WO2021065984A1

WO2021065984A1 - Method for forming sheet of cardiomyocytes

Info

Publication number: WO2021065984A1
Application number: PCT/JP2020/037084
Authority: WO
Inventors: 芳樹澤; 繁宮川; 賢二大山; 文哉大橋
Original assignee: 国立大学法人大阪大学; テルモ株式会社
Priority date: 2019-09-30
Filing date: 2020-09-30
Publication date: 2021-04-08
Also published as: JPWO2021065984A1

Abstract

The present invention addresses the problem of providing: a method for producing a high-quality sheet-like cell culture containing a variety of cells such as cardiomyocytes, in particular, cardiomyocytes derived from pluripotent stem cells, the cells highly maintaining the function thereof; a sheet-like cell culture produced using the method; and a method for treating a disease using the sheet-like cell culture. The problem is solved by a sheet-like cell culture production method comprising: (a) a step of plating a cell population including cardiomyocytes on a culture substrate; and (b) a step of culturing the plated cell population with a sheet formation medium including a cell adhesive component to form a sheet.

Description

Cardiomyocyte sheeting method

This disclosure is a commissioned research project of the Japan Medical Research and Development Organization, Regenerative Medicine Realization Center Network Program, Practical Application Research Center for Diseases and Tissues (Center A), "Center for the Creation of Myocardial Regenerative Therapy Using iPS Cells" in FY2018. It is a patent application to which Article 17 of the Development and Industrial Technology Enhancement Law applies, and transplants of various cells such as sheet-shaped cell cultures containing myocardial cells, especially pluripotent stem cell-derived myocardial cells. The present invention relates to a method for producing, a transplanted piece manufactured by using the method, a method for treating a disease using the transplanted piece, and the like.

Adult cardiomyocytes have poor self-renewal ability, and if myocardial tissue is damaged, it is extremely difficult to repair it. In recent years, in order to repair damaged myocardial tissue, attempts have been made to transplant a graft containing cardiomyocytes prepared by a cell engineering technique into an affected area (Patent Document 1, Non-Patent Document 1). Recently, attention has been paid to cardiomyocytes derived from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) as a source of cardiomyocytes used for producing such transplants. There are attempts to prepare sheet-like cell cultures containing such pluripotent stem cell-derived cardiomyocytes and therapeutic experiments in animals (Non-Patent Documents 2 to 3). However, the development of sheet-like cell cultures containing pluripotent stem cell-derived cardiomyocytes has just begun, and there are still many unclear points about their functional characteristics and factors that influence them.

When producing a clinical sheet-shaped cell culture used for regenerative medicine, it is desirable that the graft-forming medium contains as little heterologous components other than humans as possible, that is, a so-called xeno-free state. However, forming a sheet-shaped cell culture in a xeno-free state is often costly and laborious, and depending on the cells used, it is possible to form a sheet-shaped cell culture while maintaining the desired properties of the cells. It can be difficult.

Special Table 2007-528755

The present disclosure is a method for producing a high-quality implant, which comprises various cells such as cardiomyocytes, particularly cardiomyocytes derived from pluripotent stem cells, while preserving the functions of the cells, using the method. It is an object of the present invention to provide a manufactured implant, a method for treating a disease using the implant, and the like.

When preparing a graft containing cardiomyocytes to be used for transplantation, it is necessary to prepare with a xenofree graft-forming medium. In addition, when fetal bovine serum (FBS) is added to the graft-forming medium or a serum-free medium is used, problems such as cardiomyocytes not beating sufficiently and grafts not being formed well have been found. ..

The present inventors are studying the preparation of sheet-shaped cell cultures for living body transplantation using cardiomyocytes derived from pluripotent stem cells, which have sufficient functions even when prepared with a Xenofree implant-forming medium. We faced the problem that sheeting culture was not successful even when sheeted and cultured in a serum-free medium. While continuing research to solve this problem, we have found a new finding that by adding a cell adhesion component to a serum-free graft-forming medium to form a sheet, for example, a serum-free medium can be used to form a sheet. I found it. Based on this finding, further research was carried out, and it was found that it is possible to produce a high-quality graft that can withstand clinical application in the preparation of a graft containing various cells, and to complete the present invention. I arrived.

That is, the present invention relates to the following:
[1] A method for producing a graft containing somatic cells;
(A) a step of seeding the cell population containing the somatic cells on a culture substrate, and (b) a step of seeding the seeded cell population in a transplantation piece-forming medium containing a cell adhesion component. Included, said method.
[2] The method according to [1], wherein the culture substrate is further coated with a cell adhesion component and / or a masking component.
[3] The method according to [1] or [2], wherein the content of the cell adhesion component in the graft-forming medium is 1/10 to 1/100 of the amount used for coating the culture substrate. ..
[4] The method according to any one of [1] to [3], which comprises a step of removing undifferentiated cells from a cell population before (a).
[5] The method according to any one of [1] to [4], wherein the graft-forming medium does not contain serum.
[6] The method according to any one of [1] to [5], wherein the graft-forming medium contains a platelet lysate.

[7] A method for producing a sheet-like cell culture containing cardiomyocytes;
(A) A step of seeding myocardial cells at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component, and (B) sheet-forming culture using a sheet-forming medium, and sheet-like cell culture. A method for producing a sheet-shaped cell culture, which comprises a step of forming a substance.
[8] The method according to [7], wherein the sheet formation medium contains or does not contain a cell adhesion component.

[9] A method for evaluating the surface of a culture medium for producing a sheet-shaped cell culture.
(I) The step of coating the surface of the culture substrate with the cell adhesion component, thereby improving the adhesion of the cells to the surface of the culture substrate.
(Ii) Further, a step of coating the surface of the culture substrate with a masking component, thereby reducing the adhesion of cells to the surface of the culture substrate,
(Iii) A step of seeding somatic cells at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component.
The above-mentioned step including (iv) a step of forming a sheet-like cell culture by sheet-forming culture in a sheet-forming medium, and (v) a step of evaluating a peeling state of the sheet-like cell culture from the surface of a culture substrate. Method.

[10] The method according to any one of [7] to [9], wherein the sheeting medium does not contain serum.
[11] The method according to any one of [7] to [10], wherein the sheeting medium contains a platelet lysate.
[12] The method according to any one of [1] to [11], wherein the masking component is a blood-derived component.
[13] The method according to any one of [1] to [12], wherein the masking component is serum, platelet lysate or albumin.
[14] The method according to any one of [1] to [13], wherein the masking component is FBS having a concentration of 0.05% or more.
[15] The method according to any one of [1] to [14], wherein the somatic cell is a cardiomyocyte derived from a pluripotent stem cell.
[16] A therapeutically effective amount of the graft produced by the method according to any one of [1] to [6] or the sheet-shaped cell culture produced by the method according to [7] or [8]. A method for treating a heart disease in a subject, comprising the step of administering the drug to the subject in need thereof.

According to the present invention, transplants such as sheet-like cell cultures of higher quality than before can be efficiently produced from a cell population containing various cells, for example, cardiomyocytes, particularly cardiomyocytes induced to differentiate from pluripotent stem cells. Can be manufactured. In particular, in the preparation of grafts used for regenerative medicine, such as sheet-shaped cell cultures containing myocardial cells induced to differentiate from pluripotent stem cells, the graft-forming medium does not use serum, that is, it does not contain impurities derived from the manufacturing process. It is particularly useful in regenerative medicine because it enables graft formation even when the graft is formed and cultured under the conditions. Further, by adding a platelet lysate to the graft-forming medium, it is possible to prepare a graft having a desired property at a high level, and particularly to provide a very suitable graft for living-donor transplantation into humans. It becomes possible.

FIG. 1 is a photograph showing a state of sheet formation when laminin (iMatrix-511), vitronectin (VTN-N), and fibronectin (RetroNectin ^{(R)) are used as cell adhesion components.} The upper row (A and B) is for laminin, the middle row (C and D) is for vitronectin, the lower row (E and F) is for fibronectin, and the left column (A, C and E) is for use as a coating agent. When used at the recommended concentration, the right column (B, D and F) shows the result when used at 1/10 of the recommended concentration. In any case, it was confirmed that the sheet was formed. FIG. 2 shows a state in which the sheet-shaped cell culture is peeled off after sheeting with iMatrix-511 as a cell adhesion component and FBS as a masking component on a culture substrate coated with various concentrations. It is a photograph showing. By coating with FBS together with iMatrix-511, it was confirmed that peeling was possible even when iMatrix-511 with a wide concentration was used.

Hereinafter, the present invention will be described in detail.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety. In the event of a conflict between the publications referred to herein and the description herein, the description herein shall prevail.

The present disclosure is a method of producing a graft containing the desired cells;
(A) a step of seeding the cell population containing the cells on a culture substrate, and (b) a step of forming and culturing the seeded cell population in a medium containing a cell adhesion component.
The present invention relates to the above-mentioned method.

In the present disclosure, the "graft" means a structure for transplantation into a living body, and particularly means a structure for transplantation containing cells as a constituent component. In a preferred embodiment, the implant is a structure for transplantation that is free of cells and structures other than cell-derived substances (eg, scaffolds, etc.). The implants in the present disclosure include, but are not limited to, sheet-like cell cultures, spheroids, cell aggregates, and the like, preferably sheet-like cell cultures or spheroids, and more preferably sheet-like. It is a cell culture.

In the present disclosure, "sheet-shaped cell culture" refers to cells connected to each other to form a sheet. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediator is preferably derived from cells, particularly from the cells that make up the sheet-like cell culture. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminate (multilayer), for example, two layers, three layers, etc. It may be 4 layers, 5 layers, 6 layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without showing a clear layered structure of the cells. For example, in the vertical cross section of a sheet-shaped cell culture, cells may be present in a non-uniformly (for example, mosaic-like) arrangement without being uniformly aligned in the horizontal direction.

The grafts of the present disclosure, particularly sheet cell cultures, preferably do not contain scaffolds (supports). Scaffolds may be used in the art to attach cells to and / or inside the scaffold to maintain the physical integrity of the implant, such as a sheet cell culture, eg, poly. Although membranes made of vinylidene fluoride (PVDF) and the like are known, the implants of the present disclosure can maintain their physical integrity without such scaffolds. In addition, the implants of the present disclosure preferably consist only of cell-derived substances constituting the implants and do not contain any other substances.

The cell may be a heterologous cell or an allogeneic cell. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the graft is used for transplantation. For example, when the recipient is a human, cells derived from monkeys and pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not cause rejection when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cell is an autologous cell or an allogeneic cell. In one aspect of the disclosure, the cell is an autologous cell (including an autologous iPS cell). In another aspect of the disclosure, the cell is an allogeneic cell (including an allogeneic iPS cell).

The cells constituting the implant of the present disclosure are not particularly limited as long as they can form the implant, and include, for example, adherent cells (adhesive cells). Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin. Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as induced pluripotent stem (iPS) cells, It includes mesenchymal stem cells, etc.). In the present disclosure, induced pluripotent stem cells (iPS cells) are cells induced by introducing a gene. Somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells). Non-limiting examples of cells constituting the transplant include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, etc.) Endometrial, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigments) Epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), kidney cells, adrenal cells, root membrane Examples include cells, gingival cells, bone membrane cells, skin cells and the like. Non-limiting examples of iPS cell-derived adherent cells include iPS cell-derived myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, and bone membrane cells. , Skin cells, synovial cells, cartilage cells and the like.

The cells that make up the implant can be derived from any organism that can be treated with the implant. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. The number of types of cells constituting the transplant is not particularly limited, and may be composed of only one type of cells, or may be composed of two or more types of cells. When there are two or more types of cells forming a graft, the content ratio (purity) of the most abundant cells is, for example, 50% or more, preferably 60% or more, more preferably 70 at the end of formation of the sheet-like cell culture. % Or more, more preferably 75% or more.

The culture substrate is not particularly limited as long as the cells can form a cell culture on the cells, and includes, for example, containers of various materials and / or shapes, solid or semi-solid surfaces in the containers, and the like. .. The container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate. Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned. Also, the container preferably has at least one flat surface. Examples of such a container include, without limitation, a culture container having a bottom surface made of a culture substrate capable of forming a cell culture and a liquid-impermeable side surface. Specific examples of such a culture vessel include, but are not limited to, a cell culture dish, a cell culture bottle, and the like. The bottom surface of the container may be transparent or opaque. If the bottom surface of the container is transparent, cells can be observed and counted from the back side of the container. Further, the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices. The culture substrate may be prepared using the above-mentioned materials, or a commercially available one may be used.

Preferred culture substrates include, without limitation, for example, a substrate having an adhesive surface suitable for forming a sheet-like cell culture, and a substrate having a low adhesive surface suitable for forming spheroids. And / or a substrate having a uniform well-like structure and the like. Specifically, in the case of forming a sheet-like cell culture, for example, a substrate coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or hydrophilic polymer on the surface thereof, and further, collagen. , Fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan and other extracellular matrix, and base materials coated with cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface. In addition, such substrates are commercially available (eg, Corning ^(R) TC-Treated Culture Dish, Corning, etc.). In the case of spheroid formation, for example, soft agar, temperature-responsive gel obtained by cross-linking poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG), polyhydroxyethyl methacrylate (commercially available name: mebiol gel), polyhydroxyethyl methacrylate ( Examples include a base material coated with a non-cell adhesive compound such as a hydrogel such as poly-HEMA) and 2-methacryloyloxyethyl phosphorischoline (MPC) polymer and / or a base material having a uniform uneven structure on the surface. Be done. Such substrates are also commercially available (eg, EZSPHERE ^(R), etc.). The culture medium may be transparent or opaque in whole or in part.

The surface of the culture substrate may be coated with a material whose physical properties change in response to irritation, for example, temperature or light. Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc. N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacryl (Amid, etc.), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethylacrylamide, etc.), having cyclic groups (eg, N, N-dialkylacrylamide, N, N-diethylacrylamide, etc.) Meta) Acrylamide derivatives (eg 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidin, 4- (1-oxo-2-propenyl) -morpholin, 1 -(1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidin, 4- (1-oxo-2-methyl-2-propenyl) -A temperature-responsive material consisting of a homopolymer or copolymer of a vinyl ether derivative (eg, methylvinyl ether), a photoabsorbable polymer having an azobenzene group, a vinyl derivative of triphenylmethane leucohydrooxide and an acrylamide-based single amount. Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-21186 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell ^(R) ) and can be used in the production methods of the present disclosure.

The culture medium may have various shapes. The area thereof is not particularly limited, but may be, for example, about 1 cm ² to about 200 cm ² , about 2 cm ² to about 100 cm ² , about 3 cm ² to about 50 cm ² . For example, as a culture substrate, a circular culture dish having a diameter of 10 cm can be mentioned. In this case, the area is 56.7 cm ² . The culture surface may be flat or may have an uneven structure. When it has a concavo-convex structure, it is preferable that it has a uniform concavo-convex structure.

In the present disclosure, "pluripotent stem cell" is a well-known term in the art and has the ability to differentiate into three germ layers, i.e. cells of all lineages belonging to endoderm, mesoderm and ectoderm. Means cell. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear-transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Normally, when inducing differentiation of pluripotent stem cells into specific cells, pluripotent stem cells are first suspended-cultured to form aggregates of any of the above three germ layers, and then cells that form aggregates. Induce differentiation into specific cells of interest.

In the present disclosure, "pluripotent stem cell-derived differentiation-inducing cell" means any cell that has been subjected to differentiation-inducing treatment so as to differentiate from a pluripotent stem cell into a specific type of cell. Non-limiting examples of differentiation-inducing cells include muscular cells such as myocardial cells and skeletal myoblasts, neural cells such as neuron cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. Hematopoietic cells such as cells and bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells and B cells, cells constituting organs such as hepatocytes, pancreatic β cells and renal cells, In addition to cartilage cells and germ cells, it also includes precursor cells and somatic stem cells that differentiate into these cells. Typical examples of such progenitor cells and somatic stem cells include mesenchymal stem cells in myocardial cells, pluripotent cardiac progenitor cells, monopoly cardiac progenitor cells, neural stem cells in neural cells, hematopoietic cells and immunity. Examples include hematopoietic stem cells and lymphoid stem cells in related cells. Induction of differentiation of pluripotent stem cells can be performed using any known method. For example, the induction of differentiation of pluripotent stem cells into cardiomyocytes can be performed based on the methods described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO2014 / 185358. When a differentiation-inducing cell derived from a pluripotent stem cell such as an iPS-derived cardiomyocyte is used as the desired cell, the undifferentiated cell may be removed after the differentiation induction. The treatment for removing undifferentiated cells is known in the art, and the methods described in, for example, WO2017 / 038562, WO2016 / 072519, WO2007 / 088744, etc. can be used.

Further, the differentiation-inducing cell may be a cell derived from an iPS cell into which any useful gene other than the gene for reprogramming has been introduced. Non-limiting examples of such cells include, for example, iPS cells into which the chimeric antigen receptor gene described in Themeli M. et al. Nature Biotechnology, vol. 31, no. 10, pp. 928-933, 2013 has been introduced. Examples include T cells derived from. In addition, cells into which any useful gene has been introduced after induction of differentiation from pluripotent stem cells are also included in the differentiation-inducing cells of the present invention.

In the present disclosure, "cell adhesion component" means an isolated polypeptide having the property of adhering to cells, including extracellular matrix and cell adhesion factors described in the above description of the culture medium. To do. Such cell-adhesive components are generally used to coat the surface of the culture medium. Examples of cell-adhesive components include, but are not limited to, extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, and cells such as cadoherin family, selectin family, and integrin family. In addition to such as adhesion factors, these variants or functional equivalents, such as laminin variants such as laminin 511 and laminin 221 and vitronectin variants such as VTN-N, Kimizuka et al., J. Biochem. Examples thereof include variants of fibronectin such as ^{retronectin (R)} described in (1991), 110, pp.284-291 and the like. These may be contained at least one or a mixture of two or more. In a preferred embodiment, the cell adhesion components of the present disclosure do not contain blood-derived components other than cell adhesion factors and / or their functional equivalents.

The present invention will be described in detail below, taking as an example the case where the desired cell is a cardiomyocyte and the implant is a sheet-like cell culture.
One aspect of the disclosure relates to a method for producing a high quality sheet cell culture containing cardiomyocytes. The method of the present disclosure includes the following steps (a) and (b):
(A) A step of seeding a cell population containing myocardial cells on a culture substrate, and (b) a step of sheeting and culturing the seeded cell population in a sheeting medium containing a cell adhesion component.

In the present disclosure, "cardiomyocyte" means a cell having the characteristics of cardiomyocyte. The characteristics of cardiomyocytes include, but are not limited to, expression of cardiomyocyte markers, presence of autonomous pulsation, and the like. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like. .. In one embodiment, cardiomyocytes derived from pluripotent stem cells are c-TNT positive and / or CD172a positive.

The cardiomyocytes used in the method of the present disclosure may be directly obtained from a living body or derived from other cells, but are preferably derived from other cells. .. Examples of the induction into cardiomyocytes include a method of introducing a myocardial inducing factor into fibroblasts and the like, and a method of inducing differentiation of cells having the property of differentiating into cardiomyocytes such as pluripotent stem cells and cardiac progenitor cells. ..

In step (a), seeding on the culture substrate may be performed, for example, by injecting a cell suspension in which cells are suspended in a sheeting medium into a culture vessel provided with the culture substrate. For injection of the cell suspension, an instrument suitable for the injection operation of the cell suspension, such as a dropper or a pipette, can be used. The seeding density of cells is set to a density capable of forming a sheet-like cell culture, and the density may vary depending on the desired cells, but those skilled in the art select an appropriate density from methods known in the art. can do. For example, in the case of a sheet-shaped cell culture containing cardiomyocytes, it can be, for example, 2.0 × 10 ⁵ cells / cm ² or more, but may be seeded at a higher density.

Examples of higher densities include, for example, a density that reaches confluence, that is, a density at which cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, cells come into contact with each other when seeded. It can be as dense as expected, the density at which contact inhibition occurs, or the density at which cell proliferation is substantially stopped by contact inhibition or higher. The upper limit of the seeding density is not particularly limited, but if the seeding density is excessively high, more cells will die, resulting in inefficiency. In one aspect of the present disclosure, the seeding density is, for example, about 1.0 × 10 ⁶ / cm ² to about 1.0 × 10 ⁷ / cm ² , about 1.0 × 10 ⁶ / cm ² to about 5. .0 x 10 ⁶ pieces / cm ² , about 1.0 x 10 ⁶ pieces / cm ² to about 3.0 x 10 ⁶ pieces / cm ² , about 1.5 x 10 ⁶ pieces / cm ² to about 1.0 × 10 ⁷ pieces / cm ² , about 1.5 × 10 ⁶ pieces / cm ² to about 5.0 × 10 ⁶ pieces / cm ² , about 1.5 × 10 ⁶ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² , about 2.0 x 10 ⁶ pieces / cm ² to about 1.0 x 10 ⁷ pieces / cm ² , about 2.0 x 10 ⁶ pieces / cm ² to about 5.0 x 10 ⁶ pieces It can be / cm ² , about 2.0 × 10 ⁶ pieces / cm ² to about 3.0 × 10 ⁶ pieces / cm ² . In a preferred embodiment, the sowing density is from about 1.76 × 10 ⁶ / cm ² to about 2.33 × 10 ⁶ / cm ² .

The seeded cell population may contain other cells as long as they contain the desired cells (eg, myocardial cells), and when the desired cells are myocardial cells, for example, fibroblasts, vascular endothelial cells. And / or wall cells and the like may be further included. As the cell population, the cell population collected from the tissue may be used as it is, or by using, for example, the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 or WO 2014/185358. The cell population obtained by inducing differentiation from iPS cells may be used as it is, or may be used after cryopreservation, pre-culture, removal of undifferentiated cells, or the like. In a preferred embodiment, the seeded cell population is a cell population that is induced to differentiate from iPS cells, seeded on a culture substrate (preferably on a flat culture substrate), adherently cultured, and then recovered. is there. Cryopreservation and thawing may be performed before or after such adhesive culture. By performing adhesive culture, it is possible to achieve high-quality graft formation with a high probability in the subsequent formation of the graft.
In such an adhesive culture step, the culture conditions and the like may be the same as those for normal adhesive culture. For example, a commercially available culture container for adhesive culture may be used for culturing _{under 37 ° C. and 5% CO 2} conditions. The seeding density of the cells may be any density as long as it does not interfere with the adhesion between the cells and / or the formation of the adhesion between the cells and the culture substrate, and may be, for example, a subconfluent density. It may be at or above a density that reaches confluence. The culturing time may be such that adhesion between cells and / or adhesion between cells and the culture substrate is formed, and specifically, for example, 2 to 24 hours, 2 to 12 hours, and 2 to 6 hours. It may be about 2 to 4 hours.

If undifferentiated cells are included in the cell population for graft formation, there may be a risk of tumorigenesis after transplantation into the body. Therefore, in a preferred embodiment, the cell population does not include undifferentiated cells. In order to obtain a cell population that does not contain undifferentiated cells, a cell population obtained by collecting from a living body or inducing differentiation from pluripotent stem cells may be subjected to an undifferentiated cell removal operation. In the present disclosure, the "undifferentiated cell removal operation" refers to undifferentiated cells having tumorigenicity from a cell population, typically a cell population containing differentiation-inducing cells obtained by inducing differentiation of pluripotent stem cells. It means a removal operation and can be performed using any known method. Non-limiting examples of such a method include various separation methods using markers specific to undifferentiated cells (for example, cell surface markers), for example, magnetic cell separation method (MACS), flow cytometry method, affinity separation. Methods, methods for expressing selective markers (eg, antibiotic resistance genes, etc.) by specific promoters, factors necessary for the survival of undifferentiated cells (nutrient sources (nutrient sources such as methionine, maintenance of undifferentiated state such as bFGF) A method of exterminating undifferentiated cells by culturing in a medium excluding (factors, etc.), a method of culturing in the presence of factors that promote differentiation (VEGF, BMP, activin, etc.) and promoting undifferentiated cells, not yet Examples thereof include a method of treating the surface antigen of differentiated cells with a drug targeting the surface antigen. Further, the method described in WO2014 / 126146, WO2012 / 056997, the method described in WO2012 / 147992, the method described in WO2012 / 133674, WO2012. / 012803 (Special Table 2013-535194), WO2012 / 078153 (Special Table 2014-501518), Japanese Patent Application Laid-Open No. 2013-143966 and Tohyama S. et al., Cell Stem Cell Vol.12 January 2013 , Page 127-137, Lee MO et al., PNAS 2013 Aug 27; 110 (35): E3281-90, WO2016 / 072519, WO2013 / 10080, Examples thereof include the method described in JP-A-2016-093178 and the method using the heat treatment described in WO2017 / 038526. Preferably, the operation for removing undifferentiated cells is performed by culturing in a sugar-free medium as described in WO2007 / 088874. , A method using a specific antibody as described in WO2016 / 072519, a method using a heat treatment as described in WO2017 / 038526, and the like.

The culture base material to be seeded is as described in detail above, but in a preferred embodiment, it is a culture base material whose surface is coated with a cell adhesion component such as an extracellular matrix or a cell adhesion factor. The cell-adhesive component is not limited to this, for example, extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, and cell adhesion factors such as cadoherin family, selectin family, and integrin family. In addition to these, these variants may be, for example, laminin 511 (a variant of laminin), VTN-N (a variant of vitronectin), retronectin ^(R) (a variant of fibronectin). Coating of the cell adhesion component can be achieved by contacting and incubating a medium containing the cell adhesion component with the culture substrate. The amount of the cell adhesion component that can be contained in such a medium may vary depending on the area of the substrate to be coated, the cell adhesion component used, the cell type to be cultured, and the like. A person skilled in the art can set the optimum amount or concentration according to the product protocol, etc., and the amount or concentration can be set when coating with only the adhesive component without using the masking component, for example, laminin 511 or VTN-. For N, it is about 0.1 to 1.0 μg / cm ² , and for retronectin ^(R) , it is about 4 to 20 μg / cm ² .

In yet another embodiment, it is a culture substrate in which the surface is coated with a masking component such as a blood-derived component in addition to a cell adhesion component such as an extracellular matrix or a cell adhesion factor. The "masking component" means a component capable of reducing the cell adhesion on the surface of the culture medium coated with the cell adhesion component. Such components are typically blood-derived components, such as normal serum (eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.), as well as albumin and platelet lysates contained in normal serum. , Skim milk, albumin substitutes such as polyvinyl alcohol, and the like.

Coating of the medium containing the masking component may be performed simultaneously with or separately from the coating of the cell adhesion component, and is achieved by contacting and incubating the medium containing the cell adhesion component and / or the masking component with the culture substrate. obtain. The concentration of the masking component that can be contained in such a medium may vary depending on the concentration of the cell adhesion component or the cell adhesion component used, the area of the substrate to be coated, the cell type to be cultured, and the like.

For example, in the case of FBS, the concentration of the masking component is 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.16%, 0.2% in the coating medium. , 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0% 2.25%, 2.5%, 2.75%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 7.5% , 9%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 30%, 40% or more, and the upper limit is not particularly limited, but 45. % Or less 30%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 9%, 7.5%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.75%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, It may be 0.8%, 0.6%, 0.5%, 0.31%, 0.16%, 0.125%, 0.1% or less.

When coated in combination with laminin 511 as a cell adhesion component, the amount or concentration of laminin 511 is 0.01 μg / cm ² to 100 μg / cm ^{2 with} respect to 0.1% to 30% FBS, laminin 511 0. ^{05μg / cm 2 ~ 80μg / cm} 2 with respect to 0.15% to 25% of the FBS, 0.7.5% 20% with respect to ^{0.05μg / cm 2 ~ 100μg / cm} 2 laminin 511, laminin 0.05% to 25% FBS with respect to 0.05 μg / cm ² to 1.0 μg / cm ² ^{of 511, 0.5 μg / cm 2} to 80 μg / cm of laminin 511 from the viewpoint of peeling by peeling work 0.25% to 7.5% FBS for ^two, more preferably from 0.75% to 12.5% FBS relative to ^{0.5μg / cm 2 ~ 80μg / cm} 2 laminin 511. When coated with heterologous serum such as FBS, it is preferable to wash the culture substrate by any method to a level that does not cause clinical damage.

The incubation time is not particularly limited as long as the masking component can adhere to the culture substrate, for example, 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, still more preferably 6 to 12 hours. It's time. The incubation temperature is also not particularly limited as long as the masking component can adhere to the culture substrate, and is, for example, 0 to 60 ° C., preferably 4 to 45 ° C., more preferably room temperature to 40 ° C.

In step (b), the seeded cells are sheet-cultured. In the present specification, the culture for forming the seeded cells as a transplant is referred to as "transplant formation culture", and the transplant is a sheet-like cell culture, and the culture for forming the seeded cells into a sheet is referred to as a culture. In particular, it is referred to as "sheet culture". Sheeting of seeded cells can be performed by any known method and condition. Non-limiting examples of such a method are described in, for example, Japanese Patent Application Laid-Open No. 2010-081829, Japanese Patent Application Laid-Open No. 2010-226991, Japanese Patent Application Laid-Open No. 2011-110368, Japanese Patent Application Laid-Open No. 2011-172925, WO2014 / 185517 and the like. Cell graft formation (eg, sheet formation) is believed to be achieved by the cells adhering to each other via adhesion molecules or intercellular adhesion mechanisms such as extracellular matrix. Thus, the step of forming a graft of seeded cells can be accomplished, for example, by culturing the cells under conditions that form cell-cell adhesions. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at about 37 ° C. and 5% CO _2. In addition, the culture can be carried out under normal pressure (atmospheric pressure, non-pressurization). In the graft-forming culture (sheet-forming culture), it is sufficient that cell-cell adhesion is formed, so that the cells do not necessarily have to proliferate. In a preferred embodiment, the graft-forming culture (sheeted culture) is performed without cell proliferation. Culturing can be carried out in containers of any size and shape. For the size and shape of the sheet-shaped cell culture, adjust the size and shape of the cell adhesion surface of the culture vessel, or install a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing cells inside the cell.

The time for sheeting culture can vary depending on the type of cells to be seeded and the cell density. For example, when cardiomyocytes are prepared from iPS cells and made into a sheet, the sheet may be formed by ^{seeding at a density of, for example, about 2.1 × 10 5} cells / cm ² and culturing for 4 days or more. Further, when the seeding density reaches the confluence, that is, when the seeding is performed at a higher density, the period of sheeting culture can be shortened, and the culture time may be 2 to 4 days, more preferably 2 to 3 days. ..

The medium used for graft formation (for example, sheet formation) (the graft-forming medium, which may be referred to as a sheet-forming medium when the graft formation is sheet-forming) contains a cell adhesion component and transplants cells. It is not particularly limited as long as it enables fragment formation, and for example, physiological saline, various physiological buffer solutions (for example, PBS, WBSS, etc.), various basal medium-based liquids for cell culture, and the like can be used. You may use it. Such basal medium is not limited to, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM. / F12 and the like are included. Many of these basal media are commercially available, and their compositions are also known. The basal medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased. Transplantation media may also include additives such as normal serum (eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.). However, when the sheet-shaped cell culture is produced under xenofree conditions, it is particularly preferable that it does not contain heterologous sera such as bovine serum and horse serum. The present disclosure is characterized in that a graft-forming culture is performed using a graft-forming medium containing a cell adhesion component, whereby a high-quality graft can be formed even if the graft-forming medium is serum-free. It is effective.
Therefore, in a preferred embodiment, the graft-forming medium is serum-free.
The concentration of the cell adhesive component contained in the sheeting medium is not particularly limited as long as it does not adversely affect the cells, but may be equal to or less than the concentration used in the above coating. Such a concentration is, for example, about 0.01 to 10 μg / mL for laminin 511 or VTN-N, preferably about 0.1 to 1 μg / mL, and about 0.2 to 100 ^{μg / mL for retronectin (R).} , Preferably about 2-10 μg / mL and the like.

In another preferred embodiment, the sheeting medium further comprises a platelet lysate. In the present disclosure, "platelet lysate" (PL) refers to a composition rich in growth factors and the like, which is obtained by repeatedly freezing and thawing platelets. Such a composition is commercially available as a medium additive for cell culture, is known in the art, and can be prepared by, for example, the method described in Bieback et al., STEM CELLS, 2009; 27: 2331-2341. Is. In recent years, it is known to promote the proliferation of mesenchymal stem cells. In the production of sheet-shaped cell cultures containing cardiomyocytes, the present inventors have observed that by incorporating platelet lysates into the sheeting medium, strong autonomous pulsation is observed in a faster culture time than before. I found it for the first time.

The concentration of platelet lysate contained in the graft-forming medium may be such that it is usually used in the art, for example, 1%, 2.5%, 5%, 10%, 15%, 20% and the like. It may be there. In a preferred embodiment, the platelet lysate is contained in the graft-forming medium in an amount of 1% to 20%, more preferably 2% to 10%, still more preferably 2.5% to 10%.

The graft-forming medium may be appropriately replaced during the graft-forming culture. In addition, the composition of the medium may be changed according to the progress of graft formation. In the production of sheet-like cell cultures containing cardiomyocytes derived from iPS cells, the present inventors use a sheeted medium to which a Rho-kinase (ROCK) inhibitor is added as a medium for the first day of sheeted culture. As a result, it was newly found that sheet-shaped cell cultures are effectively formed. Therefore, in a preferred embodiment of the present disclosure, the sheeting medium used for the sheeting culture on day 1 comprises a Rho-kinase inhibitor. In such an embodiment, the sheeting medium after the second day may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.

The culture substrate may or may not be coated with a cell adhesive component. When the culture substrate is coated with the cell adhesion component, the cell adhesion component coating the culture substrate may be the same as or different from the cell adhesion component contained in the sheeting medium. It may be, but preferably the same cell adhesion component. In one embodiment, the culture substrate is coated with only the cell adhesive component, preferably only the cell adhesive component contained in the sheeting medium. Therefore, in such an embodiment, the culture medium does not contain components other than the cell adhesive component, such as serum. In addition, the culture substrate may be coated with another component in place of or in addition to the cell adhesion component. Examples of such other components include coating components exemplified in the above description of the culture medium, such as temperature-responsive materials. In one aspect, the culture medium of the present disclosure is coated with serum, FBS or albumin in addition to the cell adhesive components.

The concentration of the cell adhesion component contained in the graft-forming medium may differ depending on the type of the cell adhesion component contained and the state of the cells forming the graft. For example, when cells with low viability, that is, weak activity are used, the content of the cell adhesion component should be low. The concentration of the cell adhesion component contained in the sheeting medium is about 0.1%, about 0.%, based on the concentration used when the same cell adhesion component is used as the coating agent for the culture substrate (100%). It may be 5%, about 1%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, and the like. Therefore, in a preferred embodiment, the concentration range of the cell adhesion component contained in the sheeting medium is about 0 based on the concentration used when the same cell adhesion component is used as the coating agent for the culture substrate (100%). .1% to about 100%, about 0.1% to about 100%, about 0.1% to about 50%, about 0.1% to about 25%, about 0.1% to about 20%, about 0 .1% to about 10%, about 1% to about 100%, about 1% to about 100%, about 0.5% to about 100%, about 0.5% to about 100%, about 0.5% to About 50%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 10%, about 1% to about 50%, about 1% to about 25%, About 1% to about 20%, about 1% to about 10%, about 0.5% to about 100%, about 5% to about 100%, about 5% to about 50%, about 5% to about 25%, It may be about 5% to about 20%, about 5% to about 10%, and the like.

As described above, the cardiomyocytes used in the method of the present disclosure may be directly obtained from a living body or derived from other cells, but are preferably derived from other cells. It is a thing. Examples of other cells to be induced include fibroblasts such as myocardial fibroblasts, progenitor cells that differentiate into myocardial cells such as cardiac progenitor cells, and pluripotent stem cells that can differentiate into arbitrary cells, which are preferable. Are pluripotent stem cells, more preferably iPS cells, and even more preferably human iPS cells.

Another aspect of the disclosure relates to a method of producing a sheet-like cell culture containing desired somatic cells, such as cardiomyocytes. Such a method includes the following steps:
(A) A step of seeding a cell population containing desired somatic cells (hereinafter, may be referred to as sheet-forming cells) at a density reaching confluence on a culture substrate coated with a cell-adhesive component and a masking component; And (B) a step of forming a sheet-like cell culture by sheet-forming and culturing the seeded cell population in a sheet-forming medium.
In step (A), a cell population containing the desired somatic cells (sheet-forming cells) for forming a sheet-like cell culture is seeded on a culture substrate coated with a cell adhesion component. The seeding on the culture substrate is as described in detail in the above-mentioned method for producing the implant. As the sheet-forming cells, the cells described in detail in the cells constituting the implant can be used.

The culture medium is as described in detail above. The method and concentration of coating with the cell adhesion component and the masking component are as detailed in the coating of the medium containing the masking component.

In step (B), a cell population containing sheet-forming cells seeded on a culture substrate is sheet-cultured in a sheet-forming medium. Here, the sheet-forming culture is as described in detail in the above-mentioned graft-forming culture. The sheeting medium in the method of this aspect is as detailed above, except that it may or may not contain cell adhesion components. In one embodiment, the sheeting medium comprises a cell adhesive component. In another embodiment, the sheeting medium is free of cell adhesive components.

Another aspect of the present disclosure relates to a method of evaluating the surface of a culture medium for producing a sheet-like cell culture. Such a method includes the following steps:
(I) The step of coating the surface of the culture substrate with the cell adhesion component, thereby improving the adhesion of the cells to the surface of the culture substrate.
(Ii) Further, a step of coating the surface of the culture substrate with a masking component, thereby reducing the adhesion of cells to the surface of the culture substrate,
(Iii) A step of seeding cardiomyocytes at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component.
(Iv) A step of forming a sheet-like cell culture by sheet-forming culture in a sheet-forming medium, and (v) a step of evaluating a peeling state of the sheet-like cell culture from the surface of the culture substrate.

In the method of this aspect, "evaluating the surface of the culture substrate" means that the sheet-like cell culture is cultured in a sheet using a culture medium coated with a medium containing a cell adhesive component and a masking component. It means that it is evaluated whether or not it has an appropriate substrate surface as a sheeted culture substrate through the evaluation of the peeled state. Since the cells used for producing the sheet-shaped cell culture have different peeling states from the culture base material for each lot, it is necessary to evaluate the surface of the culture base material for each lot of cells. According to the method of this aspect, the surface of the culture medium can be evaluated easily and accurately.

In step (i), the surface of the culture substrate is coated with a cell adhesive component. The cell adhesion component and coating are as described in detail in the above-mentioned method for producing a graft.
In step (ii), the surface of the cell substrate coated in step (i) is further coated with a masking component.

Steps (iii) and (iv) are as described in detail in the method for producing a sheet-shaped cell culture.
In step (v), the exfoliation state of the sheet-shaped cell culture from the surface of the culture substrate is evaluated. "Evaluating the exfoliation state" means evaluating the presence or absence of exfoliation of the sheet-like cell culture from the culture substrate and the type of exfoliation when sheet-forming culture and / or exfoliation work is performed using the coated culture substrate. Means to do. By evaluating such a peeled state, the surface of the culture medium can be evaluated. The types of exfoliation mainly include spontaneous exfoliation and exfoliation by exfoliation work, and those that can be exfoliated by exfoliation work are more likely to cause shrinkage of the sheet-like cell culture due to the intercellular binding force. It can be evaluated as a preferable surface of the culture substrate. For the evaluation, a person skilled in the art can select any method such as visual inspection or image analysis.

Another aspect of the present disclosure relates to sheet cell cultures produced by the production methods of the present disclosure. In one aspect of the invention, the sheet cell culture produced by the production method of the present disclosure comprises cardiomyocytes. In another aspect of the invention, the sheet cell culture produced by the production method of the present disclosure comprises cardiomyocytes, fibroblasts, vascular endothelial cells and / or parietal cells.
The sheet cell cultures of the present disclosure are useful in treating diseases that are ameliorated by the application of sheet cell cultures, such as various diseases associated with tissue abnormalities. Therefore, in one aspect, the sheet cell cultures of the present disclosure are intended for use in the treatment of diseases that are ameliorated by the application of sheet cell cultures, particularly those associated with tissue abnormalities. The sheet-shaped cell cultures of the present disclosure have similar properties peculiar to constituent cells except that they have higher mechanical strength than conventional sheet-shaped cell cultures, and therefore at least conventional myoblasts. It can be applied to tissues and diseases that can be treated with sheet-like cell cultures containing cells or fibroblasts. The tissues to be treated include, for example, myocardium, cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, stomach, etc. Examples include the gastrointestinal tract such as the small intestine, duodenum, and large intestine. In addition, the diseases to be treated are not limited, for example, heart diseases (for example, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.), corneal diseases (for example, corneal epithelial stem cell exhaustion, corneal membrane). Injury (heat / chemical corrosion), corneal ulcer, corneal opacification, corneal perforation, corneal scar, Stevens Johnson syndrome, ocular herbitis, etc.), retinal disease (eg, retinal pigment degeneration, age-related yellow spot degeneration, etc.) , Esophageal disease (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin disease (eg, skin injury (trauma, burn), etc.), joint disease (eg, osteoarthritis) , Cartilage disease (eg, cartilage damage), Liver disease (eg, chronic liver disease), Pancreatic disease (eg, diabetes), Dental disease (eg, periodontal disease, etc.), Kidney disease (eg, renal failure) , Renal anemia, renal osteodystrophy, etc.), thyroid disease (eg, hypothyroidism), muscle disease (eg, muscle injury, myitis, etc.), middle ear disease (eg, middle ear inflammation, etc.), bone marrow disease (For example, leukemia, poor regeneration anemia, immunodeficiency disease, etc.). The usefulness of the sheet-shaped cell culture of the present disclosure for the above-mentioned diseases is described in, for example, Patent Document 1, Non-Patent Document 1, Tanaka et al., J Gastroenterol. 2013; 48 (9): 1081-9. Are listed. The sheet-like cell cultures of the present disclosure can also be fragmented to an injectable size and injected at the site requiring treatment for greater efficacy than injection with a single cell suspension (Wang et. al., Cardiovasc Res. 2008; 77 (3): 515-24). Therefore, such a use is also possible for the sheet-shaped cell culture of the present disclosure.

Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, including applying an effective amount of the implant produced by the method of the present disclosure to a subject in need thereof. The diseases to be treated are as described above.

In the present disclosure, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of disease, etc. For example, the term "treatment" is medically acceptable for a variety of purposes, including delaying or stopping the progression of a disease associated with a tissue abnormality, regressing or eliminating a lesion, preventing the onset or recurrence of the disease, and the like. Including interventions to be performed.

In the treatment method of the present disclosure, an ingredient that enhances the viability, engraftment and / or function of the implant, other active ingredients useful for treating the target disease, etc. are used in combination with the implant of the present disclosure. be able to.

The treatment method of the present disclosure may further include the step of producing the implant of the present disclosure according to the production method of the present disclosure. The treatment method of the present disclosure serves as a cell (for example, skin cells, blood cells, etc. when using iPS cells) or a source of cells for producing a graft from a subject before the step of producing the graft. It may further include the step of collecting tissue (for example, skin tissue, blood, etc. when using iPS cells). In one embodiment, the subject from which the cell or tissue from which the cell is source is collected is the same individual as the subject to whom the cell culture, composition, implant, or the like is administered. In another embodiment, the subject from which the cell or tissue from which the cell is sourced is harvested is a separate entity of the same species as the subject receiving the administration, such as a cell culture, composition, or implant. In another embodiment, the subject from which the cell or tissue that is the source of the cell is collected is an individual different from the subject to which the implant or the like is administered.

In the present disclosure, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-like cell cultures, etc.). The amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissues. The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained. When applied to a tissue, the cell culture, composition, sheet-like cell culture or the like of the present invention may be fixed to the target tissue by a locking means such as a suture or a staple.

The present invention will be described in more detail with reference to the following examples, but these show specific specific examples of the present invention, and the present invention is not limited thereto.

In the following examples, clinical human iPS cells established at the Center for iPS Cell Research and Application (CiRA), Kyoto University were used as pluripotent stem cells. Human iPS cells were maintained by the feeder-free method with reference to M. Nakagawa et al., Scientific Reports, 4: 3594 (2014). Then, referring to the description of Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014 / 185358 and WO2017 / 038562, the embryoid body is induced to differentiate human iPS cells into cardiomyocytes. Got Specifically, human iPS cells maintained and cultured in a culture medium containing no feeder cells are cultured on EZSphere (Asahi Glass) for one day in StemFit AK03 medium (Ajinomoto) containing 10 μM Y27632 (Wako Pure Chemical Industries). Then, the obtained embryo-like body was cultured in a culture medium containing Actibin A, bone-forming protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further, Wnt inhibitor (IWP3) and BMP4 inhibition were performed. Human myocardial cells derived from iPS cells were obtained by culturing in a culture medium containing a drug (Dorsomorphin) and a TGFβ inhibitor (SB431542) and then in a culture medium containing VEGF and bFGF. The proportion of cardiomyocytes in the resulting cell population ranged from 50% to 90%.

Example 1. Comparison between FBS-containing medium and PL-containing medium Using the cell population obtained above containing cardiomyocytes induced to differentiate from human iPS cells, the sheeting culture conditions were examined. 20% FBS or 5% human platelet lysate was added to DMEM / F12 medium as a sheeting medium. Laminin (iMatrix-511) as a cell adhesion component was further added to the sheeting medium containing the platelet lysate at 0.1 μg / mL, 0.25 μg / mL or 0.5 μg / mL, respectively. In addition, the Rho-kinase inhibitor Y27632 was added to the sheeting medium only on the first day of the sheeting culture. The cell population containing cardiomyocytes was seeded in a temperature-responsive culture dish (UpCell ^(R) ^{, CellSeed) at a density of 1.5 × 10 6} cells / cm ² and placed in an environment of 37 ° C. and 5% CO ₂ for 3 days. It was cultured. As the temperature-responsive culture dish, the same one as the culture solution (however, Y27632 was not included) was put therein, and the culture dish was incubated overnight at 37 ° C. and coated. After culturing, the sheet-shaped cell culture containing cardiomyocytes was exfoliated from the culture dish.

The results are shown in the table below.

When a sheeted medium containing FBS was used, no pulsation was observed at the time of sheeting and culturing for 3 days, and even if it was observed, the pulsation was weak, whereas laminin and platelet lysate were observed. Strong pulsation was observed in all the media using the sheet-containing medium containing the above, after culturing for 3 days.

Example 2. Sheeting in serum-free medium Similar to Test Example 1, 0.1 μg / mL of standard medium with 20% FBS added to DMEM / F12 medium and laminin (iMatrix-511) without adding blood-derived components was added. Sheet culture was performed using each of the medium (E6) as a sheet medium.
The results are shown in the table below.

When sheeted with a serum-free medium containing laminin (medium containing no blood-derived components), pulsation was not observed until the second day of culture, but autonomous pulsation was observed and sheeted on the third day. We were able to. It was clarified that a cardiomyocyte sheet-like cell culture that beats independently can be obtained by containing a cell adhesion component (laminin) even if the medium does not contain a blood-derived component such as serum or platelet lysate.

Example 3. Examination of cell adhesion components In order to investigate whether sheeting is possible even when cell adhesion components other than laminin are used, laminin (iMatrix-511) and vitronectin (VTN) are used as cell adhesion components. Sheeting culture was performed using -N) and fibronectin ( ^{R) (Takarabio).} When iMatrix-511, VTN-N and RetroNectin ^(R) are used for coating the culture substrate, the recommended concentrations are 0.5 μg / cm ² , 0.5 μg / cm ² and 8 μg / cm ² , respectively. In, sheet-forming culture was carried out using the recommended concentration and a concentration of 1/10 of the recommended concentration. The above cell adhesion components were added in place of laminin 0.1 μg / cm ² in the E6 medium used in Example 2, and sheet-forming culture was carried out in the same manner as in Example 2.
The results are shown in FIG. Sheet-shaped cell cultures could be prepared regardless of which medium was used.

Example 4. Examination of cell condition and optimum concentration We investigated how the cell adhesion component affects the step of removing undifferentiated cells that can affect the activity of cells. In order to confirm whether the optimum concentration of the cell adhesion component that can be used for sheeting culture changes depending on the state of the cells, three types of cells were used for examination. As the highly active cells, a cell population containing myocardial cells induced to differentiate from the human iPS cells obtained above (no step of freezing and thawing the cells, no step of removing undifferentiated cells) was used as the above-mentioned medium-active cells. The cell population containing the myocardial cells induced to differentiate from the human iPS cells obtained in 1 above was once frozen and thawed, and the cells containing the myocardial cells induced to differentiate from the human iPS cells obtained above were regarded as low-activity cells. Undifferentiated cells were removed from the population, then frozen and thawed. The treatment for removing undifferentiated cells was carried out in order by using the heat treatment described in WO2017 / 038562, the sugar-free medium culture method described in WO2007 / 0888874, and the anti-CD30 antibody-binding drug treatment described in WO2016 / 072519.

Using the above three types of cells, sheet culture was carried out in the same manner as in Example 2 using E6 medium to which iMatrix-511 was added at a concentration of 1/10 of the recommended concentration as a cell adhesion component.
As a result, when highly active cells were used, the cells could be formed into sheets, but the cell-cell adhesion became stronger and the sheet-like cell cultures spontaneously exfoliated. When medium-active cells were used, they could be made into sheets and could be peeled off by peeling work. When low-activity cells were used, the cell-cell adhesion was weak when the peeling operation was performed, and the cells could not be peeled as a sheet. Therefore, when iMatrix-511 was similarly sheeted and cultured using E6 medium to which 1/100 of the recommended concentration was added using low-activity cells, it was possible to form a sheet and peel it off by peeling work. It was.

From the above, it was found that the cell-cell adhesion force changes depending on the cell activity, so it is necessary to add a cell adhesion component at a concentration corresponding to that. That is, it was clarified that it is necessary to supplement the cell adhesion component because the cell activity is reduced and the cell-cell adhesion is reduced when the freezing / thawing step and the step of removing undifferentiated cells are performed. ..

Example 5. Examination of coating with cell adhesion component and masking component DMEM / F12 investigated how the peeling state of sheet-shaped cell culture changes when the cell culture dish is coated with a masking component such as FBS together with the cell adhesion component. 0%, 0.31%, 0.63%, 1.25%, 2.5%, 5%, 10%, 20% FBS and 0.076 μg / cm ² , 0.76 μg / cm ² , 7 A total of 40 coating solutions were prepared with .6 μg / cm ² and 76 μg / cm ² of iMatrix-511, which were added to each well of a temperature responsive culture dish and incubated overnight at 37 ° C. for coating. After removing the coating solution, 1 mL of HBSS (manufactured by Invitrogen) was added to the culture dish, and the mixture was slowly stirred and washed for 20 seconds and then discarded by pipetting, and this was repeated twice. Sheets similar to Example 2 using a culture dish coated with E6 medium containing iMatrix-511 at a concentration of 1/10 of the recommended concentration using low-activity cells treated to remove undifferentiated cells of Example 4. Chemical culture was performed.

The culture solution was removed, HBSS (+) was added, and the mixture was allowed to stand at room temperature for 1 hour and 30 minutes for peeling work. The exfoliated state of the sheet-shaped cell culture is shown in Table 3 and FIG. “Peeling” of the colorless cells in Table 3 indicates spontaneous peeling, and “peeling” of the gray cells indicates peeling by the peeling operation. X indicates that the material was not peeled off.

It was clarified that by containing a masking component such as FBS, it can be peeled off even if it is coated with a solution containing a high concentration of cell adhesion component. It is presumed that this is because the cell adhesion action on the surface of the culture dish due to the cell adhesion component was reduced by the masking component. In Example 4, the low-activity cells could not be exfoliated without the addition of the cell adhesion component, but could be exfoliated by coating the cell adhesion component and the masking component. Therefore, it was clarified that the sheet-like cell culture can be exfoliated without supplementing the cell adhesion component at a concentration corresponding to the cell activity by the coating. Furthermore, it was clarified that the peeling state of the sheet-shaped cell culture can be controlled so that the natural peeling can be suppressed by adjusting the concentrations of the masking component and the cell adhesion component, and the peeling can be performed only by the peeling operation.

According to the present invention, it is possible to obtain a high-quality sheet-shaped cell culture when forming a sheet-shaped cell culture using cells or the like induced to differentiate from pluripotent stem cells. In particular, even in the production of a xeno-free sheet-shaped cell culture used for clinical use, it becomes possible to easily form a high-quality sheet-shaped cell culture.

Claims

A method of producing a graft containing somatic cells;
(A) a step of seeding the cell population containing the somatic cells on a culture substrate, and (b) a step of culturing the seeded cell population in a graft-forming medium containing a cell adhesion component.
The method described above.
The method according to claim 1, wherein the culture substrate is further coated with a cell adhesion component and / or a masking component.
The method according to claim 1 or 2, wherein the content of the cell adhesion component in the graft-forming medium is 1/10 to 1/100 of the amount used for coating the culture substrate.
The method according to any one of claims 1 to 3, which comprises a step of removing undifferentiated cells from the cell population before (a).
The method according to any one of claims 1 to 4, wherein the graft-forming medium does not contain serum.
The method according to any one of claims 1 to 5, wherein the graft-forming medium contains a platelet lysate.
A method for producing sheet-like cell cultures containing cardiomyocytes;
(A) A step of seeding cardiomyocytes at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component, and (B) sheet-forming culture using a sheet-forming medium, and sheet-like cell culture. The process of forming things,
A method for producing a sheet-shaped cell culture, including.
The method according to claim 7, wherein the sheet formation medium contains or does not contain a cell adhesive component.
A method for evaluating the surface of a culture medium for producing a sheet-like cell culture.
(I) The step of coating the surface of the culture substrate with the cell adhesion component, thereby improving the adhesion of the cells to the surface of the culture substrate.
(Ii) Further, a step of coating the surface of the culture substrate with a masking component, thereby reducing the adhesion of cells to the surface of the culture substrate,
(Iii) A step of seeding somatic cells at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component.
(Iv) A step of forming a sheet-like cell culture by sheet-forming culture in a sheet-forming medium, and (v) a step of evaluating the peeling state of the sheet-like cell culture from the surface of the culture substrate.
The method described above.
The method according to any one of claims 7 to 9, wherein the sheeting medium does not contain serum.
The method according to any one of claims 7 to 10, wherein the sheeting medium contains a platelet lysate.
The method according to any one of claims 1 to 11, wherein the masking component is a blood-derived component.
The method according to any one of claims 1 to 12, wherein the masking component is serum, platelet lysate or albumin.
The method according to any one of claims 1 to 13, wherein the masking component is FBS having a concentration of 0.05% or more.
The method according to any one of claims 1 to 14, wherein the somatic cell is a cardiomyocyte derived from a pluripotent stem cell.
A therapeutically effective amount of the implant produced by the method according to any one of claims 1 to 6 or the sheet cell culture produced by the method according to claim 7 or 8 is required. A method of treating a heart disease in a subject, comprising the step of administering to the subject.