2, wherein: The method further comprises the step of (D) allowing the isolated fibroblast-like cells to proliferate.

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

5. The method of claim 2, wherein: The culturing time in the step (c) is 60 days or longer.

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

7. A fibroblast-like cell, wherein: PDGFRA and CD81 were positive, and Foxl1 and GREM1 were expressed.

8. The fibroblast-like cell according to claim 7, wherein It was positive for PDGFRA and CD81, negative for CD34, and expressed Foxl1 and GREM1.

9. The fibroblast-like cell according to claim 7, wherein The fibroblast-like cells are derived from pluripotent stem cells or from a culture of an intestinal structure, wherein the intestinal structure is made from pluripotent stem cells and contains endoderm-derived cells and mesoderm-derived cells.

10. The fibroblast-like cell according to claim 9, wherein The intestinal structure is prepared by a method comprising the following steps: (a) preparing a cell culture substrate, the cell culture substrate comprising a substrate and 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 culture medium.

11. The fibroblast-like cell according to claim 9, wherein The pluripotent stem cells are derived from humans.

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 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 proliferating the cell by planar culture. 14 . The feeder cell according to claim 6 or the fibroblast-like cell according to claim 7 , which is a feeder cell capable of passage the cell three or more times. 15 . A cell sheet comprising the feeder cell according to claim 6 or the fibroblast-like cell according to claim 7 . 16 . A co-culture comprising at least one cell selected from the group consisting of small intestinal enteric epithelial cells and hepatocytes, and the feeder cells according to claim 6 or the fibroblast-like cells according to claim 7 .

17. The co-culture of claim 16, wherein The cells were passaged for more than 3 times. A cell sheet comprising the co-culture according to claim 16 .

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

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

21. The cell sheet according to claim 20, wherein Also included is a layer of feeder cells.

22. A transplant material comprising the cell sheet according to claim 15 or 18. 23 . A method for maintaining or proliferating cells, comprising the step of culturing the cells in the presence of the feeder cells according to claim 6 or the fibroblast-like cells according to claim 7 .

24. The method of claim 23, wherein: The cells are cultured in a planar manner.

25. The method of claim 23 or 24, wherein: The cell is at least one cell 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 from the same individual.

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

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

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