Methods to enhance the activity of grafts

Abstract

To provide a method for enhancing the activity selected from the group consisting of cytokine production ability, proliferation ability, engraftment ability, blood vessel-inducing ability, and tissue regeneration ability of a sheet-like cell culture comprising a graft, particularly a differentiation-inducing cell derived from a pluripotent stem cell.SOLUTION: The foregoing problem is solved by a method comprising a step of incubating a graft at 25°C or higher.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This disclosure relates to a method for enhancing the activity of a graft, a method for manufacturing a graft including said method, a graft manufactured by said manufacturing method, and a method for treating a disease using said graft. [Background technology]

[0002] In recent years, various attempts have been made to transplant cells to repair damaged tissues. For example, fetal cardiomyocytes, skeletal muscle blasts, mesenchymal stem cells, cardiac stem cells, ES cells, and iPS cells have been attempted to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction (Non-Patent Literature 1).

[0003] As part of these efforts, cell structures formed using scaffolds and sheet-like cell cultures formed from cells have been developed (Patent Document 1, Non-Patent Document 2). Regarding the application of such sheet-like cell cultures and other grafts to treatment, studies are underway on the use of cultured epidermal sheets for skin damage caused by burns, the use of corneal epithelial sheet-like cell cultures for corneal damage, and the use of oral mucosa sheet-like cell cultures for endoscopic resection of esophageal cancer, and some of these have entered the stage of clinical application. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2007-528755 [Non-patent literature]

[0005] [Non-Patent Document 1] Haraguchi et al., Stem Cells Transl Med. 2012 Feb;1(2):136-41 [Non-Patent Document 2] Sawa et al., Surg Today. 2012 Jan;42(2):181-4

Summary of the Invention

Problems to be Solved by the Invention

[0006] As the clinical application of grafts such as sheet-like cell cultures progresses, there has been a growing demand for grafts that are of higher quality, easier to handle, and can be simply manufactured. It is known that various cytokines produced by cells in the graft act on the tissue at the transplantation site to promote the regeneration of the tissue. However, in the research on improving the graft, the inventors faced the problem that when the graft is stored, the activities such as cytokine production ability and the proliferation ability of the cells in the graft decrease.

[0007] Continuing the research to solve such problems, it was found that by incubating the sheet-like cell culture at 25°C or higher, the cytokine production ability and the activity of the cells in the graft can be improved. Further research was carried out, and it was found that when using a graft having a scaffold, the cytokine production ability can be improved while maintaining the shape of the graft, and the present invention was thus completed.

[0008] That is, the present invention relates to the following: <1> A method for enhancing an activity selected from the group consisting of cytokine production ability, proliferation ability, engraftment ability, angiogenesis ability, and tissue regeneration ability of a graft containing differentiation-induced cells derived from pluripotent stem cells, the method comprising the step of incubating the graft at 25°C or higher. <2> The method according to claim 1, wherein the graft has a scaffold. <3> The method according to claim 2, wherein the scaffold is a gel containing fibrin, gelatin, or collagen. <4> A method for manufacturing a graft, the method comprising the method according to any one of <1> to <3>. <5> The method according to any one of <1> to <4>, wherein the graft is a sheet-like cell culture. <6> <1> ~ <3> A graft obtained by the method described in any one of the items or <4> or <5> A graft produced by the method described above. <7> To treat heart disease <6> The graft described above. <8> A method for treating a disease that is improved by the application of a graft, <6> or <7> The method comprising the step of applying the graft described above to a target that requires it. [Effects of the Invention]

[0009] The grafts according to the present invention exhibit high activity, including cytokine production and proliferation, and superior engraftment into tissue, subsequent viability, functionality, and functional sustainability. Therefore, they enable efficient treatment of various diseases, as well as long-term storage and transport. Consequently, it becomes possible to provide grafts that maintain high quality. [Brief explanation of the drawing]

[0010] [Figure 1] This shows the time course of cell survival rates when sheet-like cell cultures are stored at 4°C and 25°C. [Figure 2] This shows the changes in cytokine production when sheet-like cell cultures are stored at 4°C and 25°C. [Figure 3] The images show a sheet-like cell culture with an isolated fibrin gel layer formed (Example 1(A)) and a sheet-like cell culture with the gaps filled with fibrin gel before isolation (Example 2(B)). [Figure 4] The cell counts of a sheet-like cell culture with a fibrin gel layer (Example 1) when stored at 4°C, 25°C, 30°C, 35°C, 37°C, and 39°C are shown. [Figure 5] The cytokine production levels of a sheet-like cell culture with a fibrin gel layer (Example 1) when stored at 25°C, 30°C, 35°C, 37°C, and 39°C are shown. [Figure 6]The images show the sheet-like cell cultures prepared in Comparative Example 3 stored at 4°C (A) and 25°C (B), and the sheet-like cell cultures with a fibrin gel layer formed in Example 4 stored at 4°C (C) and 25°C (D). [Modes for carrying out the invention]

[0011] The details of this disclosure are described below. Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. All patents, applications, published applications, and other publications referenced herein are incorporated herein by reference in their entirety. In the event of any conflict between the publications referenced herein and the statements herein, the statements herein shall prevail.

[0012] <Methods to enhance the activity of grafts> This disclosure provides a method for enhancing the activity of a graft, selected from the group consisting of cytokine production ability, proliferation ability, engraftment ability, angiogenesis ability, and tissue regeneration ability, and includes the step of incubating a sheet-like cell culture at 25°C or higher.

[0013] In this disclosure, “graft” means a structure for implantation into a living organism, and more particularly a transplant structure containing cells as a component. The cells constituting the graft may be connected to one another directly (including via cellular elements such as adhesion molecules) and / or via intervening substances (also referred to herein as “scaffold”). The cells are connected at least physically (mechanically), but may also be connected functionally, for example, chemically or electrically. Examples of grafts in this disclosure, but not limited to, include sheet cell cultures, cell aggregates, spheroids, and organoids (hereinafter, “cell aggregates,” “spheroids,” and “organoids” are collectively referred to herein as “cell aggregates”), preferably sheet cell cultures or spheroids, more preferably sheet cell cultures.

[0014] In one embodiment of the present invention, the graft includes a scaffold. The scaffold is used in the art to maintain the physical integrity and provide strength to the graft by attaching or embedding cells on its surface and / or inside. The scaffold is not particularly limited as long as it is a material that can connect cells at least physically (mechanically), and may be a cell-derived material or a non-cell-derived material. Examples of scaffolds include biocompatible films or gels. Examples include, but are not limited to, films such as amniotic membrane, PVDF, polytetrafluoroethylene (PTFE), polyurethane, polypropylene, polyester, vinyl chloride, polycarbonate, acrylic, silicone, MPC (2-methacryloyloxyethyl phosphorylcholine), polylactic acid, polyglycolic acid, and lactic acid-glycolic acid copolymer (PLGA); temperature-responsive gels (commercial name: Mebiol Gel) made by crosslinking poly(N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG); and gels containing hyaluronic acid, glycosaminoglycans, proteoglycans, chondroitin, cellulose, agarose, carboxymethylcellulose, chitin, chitosan, gelatin, atelocollagen, elastin, fibronectin, pronectin, laminin, tenascin, fibroin, entactin, thrombospongin, retronectin, dextrin, and trehalose. The scaffold is preferably a gel containing fibrin gel, gelatin, or collagen, and more preferably a gel containing fibrin gel. Examples of grafts having a scaffold include, but are not limited to, grafts having a film on the surface or inside, grafts with a gel layer formed on the surface or inside, or grafts in which coherence is provided by a gel that fills the gaps between cells, cell aggregates, or sheet-like cell cultures.

[0015] In this disclosure, “sheet-like cell culture” means a sheet-like structure in which cells are linked together. The cells may be linked together directly (including via cellular elements such as adhesion molecules) and / or via scaffolds. Therefore, in this disclosure, "sheet-like cell culture" includes not only cells cultured in a sheet-like manner, but also cells molded into a sheet-like shape. A sheet-like cell culture may consist of one cell layer (monolayer) or two or more cell layers (e.g., two, three, four, five, six layers, etc.). Furthermore, a sheet-like cell culture may have a three-dimensional structure with a thickness exceeding the thickness of a single cell, even if the cells do not exhibit a clear layered structure. For example, in a vertical cross-section of a sheet-like cell culture, the cells may not be uniformly aligned horizontally but rather unevenly arranged (e.g., in a mosaic pattern), and scaffolds such as films or gels may be included between the cells.

[0016] The cells may be heterologous or homologous. Here, "heterologous cells" refers to cells derived from an organism of a different species than the recipient when the graft is used for transplantation. For example, if the recipient is human, cells derived from monkeys or pigs would be considered heterologous cells. "Homologous cells" refers to cells derived from an organism of the same species as the recipient. For example, if the recipient is human, human cells would be considered homologous cells. Homologous cells include autologous cells (also called self-derived cells or autogenous cells), i.e., cells derived from the recipient, and allogeneic non-self-derived cells (also called allogeneic cells). Autologous cells are preferred in this disclosure because they do not cause rejection when transplanted. However, it is also possible to use heterologous cells or allogeneic non-self-derived cells. When using heterologous cells or allogeneic non-self-derived cells, immunosuppressive treatment may be necessary to suppress rejection. In this specification, cells other than autologous cells, i.e., heterologous cells and allogeneic non-self-derived cells, may be collectively referred to as non-self-derived cells. In one aspect of this disclosure, the cells are autologous or allogeneic cells. In one aspect of this disclosure, the cells are autologous cells (including autologous iPS cells). In another aspect of this disclosure, the cells are allogeneic cells (including allogeneic iPS cells).

[0017] The cells constituting the graft in this disclosure are cells differentiated from pluripotent stem cells by gene transfer and are not particularly limited as long as they can form a graft, especially a sheet-like cell culture, and include, for example, adherent cells. Adherent cells include, for example, somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (e.g., tissue stem cells such as myoblasts and cardiac stem cells, embryonic stem cells, pluripotent stem cells such as induced pluripotent stem cells (iPS (induced pluripotent stem) cells), mesenchymal stem cells, etc.). iPS cells are cells induced by gene transfer. Non-limiting examples of cells that make up the graft include, for example, myoblasts (e.g., skeletal myoblasts), mesenchymal stem cells (e.g., those derived from bone marrow, adipose tissue, peripheral blood, skin, hair follicles, muscle tissue, endometrium, placenta, umbilical cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (e.g., oral mucosal epithelial cells, retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (e.g., vascular endothelial cells, etc.), hepatocytes (e.g., hepatocytes, etc.), pancreatic cells (e.g., islet cells, etc.), renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, etc. Non-limiting examples of iPS cell-derived adherent cells include iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.

[0018] In this disclosure, "pluripotent stem cell" is a well-known term in the art and refers to a cell that has the ability to differentiate into all lineages of cells belonging to the three germ layers, namely the endoderm, mesoderm, and ectoderm. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and induced pluripotent stem cells (iPS cells). Typically, when differentiating pluripotent stem cells into specific cells, the pluripotent stem cells are first cultured in suspension to form aggregates of cells from one of the three germ layers, and then the cells forming the aggregates can be differentiated into the target cells.

[0019] In this disclosure, “differentiation-inducing cells derived from pluripotent stem cells” means any cells that have been differentiated from pluripotent stem cells into a specific type of cell. Non-limiting examples of differentiation-inducing cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neurons, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, hematopoietic cells such as blood cells and bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, organ cells such as hepatocytes, pancreatic β-cells, and kidney cells, chondrocytes, germ cells, and other cells, as well as progenitor cells and somatic stem cells that differentiate into these cells. Typical examples of such progenitor cells and somatic stem cells include, for example, mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in nervous system cells, and hematopoietic stem cells and lymphoid stem cells in hematopoietic cells and immune-related cells. Differentiation induction of pluripotent stem cells can be carried out using any known method. For example, differentiation induction from 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 differentiation-inducible cells derived from pluripotent stem cells, such as iPS-derived cardiomyocytes, are used as the desired cells, undifferentiated cells may be removed after differentiation induction. Methods for removing undifferentiated cells are known in the art and can be used, for example, as described in WO2017 / 038562, WO2016 / 072519 and WO2007 / 088874.

[0020] Furthermore, the differentiated cells may be cells derived from iPS cells into which any useful gene other than the gene for reprogramming has been introduced. Non-limiting examples of such cells include, for example, T cells derived from 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. Cells that have been differentiated from pluripotent stem cells and then into which any useful gene has been introduced are also included in the differentiated cells of the present invention.

[0021] In this disclosure, "cardiomyocyte" means a cell having the characteristics of a cardiomyocyte, and these characteristics include, but are not limited to, the expression of cardiomyocyte markers and the presence of autonomous beating. Examples of non-limited cardiomyocyte markers include c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, and VCAM. Cardiomyocytes derived from iPS cells are preferably exemplified as cardiomyocytes. In the present invention, the cells constituting the graft are preferably cardiomyocytes, hepatocytes, fibroblasts, myoblasts, pancreatic cells, renal cells, vascular endothelial cells, or corneal epithelial cells, more preferably cardiomyocytes, and most preferably iPS cell-derived cardiomyocytes.

[0022] The cells constituting the graft may originate from any organism that can be treated with the graft. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, and the like. Furthermore, the number of cell types constituting the sheet-like cell culture is not particularly limited; it may consist of only one type of cell, or it may use two or more types of cells. When there are two or more types of cells forming the sheet-like material, the content ratio (purity) of the most abundant cell is 50% or more, preferably 60% or more, more preferably 70% or more, and even more preferably 75% or more at the end of the formation of the sheet-like cell culture.

[0023] The graft can be one that has been prepared by any known method. In one embodiment of the present invention, if the graft is a sheet-like cell culture, the sheet-like cell culture can be one that has been prepared by a method typically comprising the steps of seeding cells onto a substrate and forming the seeded cells into a sheet. In one embodiment of the present invention, the graft having a scaffold can be one that has been manufactured by any known method. In one embodiment of the present invention, if the graft has a scaffold, a sheet-like cell culture with a gel layer can be used, which is produced by a method that includes, for example, the step of forming a gel layer after the step of sheeting the seeded cells. The step of forming a gel layer on the sheet-like cell culture can be carried out using known methods, for example, by spraying thrombin solution and then allowing the sheet-like cell culture to stand for a certain period of time (see Japanese Patent No. 6495603).

[0024] In one embodiment of the present invention, if the graft has a scaffold, a gel-integrated graft can be used, which is manufactured by a method comprising the steps of arranging cells, cell aggregates and / or sheet-like cell cultures on a substrate with gaps between them, and filling the gaps between the cells with gel to form a sheet. In yet another embodiment of the present invention, if the graft has a scaffold, a gel-integrated graft can be used, which is manufactured by a method comprising the steps of settling cells, cell aggregates and / or sheet cell cultures onto a substrate, and forming the cells, cell aggregates and / or sheet cell cultures into a sheet using a gel. The step of filling the gaps with gel to form a sheet, or forming a sheet using gel, is carried out, for example, by gently adding gel to a substrate on which cells are arranged on its surface. The amount added may be any amount, but preferably an amount that results in a sheet with a thickness of, for example, 10 μm to 2000 μm, preferably 10 μm to 500 μm, and more preferably 50 μm to 200 μm. In one embodiment, the gel is formed by mixing two liquids, and any known method can be used to gel such a gel. Such methods include, for example, a method of simultaneously adding fibrinogen solution and thrombin solution, or a method of forming a fibrin gel by dropping fibrinogen solution onto cells and then spraying thrombin solution (Japanese Patent Publication No. 6495603), but are not limited to these.

[0025] In one embodiment of the present invention, when a gel is used as a scaffold, the gel is biodegradable, meaning it is broken down in the body, absorbed, metabolized, and excreted. Examples include fibrin gel obtained by mixing fibrinogen solution and thrombin solution, and anti-adhesion agents obtained by mixing NHS-modified CM dextrin and trehalose aqueous solutions with sodium carbonate and sodium bicarbonate aqueous solutions. These are commercially available, for example, as Volheal® for tissue adhesion (manufactured by Teijin Pharma), Beriplast® for tissue adhesion (manufactured by CSL Behring), and Adspray® (manufactured by Terumo), and can be used in the present invention.

[0026] This disclosure includes the step of incubating the graft at 25°C or above. The incubation time is not particularly limited, as long as the activity of the sheet-like cell culture does not decrease, and is about 1 hour to about 96 hours, preferably about 4 hours to about 72 hours, more preferably about 5 hours to about 72 hours, and even more preferably about 8 hours to about 48 hours.

[0027] The incubation temperature is not particularly limited as long as the activity of the graft does not decrease, and is, for example, approximately 20°C to approximately 45°C. The lower limit of the incubation temperature is, for example, approximately 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C or higher, 25°C or higher, approximately 26°C or higher, approximately 27°C or higher, approximately 28°C or higher, approximately 29°C or higher, approximately 30°C or higher, approximately 31°C or higher, approximately 32°C or higher, approximately 33°C or higher, approximately 34°C or higher, approximately 35°C or higher, approximately 36°C or higher, approximately 37°C or higher, approximately 38°C or higher, approximately 39°C or higher, approximately 40°C or higher, approximately 41°C or higher, approximately 42°C or higher, approximately 43°C or higher, approximately 44°C or higher, and approximately 45°C or higher.

[0028] The upper limit of the incubation temperature is, for example, approximately 26°C or lower, approximately 27°C or lower, approximately 28°C or lower, approximately 29°C or lower, approximately 30°C or lower, approximately 31°C or lower, approximately 32°C or lower, approximately 33°C or lower, approximately 34°C or lower, approximately 35°C or lower, approximately 36°C or lower, approximately 37°C or lower, approximately 38°C or lower, approximately 39°C or lower, approximately 40°C or lower, approximately 41°C or lower, approximately 42°C or lower, approximately 43°C or lower, approximately 44°C or lower, and approximately 45°C or lower, preferably 39°C or lower, and any combination of the upper and lower limits exemplified above. That is, for example, about 20°C to about 45°C, about 25°C to about 45°C, about 26°C to about 44°C, 27°C to about 43°C, 28°C to 42°C, about 29°C to about 41°C, about 30°C to about 40°C, about 31°C to about 39°C, about 32°C to about 38°C, about 33°C to about 37°C, about 34°C to about 36°C, preferably 27°C to about 43°C, more preferably about 30°C to about 40°C, even more preferably about 32°C to about 39°C, even more preferably about 33°C to about 39°C, and particularly preferably about 34°C to about 39°C.

[0029] The incubation step at 25°C or higher in the present invention can be performed at any time after the production of the graft. For example, it can be performed immediately after the production of the graft, or after storing the graft at 4°C, for example, and when transferring the graft to a different facility, or within the facility after the transfer. However, it is preferable to perform it continuously from immediately after production. In one embodiment of the present invention, if a step of forming a gel layer is added before the incubation step, it is preferable that the step of forming the gel layer be performed before storing the graft at, for example, 4°C.

[0030] The graft used in the present invention may remain attached to a substrate (e.g., a culture substrate) or may be detached from the substrate. If the graft remains attached to the substrate, the incubation step can be performed after adding any medium to the substrate (or to the container if the substrate is the surface of a container). If the graft is detached from the substrate, the incubation step can be performed after adding the medium to the container containing the graft. The medium is not particularly limited as long as it can maintain the survival of the cells constituting the graft, and may be, for example, physiological saline, various physiological buffers (e.g., PBS, HBSS, etc.), or based on various basal culture media for cell culture. Such basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. Many of these basal media are commercially available, and their compositions are publicly known. The basal media may be used in its standard composition (e.g., as commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. The medium may usually contain additives such as serum (e.g., bovine serum such as fetal bovine serum (FBS), horse serum, human serum, etc.) and various growth factors (e.g., FGF, EGF, VEGF, HGF, etc.). A preferred medium is DMEM, and DMEM containing 10% FBS is particularly preferred.

[0031] The method of the present invention may be used to maintain or enhance the activity of grafts, such as cytokine activity and proliferative capacity, or to restore, maintain, or enhance the activity of sheet-like cell cultures or the like, whose activity has decreased due to storage or transport. Furthermore, it may be used to restore, maintain, or enhance the activity of grafts that have been partially damaged during graft production, for example, after adding a step to form a gel layer.

[0032] Grafts subjected to the method of the present invention have higher activity than grafts that have not undergone the incubation step of the present invention (hereinafter sometimes referred to as control grafts). Such activity includes, but is not limited to, cytokine production ability, proliferation ability, engraftment ability, angiogenesis ability, and tissue regeneration ability. Here, "high activity" means that the activity is 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 300% or more, or 400% or more higher than the activity of the control graft.

[0033] In one embodiment of the present invention, cytokines are beneficial for the engraftment of grafts into the recipient tissue. In one embodiment of the present invention, cytokines are beneficial for angiogenesis. In one embodiment of the present invention, cytokines are beneficial for the induction of bone marrow mesenchymal stem cells. In one embodiment of the present invention, cytokines are beneficial for tissue regeneration. Such cytokines are known in the art, and those skilled in the art can determine appropriate cytokines based on known information. In one embodiment of the present invention, cytokines include growth factors. In one embodiment of the present invention, cytokines are selected from the group consisting of VEGF, HGF, SDF-1, FGF, and SCF. Therefore, in one embodiment of the present invention, cytokine production capacity is the capacity to produce growth factors. Also, in one embodiment of the present invention, cytokine production capacity is the capacity to produce growth factors selected from the group consisting of VEGF, HGF, SDF-1, FGF, and SCF.

[0034] The activity of the graft can be quantified using various methods. If the activity is cytokine production capacity, for example, the cytokine production capacity can be quantified by culturing the graft obtained by the method of the present invention in a predetermined culture medium for a predetermined time and measuring the amount of cytokine secreted into the culture medium, or by measuring the amount of cytokine genes expressed in the graft. Methods known in the art can be used to measure the amount of specific proteins or the level of gene expression.

[0035] If the activity is proliferative capacity, the proliferative capacity can be quantified, for example, by measuring the number of viable cells in the graft constituting the graft obtained by the method of the present invention, or by measuring the number of surviving cells in the graft after it has been stored in a culture medium for a predetermined time. Methods known in the art can be used to measure the number of viable cells in the graft.

[0036] If the activity is engraftment ability, the engraftment ability can be quantified by applying the graft or sheet-like cell culture to the tissue, observing the state of the applied graft after a predetermined time has elapsed, for example, adhesion to the tissue, size, color, and morphology of the remaining graft, and scoring these. If the activity is angiogenic ability, the angiogenic ability can be quantified by applying the graft to the tissue, observing the state of the applied graft and the tissue at the application site after a predetermined time has elapsed, for example, the presence or absence of angiogenesis, and scoring these. Furthermore, if the activity is tissue regeneration ability, the tissue regeneration ability can be quantified by applying the graft to the tissue, observing the state of the tissue at the application site (graft recipient) after a predetermined time has elapsed, for example, tissue size, tissue microstructure, ratio of damaged tissue to normal tissue, degree of induction of cardiomyocytes from bone marrow mesenchymal stem cells, and tissue function, and scoring these.

[0037] In one embodiment of the present invention, grafts having a scaffold are less prone to twisting and / or shrinking than grafts without a scaffold, provided that the incubation step is performed under the same conditions as in the incubation step of the method of the present invention. In this disclosure, "twisting" and "shrinking" of the graft refer to the twisting of the outer edge of the graft (e.g., a sheet of cell culture) and the curling of the graft, respectively. Such "twisting" and "shrinking" include not only those caused by intercellular adhesion forces but also wrinkles and folds that occur physically during transport. The degree of "twisting" and "shrinking" may be quantified, for example, by observing the shape, size, and morphology of the graft using known methods and scoring them.

[0038] In one embodiment, the method of the present invention is carried out entirely in vitro. In another embodiment, the production method of the present invention includes steps carried out in vivo, without limitation, such as collecting cells or tissues that will serve as a source of cells from a subject. In one embodiment, the method of the present invention is carried out entirely under sterile conditions.

[0039] <Method for producing highly active grafts> Another aspect of the present invention relates to grafts obtained by a method for producing grafts, which includes the method of the present invention. In one embodiment of the present invention, the production of a graft includes, but is not limited to, the steps of seeding cells on a substrate, forming the seeded cells into a sheet, and incubating at 25°C or higher. Furthermore, after the sheeting step, the sheeted graft may be peeled off and then incubated, or the graft may be incubated while still attached to the culture substrate. Additionally, after the sheeting step of the seeded cells, a gel layer may be formed on the graft. In this case, the gel layer may be formed after peeling or while still attached to the substrate.

[0040] In one embodiment of the present invention, the production of a graft includes, but is not limited to, the steps of arranging cells, cell aggregates and / or sheet-like cell cultures on a substrate with gaps between them, filling the gaps between the cells with gel to form a sheet, and incubating at 25°C or higher. In yet another embodiment of the present invention, the production of a graft includes, but is not limited to, the steps of: settling cells, cell aggregates and / or sheet-like cell cultures onto a substrate; forming the cells, cell aggregates and / or sheet-like cell cultures into a sheet using a gel; and incubating at 25°C or higher. In this embodiment, after the step of embedding with the gel to form a sheet or the step of forming a sheet-like cell culture into a sheet using the gel, the step of peeling off the sheet-like graft may be performed, followed by the incubation step, or the step of incubation may be performed with the graft still attached to the culture substrate. Each of these steps can be carried out by any known method suitable for the production of various grafts. The method of the present invention may further include a step of producing a graft, in which case the step of producing a graft may include any one or more steps.

[0041] The substrates that can be used to manufacture grafts, such as culture substrates, are not particularly limited as long as cells can form grafts on them, and include, for example, containers of various materials and / or shapes, and solid or semi-solid surfaces within the containers. The containers are preferably made of a structure and material that does not allow liquids such as culture media to pass through. Such materials, without limitation, include, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethylacrylamide, and metals (e.g., iron, stainless steel, aluminum, copper, brass). Furthermore, the containers are preferably made of at least one flat surface. Examples of such containers, without limitation, include, for example, a culture vessel having a bottom surface made of a substrate on which cell cultures can be formed and liquid-impermeable sides. Specific examples of such culture vessels, without limitation, include cell culture dishes and cell culture bottles. The bottom surface of the container may be transparent or opaque. If the bottom of the container is transparent, it becomes possible to observe and count cells from the back of the container. The container may also have a solid or semi-solid surface inside. Examples of solid surfaces include plates and containers made of various materials as described above, while examples of semi-solid surfaces include gels and soft polymer matrices. The base material may be made using the above materials, or a commercially available one may be used.

[0042] Preferred substrates are not limited to, for example, suitable for graft formation. Examples include substrates having an adhesive surface, substrates having a low-adhesion surface, and / or substrates having a uniform well-like structure. Specifically, in the case of forming sheet-like cell cultures, examples include polystyrene treated with corona discharge, substrates coated on their surface with hydrophilic compounds such as collagen gel or hydrophilic polymers, and substrates coated on their surface with extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycans, glycosaminoglycans, or cell adhesion factors such as the cadherin family, selectin family, or integrin family. Such substrates are commercially available (e.g., Corning(R) TC-Treated Culture Dish, Corning, etc.). In the case of spheroid formation, examples of substrates include those coated with non-cell adhesive compounds such as soft agar, a temperature-responsive gel made of poly(N-isopropylacrylamide) (PIPAAm) crosslinked with polyethylene glycol (PEG) (commercial name: Mebiol Gel), polyhydroxyethyl methacrylate (PolyHEMA), and hydrogels such as 2-methacryloyloxyethyl phosphorischoline (MPC) polymer, and / or substrates having a uniform uneven surface structure. Such substrates are also commercially available (e.g., EZSPHERE(R)). The substrate may be transparent or opaque in whole or in part.

[0043] The substrate may be coated on the surface with a material whose physical properties change in response to stimuli, such as temperature or light. Such materials are not limited to, but include, for example, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (e.g., N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), and (meth)acrylamide derivatives having a cyclic group (e.g., 1-(1- Known materials such as temperature-responsive materials consisting of homopolymers or copolymers of oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, or vinyl ether derivatives (e.g., methyl vinyl ether), light-absorbing polymers having azobenzene groups, copolymers of vinyl derivatives of triphenylmethane leucohydrooxide and acrylamide monomers, and N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, Japanese Patent Publication No. 2-211865 and Japanese Patent Publication No. 2003-33177). By applying a predetermined stimulus to these materials, their physical properties, such as hydrophilicity or hydrophobicity, can be altered, thereby promoting the detachment of cell cultures attached to the material. Culture dishes coated with temperature-responsive materials are commercially available (for example, UpCell® from CellSeed Co., Ltd. and Cepallet® from DIC Corporation), and these can be used in the present invention.

[0044] The base material may be of various shapes. Furthermore, its area is not particularly limited, but for example, approximately 1 cm². 2 ~about 200cm 2 Approximately 2cm 2 ~about 100cm 2 , about 3cm 2 ~about 50cm 2 For example, a circular culture dish with a diameter of 10 cm can be used as a substrate. In this case, the area is 56.7 cm². 2 The culture surface may be flat or may have an uneven surface. If it has an uneven surface, a uniform uneven surface is preferable.

[0045] The substrate may be coated with serum. Using a serum-coated substrate allows for the formation of denser grafts, particularly sheet-like cell cultures. "Serum-coated" means that serum components are attached to the surface of the substrate. This can be achieved, for example, by treating the substrate with serum. Treatment with serum includes bringing the serum into contact with the substrate and, if necessary, incubating it for a predetermined period.

[0046] As serum, heterologous and / or homologous serum can be used. Heterologous serum refers to serum derived from an organism of a different species than the recipient when a graft, especially a sheet of cell culture, is used for transplantation. For example, if the recipient is human, serum derived from cattle or horses, such as fetal bovine serum (FBS, FCS), calf serum (CS), or horse serum (HS), would be considered heterologous serum. Homologous serum refers to serum derived from an organism of the same species as the recipient. For example, if the recipient is human, human serum would be considered homologous serum. Homologous serum includes autologous serum (also called autoseroserum), i.e., serum derived from the recipient, and homologous allogeneic serum derived from an individual of the same species other than the recipient. In this specification, serum other than autologous serum, i.e., heterologous serum and homologous allogeneic serum, may be collectively referred to as non-autologous serum.

[0047] Serum for coating the substrate can be commercially available or prepared by standard methods from blood collected from the desired organism. Specifically, for example, the collected blood can be left at room temperature for about 20 to 60 minutes to allow it to coagulate, then centrifuged at about 1000 × g to 1200 × g, and the supernatant can be collected.

[0048] When incubating on a substrate, serum may be used undiluted or diluted. Dilution can be performed in any medium, for example, water, physiological saline, various buffers (e.g., PBS, HBSS, etc.), various liquid culture media (e.g., DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.), etc. The dilution concentration is not particularly limited as long as the serum components can adhere to the substrate, and is, for example, about 0.5% to about 100% (v / v), preferably about 1% to about 60% (v / v), more preferably about 5% to about 40% (v / v).

[0049] The incubation time is not particularly limited as long as the serum components can adhere to the substrate, and is, for example, about 1 hour to about 72 hours, preferably about 2 hours to about 48 hours, more preferably about 2 hours to about 24 hours, and even more preferably about 2 hours to about 12 hours. The incubation temperature is also not particularly limited as long as the serum components can adhere to the substrate, and is, for example, about 0°C to about 60°C, preferably about 4°C to about 45°C, and more preferably room temperature to about 40°C.

[0050] The serum may be discarded after incubation. Conventional liquid disposal methods such as aspiration with a pipette or decantation can be used to discard the serum. In a preferred embodiment of this disclosure, the substrate may be washed with a serum-free washing solution after serum disposal. The serum-free washing solution is not particularly limited as long as it does not contain serum and does not adversely affect serum components attached to the substrate. For example, it can be water, physiological saline, various buffers (e.g., PBS, HBSS, etc.), various liquid culture media (e.g., DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.), etc. Conventional substrate washing methods can be used, for example, by adding the serum-free washing solution to the substrate, stirring for a predetermined time (e.g., about 5 seconds to about 60 seconds), and then discarding it.

[0051] In this disclosure, the substrate may be coated with a growth factor. Here, “growth factor” means any substance that promotes cell proliferation compared to the absence of such a substance, and includes, for example, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc. The method for coating the substrate with a growth factor, the disposal method, and the washing method are basically the same as those for serum, except that the dilution concentration during incubation is, for example, about 0.0001 μg / mL to about 1 μg / mL, preferably about 0.0005 μg / mL to about 0.05 μg / mL, and more preferably about 0.001 μg / mL to about 0.01 μg / mL.

[0052] In this disclosure, the substrate may be coated with a steroid. Here, "steroid" refers to a compound having a steroid nucleus that can have adverse effects on the body, such as adrenal insufficiency and Cushing's syndrome. Such compounds include, but are not limited to, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, and the like. The method for coating the substrate with a steroid, the disposal method, and the washing method are basically the same as those for serum, except that the dilution concentration during incubation is, for example, about 0.1 μg / mL to about 100 μg / mL, preferably about 0.4 μg / mL to about 40 μg / mL, and more preferably about 1 μg / mL to about 10 μg / mL as dexamethasone.

[0053] The substrate may be coated with serum, growth factors, or steroids, or with any combination thereof, namely serum and growth factors, serum and steroids, serum, growth factors and steroids, or growth factors and steroids. When coating with multiple components, these components may be mixed and coated simultaneously, or coated in separate steps.

[0054] The culture medium used in the manufacturing method of the present invention is not particularly limited as long as it can maintain cell viability, but typically, a medium mainly composed of amino acids, vitamins, and electrolytes can be used. In one embodiment of the present invention, the culture medium is based on a basal culture medium for cell culture. Such basal culture media include, but are not limited to, DMEM, MEM, F12, DMEM / F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc. Many of these basal culture media are commercially available and their compositions are well known. However, when used in the manufacturing method of the present invention, their composition may be appropriately changed depending on the cell type and cell conditions.

[0055] Cells containing graft-forming cells are seeded on a substrate. The graft-forming cells are not particularly limited as long as they are the cells described above as cells that can constitute a graft (for example, a sheet-like cell culture). The cells contain at least one type of graft-forming cell (for example, sheet-forming cells), but may contain two or more types of graft-forming cells, or may contain cells other than graft-forming cells. In another aspect of the present disclosure, at least one type of graft-forming cell contained in the cell population is a cardiomyocyte. In such an aspect, the cells may further contain vascular endothelial cells. That is, a graft containing cardiomyocytes and vascular endothelial cells as graft-forming cells can be mentioned. In another aspect of the present disclosure, fibroblasts, vascular endothelial cells, and / or mural cells, etc. are included as graft-forming cells. For example, the graft may contain about 30 to 70% cardiomyocytes, 0.1% to about 20% vascular endothelial cells, and about 1% to about 40% mural cells. In yet another aspect of the present disclosure, at least one type of graft-forming cell contained in the cell population is a mesenchymal stem cell. In such an aspect, the cells may further contain vascular endothelial cells.

[0056] The seeding density of the cells to be seeded is not particularly limited as long as it is a density that can form a graft. However, in a preferred aspect, the cell population is seeded at a density that reaches confluence or a density higher than that. In the present disclosure, the "density that reaches confluence" refers to a density at which, when the cells are seeded, the entire adhesion surface of the culture vessel is covered without gaps by the seeded cells. For example, it is a density at which the cells are expected to contact each other when seeded, a density at which contact inhibition occurs, or a density at which the growth of the cells is substantially stopped due to contact inhibition.

[0057] Non-limiting examples of the seeding density of the cell population are about 7.1×10 5 cells / cm 2 ~ about 3.0×10 6 cells / cm 2 , about 7.3×10 5 cells / cm 2 ~ about 2.8×10 6 cells / cm 2 , about 7.5×10 5 cells / cm 2 ~ about 2.5×106 pieces / cm 2 , about 7.5×10 5 pieces / cm 2 ~Approx. 3.0×10 6 pieces / cm 2 , about 7.8×10 5 pieces / cm 2 ~Approx. 2.3×10 6 pieces / cm 2 , about 8.0×10 5 pieces / cm 2 ~Approx. 2.0×10 6 pieces / cm 2 , about 8.5×10 5 pieces / cm 2 ~Approx. 1.8×10 6 pieces / cm 2 , about 9.0×10 5 pieces / cm 2 ~Approx. 1.6×10 6 pieces / cm 2 This includes densities such as those mentioned above. Unless otherwise specified, these densities refer to the total density of all cells contained within the cell population.

[0058] In yet another embodiment, seeding can be carried out in a cell culture medium substantially free of growth factors at a density in which at least one type of graft-forming cell that may be present in the cell population is substantially inhibited from proliferating. In such an embodiment, other cells that may be present in the cell population may be present at a density in which they can proliferate, albeit with inhibited growth. The substrate used in the method of the present disclosure is as described above. In one preferred embodiment, the substrate may be coated with serum. In another preferred embodiment, the substrate may be coated with a temperature-responsive material. In a further preferred embodiment, the substrate may be coated with a temperature-responsive material and serum.

[0059] <Kit> In another aspect of the present invention, the present invention relates to a kit for producing grafts, comprising some or all of the elements used in the production of grafts, particularly in the production of grafts without proliferation culture. The kit of the present invention may, without limitation, include, for example, materials for forming a gel used in the step of forming a gel layer, such as a solution containing fibrin and a solution containing thrombin, as well as cells constituting the graft (e.g., cryopreserved cells, cells recovered by the recovery method of the present invention, etc.), culture medium, culture dish, equipment (e.g., pipette, dropper, forceps, etc.), instructions on how to manufacture the graft (e.g., instructions for use, a medium recording information on the manufacturing method and the method for recovering cryopreserved cells of the present invention, such as a flexible disk, CD, DVD, Blu-ray disc, memory card, USB memory, etc.).

[0060] <Highly active grafts> Another aspect of the present invention relates to grafts obtained by the method of the present invention, or grafts obtained by a method for producing grafts that includes the method of the present invention. In one aspect of the present invention, a graft produced by the method for producing the present disclosure comprises cardiomyocytes. In another aspect of the present invention, a graft produced by the method for producing the present disclosure comprises cardiomyocytes, fibroblasts, endothelial cells and / or parietal cells. The grafts obtained by the manufacturing method of the present invention are characterized by higher activity than control grafts. Details regarding the higher activity are as described above. In one embodiment, the grafts of the present invention have higher activity than control grafts, selected from the group consisting of cytokine production ability, proliferation ability, engraftment ability, angiogenesis ability, and tissue regeneration ability. In another embodiment, the grafts have higher cytokine production ability than control grafts, selected from the group consisting of HGF, SDF-1, FGF, SCF, and VEGF. Because the grafts have high activity, they show superior effects compared to control grafts in various therapeutic applications. In particular, high HGF or VEGF production ability promotes tissue engraftment, angiogenesis, and tissue regeneration, and high proliferation ability allows the grafts to function sustainably for a long period of time, thereby enhancing the therapeutic effect. In one embodiment of the present invention, the grafts obtained by the manufacturing method of the present invention are grafts having a scaffold, and are less prone to twisting and / or shrinking compared to grafts without a scaffold. Therefore, grafts with scaffolds not only improve long-term and sustained therapeutic effects compared to control grafts without scaffolds in terms of tissue engraftment, vascular induction, and tissue regeneration promotion, but they also offer significantly greater convenience because they are stronger, easier to handle, easier to apply to the affected area, and less variation in operation depending on the skill level of the practitioner, thus enabling reliable treatment of the disease.

[0061] <Methods for treating diseases> Another aspect of this disclosure relates to a method for treating a disease in a subject, which includes applying an effective amount of a graft obtained by the method of this disclosure or by a method for producing a graft including the method to the subject in need. The diseases to be treated are as described above.

[0062] In this disclosure, the term “treatment” encompasses all medically acceptable types of prophylactic and / or therapeutic interventions aimed at curing, temporarily relieving, or preventing a disease. For example, the term “treatment” encompasses a variety of medically acceptable interventions for various purposes, including delaying or halting the progression of a disease associated with tissue abnormalities, regression or disappearance of lesions, and prevention of the onset or recurrence of such disease.

[0063] In the treatment method of this disclosure, components that enhance the viability, engraftment, and / or function of the graft, or other active ingredients useful for treating the target disease, can be used in combination with the graft of this disclosure.

[0064] The processing method of the present disclosure may further include a graft whose activity has been enhanced according to the method of the present disclosure. The processing method of the present disclosure may further include, before the step of producing a graft, a step of collecting cells (for example, skin cells, blood cells, etc., if iPS cells are used) or tissue (for example, skin tissue, blood, etc., if iPS cells are used) from a subject for producing a graft. In one embodiment, the subject from which cells or tissues that will be the source of cells are collected is the same individual as the subject receiving the cell culture, composition, or graft. In another embodiment, the subject from which cells or tissues that will be the source of cells are collected is a different individual of the same species as the subject receiving the cell culture, composition, or graft. In another embodiment, the subject from which cells or tissues that will be the source of cells are collected is a different individual of the same species as the subject receiving the cell culture, composition, or graft.

[0065] In this disclosure, an effective dose is, for example, an amount that can suppress the onset or recurrence of a disease, alleviate symptoms, or delay or halt its progression (e.g., the size, weight, or number of sheet-like cell cultures), and preferably an amount that prevents the onset and recurrence of the disease or cures the disease. It is also preferable that the dose does not cause adverse effects that outweigh the benefits of administration. Such a dose can be appropriately determined, for example, by testing in experimental animals such as mice, rats, dogs, or pigs, or in disease model animals, and such testing methods are well known to those skilled in the art. The size of the tissue lesion to be treated can also be an important indicator for determining the effective dose.

[0066] Methods of administration include, for example, intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissue. The frequency of administration is typically once per treatment, but if the desired effect is not obtained, multiple administrations may be performed. When applying to tissue, the cell culture, composition, or graft of the present invention may be fixed to the target tissue by fastening means such as sutures or staples. [Examples]

[0067] The present invention will be described in more detail with reference to the following examples, which are specific examples of the present invention and are not limited thereto.

[0068] In the following examples, clinical-grade human iPS cells established at the Center for iPS Cell Research and Application (CiRA) of Kyoto University were used as pluripotent stem cells. Human iPS cells were maintained using a feeder-free method, following the instructions in M. Nakagawa et al., Scientific Reports, 4:3594 (2014). Then, Miki et al., Ce Based on the descriptions in Stem Cell 16, 699-711, June 4, 2015, WO2014 / 185358, and WO2017 / 038562, we differentiated human iPS cells into cardiomyocytes and obtained embryoid bodies. Specifically, human iPS cells maintained in a culture medium without feeder cells were cultured for 1 day on an EZ Sphere (Asahi Glass) in StemFit AK03 medium (Ajinomoto) containing 10 μM Y27632 (Wako Pure Chemical Industries). The resulting embryoid bodies were cultured in a culture medium containing activin A, bone morphogenetic protein (BMP) 4, and basic fibroblast growth factor (bFGF). Further cultures were performed in a culture medium containing a Wnt inhibitor (IWP3), a BMP4 inhibitor (Dorsomorphin), and a TGFβ inhibitor (SB431542). Subsequently, cultures were performed in a culture medium containing VEGF and bFGF to obtain iPS cell-derived human cardiomyocytes. The proportion of cardiomyocytes in the obtained cells was 50% to 90%.

[0069] Comparative Example 1: Production of Sheet-like Cell Cultures Cardiomyocytes cryopreserved in cell freezing solution (MCDB medium containing 10% DMSO) were thawed at 37°C and washed twice with physiological buffer containing 0.5% serum albumin. Washed cells: 6.0 × 10⁶ 7 The cells were suspended in 10 mL of DMEM medium containing 20% ​​human serum and seeded in a 10 cm diameter cell culture dish (UpCell(R) 10 cm dish, CS3005, manufactured by CellSeed Co., Ltd.). After seeding, the cells were cultured for 20 hours in an incubator (BNA-121D, manufactured by ESPEC Corporation) set to 37°C and 5% CO2. After culturing, the culture dish was removed from the incubator, and it was confirmed that the sheet-like cell culture adhered to the entire bottom surface of the culture dish, and the medium was discarded. Subsequently, the sheet-like cell culture was isolated from the culture dish by temperature treatment (standing at room temperature (20-25°C) for 5-30 minutes) and pipetting. The obtained sheet-like cell culture measured 47 mm × 47 mm.

[0070] Comparative Example 2: Measurement of cell count and cytokine production in sheet-like cell cultures The sheet-like cell cultures prepared in Comparative Example 1 were stored in Hanks equilibrium salt solution and DMEM medium containing 10% human serum at 4°C and 25°C for 72 hours. The change in the number of cardiomyocytes constituting the sheet-like cell cultures was confirmed using Cell Counting Kit 8 (Dojin Chemical), and the survival rate (%) was calculated as {after storage / before storage (absorbance at 450 nm)} × 100. Figure 1 shows the change in the survival rate over time. The results shown in Figure 1 clearly indicate that the survival rate of sheet-like cell cultures decreases over time. The survival rate was highest when stored in Hanks equilibrium salt solution at 4°C. Shrinkage occurred when stored at 37°C for 72 hours.

[0071] Sheet cell cultures prepared in Comparative Example 1 were stored in Hanks equilibrium salt solution and DMEM medium containing 10% human serum at 4°C and 25°C for 96 hours. The concentration of cytokines (VEGF) produced in the culture supernatant was measured by ELISA (n=3). The measurement was performed using the Human VEGF Quantikine ELISA Kit (R&D systems, catalog number DVE00) according to the manufacturer's manual. As shown in Figure 2, the amount of VEGF produced decreased over time. The decrease in VEGF production was suppressed by storage at 25°C compared to 4°C.

[0072] Comparative Example 3: Production of Sheet-like Cell Cultures Cardiomyocytes cryopreserved in cell freezing solution (MCDB medium containing 10% DMSO) were thawed at 37°C and washed twice with physiological buffer containing 0.5% serum albumin. Washed cells: 4.0 × 10⁶ 6 The cells are suspended in 2 mL of DMEM medium containing 20% ​​human serum, and then placed in a 12-well temperature-responsive substrate (UpCell). (R)The cells were seeded in a 12-well CS3003 (Cellseed Co., Ltd.) container. After seeding, the cells were cultured for 20 hours in an incubator (BNA-121D, ESPEC Corporation) set to 37°C and 5% CO2. After culturing, the culture dish was removed from the incubator, and it was confirmed that the sheet-like cell culture adhered to the entire bottom surface of the culture dish, and the culture medium was discarded. Subsequently, the sheet-like cell culture was isolated from the culture dish by temperature treatment (standing at room temperature (20-25°C) for 5-30 minutes) and pipetting.

[0073] Example 1: Production of a sheet-like cell culture with a gel layer formed. A sheet-like cell culture was obtained using the same procedure as in Comparative Example 1. The culture medium was removed from the culture dish, and fibrinogen solution (Volheal) was placed on top of the sheet-like cell culture. (R) Dissolve the contents of vial 1 (fibrinogen lyophilized powder) of tissue adhesion product (manufactured by Teijin Pharma) in the contents of vial 2 (fibrinogen dissolving solution, fibrinogen concentration 80 mg / mL, the same applies below) in 500 μL of Volheal. (R) The two-component mixing kit (approximately 6 cm long, with an apply nozzle approximately 1 mm in inner diameter, manufactured by Nipro Corporation) included with the tissue adhesion preparation kit was used to drop the mixture. Next, thrombin solution (Volheal) was added. (R) Dissolve the contents of vial 3 (thrombin freeze-dried powder) of tissue adhesion product (manufactured by Teijin Pharma) in the contents of vial 4 (thrombin dissolving solution, thrombin concentration 250 units / mL, the same applies below) in 800 μL of Volheal. (R) A spray set (manufactured by Akita Sumitomo Bakelite Co., Ltd.) was used, and the spray nozzle was positioned approximately 7 cm away from the cell sheet at a pressure of 0.03 MPa.

[0074] A fibrin gel is formed by the reaction of fibrinogen solution and thrombin solution. After standing for approximately 5 minutes, 24 mL of Hanks equilibrium salt solution (HBSS(+), Cat No. 14025, Life Technologies, Inc., hereafter the same) was added to the culture dish and immediately removed to wash the culture dish containing the sheet-like cell culture. This removes unreacted fibrinogen solution and thrombin solution. Next, 24 mL of Hanks equilibrium salt solution was added to the culture dish again and stood for approximately 15 minutes. After removing the solution from the culture dish, the fibrin gel that had solidified outside the sheet-like cell culture was trimmed with a scalpel, and the sheet-like cell culture with the fibrin gel layer was isolated. A photograph of the sheet-like cell culture after isolation is shown in Figure 3(A).

[0075] Example 2: Preparation of a sheet-like cell culture in which the intercellular spaces are filled with gel. Human cardiomyocytes derived from iPS cells, cryopreserved in cell preservation solution (MCDB medium containing 10% DMSO), were thawed at 37°C, diluted with 10% FBS / DMEM, centrifuged, and the supernatant was removed. The cells were then divided into 2 × 10⁶ cells. 6 cells / cm 2 The cells were suspended at a density in 15 mL of DMEM medium containing 20% ​​human serum and seeded onto a 6 cm diameter temperature-responsive substrate (UpCell®, 6 cm dish, CS3006, CellSeed Co., Ltd.). After seeding, the cell population was cultured for 72 hours in an incubator (BNA-121D, ESPEC Corporation) set to 37°C and 5% CO2. After culturing, the substrate was removed from the incubator, it was confirmed that the cells were adhering to the substrate, and the culture medium was discarded. After discarding the culture medium, 500 μl of fibrinogen solution (fibrinogen lyophilized powder from vial 1 of Beriplast® Tissue Adhesion (manufactured by CSL Behring) dissolved in the contents of vial 2 (fibrinogen dissolving solution), fibrinogen concentration 80 mg / mL) and 500 μl of thrombin solution (thrombin lyophilized powder from vial 3 of Beriplast® Tissue Adhesion (manufactured by CSL Behring) dissolved in the contents of vial 4 (thrombin dissolving solution), thrombin concentration 300 units / mL) were dropped onto the substrate and allowed to stand for approximately 5 minutes to form a fibrin gel. After fibrin gel formation, 10 mL of Hanks equilibrium salt solution (HBSS(+), Cat No. 14025, Life Technologies) was added, and the mixture was washed three times to remove unreacted fibrinogen and thrombin. Subsequently, the mixture was allowed to stand at room temperature (20-25°C) for 5-30 minutes for temperature treatment of the temperature-responsive material. The sheet-like material was then detached from the substrate by pipetting, and the sheet-like cell culture, in which the intercellular spaces were filled with gel, was isolated. A photograph of the sheet-like cell culture before isolation is shown in Figure 3(B).

[0076] Example 3: Measurement of cell count and cytokine production in sheet-like cell cultures The number of cells constituting the sheet-like cell cultures prepared in Examples 1 and 2 was measured immediately after isolation (day 0) and after incubation (storage) in DMEM medium containing 10% human serum at 4°C, 25°C, 30°C, 35°C, 37°C, and 39°C for 72 hours, using absorbance at 450 nm. The results of the sheet-like cell culture prepared in Example 1 are shown in Figure 4. In the sheet-like cell culture where a fibrin gel layer was formed, a decrease in cell number was observed when incubated at 4°C, but there was almost no decrease at 25°C, and a significant increase in cell number was observed at 30°C and above. Similar results were obtained for the sheet-like cell culture prepared in Example 2.

[0077] The sheet-like cell cultures prepared in Examples 1 and 2 were stored immediately after isolation and in DMEM medium containing 10% human serum at 25°C, 30°C, 35°C, 37°C, and 39°C for 72 hours. Cytokine production was measured using the same procedure as in Comparative Example 2. The results for the sheet-like cell culture prepared in Example 1 are shown in Figure 5. Adding the step of incubating the sheet-like cell culture prepared in Example 1 at 25°C suppressed the decrease in cytokine production of the sheet-like cell culture, similar to Comparative Example 2. Furthermore, it was found that storing the culture at 30°C or higher significantly enhanced cytokine production. Similar results were obtained with the sheet-like cell culture prepared in Example 2.

[0078] Example 4: Preparation of a sheet-like cell culture in which the intercellular spaces are filled with gel. Human cardiomyocytes derived from iPS cells, cryopreserved in cell preservation solution (MCDB medium containing 10% DMSO), were thawed at 37°C, diluted with 10% FBS / DMEM, centrifuged, and the supernatant was removed. The cells were then divided into 2 × 10⁶ cells. 6 cells / cm 2 The cells were suspended at a density in 3 mL of 20% human serum-containing DMEM medium and seeded onto a 12-well temperature-responsive substrate (UpCell(R) 12Well, CS3003, CellSeed Co., Ltd.). After seeding, the cell population was cultured for 72 hours in an incubator (BNA-121D, ESPEC Corporation) set to 37°C and 5% CO2. After culturing, the substrate was removed from the incubator, it was confirmed that the cells were adhering to the substrate, and the culture medium was discarded. After discarding the culture medium, 100 μl of fibrinogen solution (fibrinogen lyophilized powder from vial 1 of Beriplast® Tissue Adhesion (manufactured by CSL Behring) dissolved in the contents of vial 2 (fibrinogen dissolving solution), fibrinogen concentration 80 mg / mL) and 100 μl of thrombin solution (thrombin lyophilized powder from vial 3 of Beriplast® Tissue Adhesion (manufactured by CSL Behring) dissolved in the contents of vial 4 (thrombin dissolving solution), thrombin concentration 300 units / mL) were dropped onto the substrate and allowed to stand for approximately 5 minutes to form a fibrin gel. After fibrin gel formation, 2 mL of Hanks equilibrium salt solution (HBSS(+), Cat No. 14025, Life Technologies) was added, and the mixture was washed three times to remove unreacted fibrinogen and thrombin. Subsequently, the mixture was allowed to stand at room temperature (20-25°C) for 5-30 minutes for temperature treatment of the temperature-responsive material. The sheet-like material was then detached from the substrate by pipetting, and the sheet-like cell culture with intercellular spaces filled with gel was isolated.

[0079] Example 5: Morphological changes in sheet-like cell cultures Figure 6 shows photographs of the sheet-like cell culture prepared in Comparative Example 3 and the sheet-like cell culture with gel-filled gaps prepared in Example 4, stored at 4°C and 25°C. Figure 6 shows photographs of the sheet-like cell culture prepared in Comparative Example 3 at 4°C (A) and 25°C (B), and the sheet-like cell culture with gel-filled gaps prepared in Example 4 at 4°C (C) and 25°C (D). It can be seen that the sheet-like cell culture prepared in Example 4 does not twist or shrink even when stored at 25°C (D). Therefore, it has become clear that grafts with scaffolds can enhance cytokine production while maintaining their morphology.

[0080] The various features of the present invention described herein can be combined in various ways, and all embodiments resulting from such combinations, including combinations not specifically described herein, are within the scope of the present invention. Furthermore, those skilled in the art will understand that numerous variations are possible without departing from the spirit of the invention. Accordingly, it should be understood that the embodiments described herein are merely illustrative and are not intended to limit the scope of the present invention.

Claims

1. A method for enhancing the activity of a sheet-like cell culture containing iPS cell-derived cardiomyocytes, selected from the group consisting of cytokine production ability and proliferation ability, Cells containing iPS cell-derived cardiomyocytes and sheet-forming cells, 7.1 × 10 ５ pieces / cm ２ ~3.0 x 10 ６ pieces / cm ２ The steps include: seeding at a seeding density onto a culture substrate to provide a sheet-like cell culture containing iPS cell-derived cardiomyocytes onto the culture substrate; The steps include: detaching the sheet-like cell culture from the culture substrate, and The method comprising the step of detaching the sheet-like cell culture from the culture substrate and then incubating it in DMEM / serum medium at 25°C for 1 or 3 days, or at 25°C to 39°C for 72 hours.

2. The method according to claim 1, wherein the sheet-like cell culture has a scaffold.

3. The method according to claim 2, wherein the scaffold is a gel comprising fibrin, gelatin, or collagen.

4. A method for producing a sheet-like cell culture, comprising the method according to any one of claims 1 to 3.

Images