JP7355577B2 - Method for producing sheet-like material containing differentiation-induced cells derived from pluripotent stem cells - Google Patents

Description

The present invention relates to a method for producing a sheet containing differentiation-induced cells derived from pluripotent stem cells, a sheet produced by the method, a kit for producing the sheet, and a sheet Concerning how to apply things.

In recent years, attempts have been made to transplant various cells to repair damaged tissues. For example, use of fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. to repair myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction. has been attempted (Non-Patent Document 1).

Examples of such attempts include cell structures formed using scaffolds (Patent Document 1) and sheet-shaped cell cultures in which cells are formed into sheets (Non-Patent Document 2) for the purpose of transplantation. cell preparations have been developed, some of which have entered the stage of clinical application.
Gels have been used for various purposes in the production of such cell preparations. For example, a gel layer is layered on a sheet-shaped cell culture to improve operability (Patent Document 2), and a cell culture layer is prepared by embedding cardiomyocytes, smooth muscles, and fibroblasts in a hydrogel and then culturing the cells. Cardiopatch (Non-Patent Document 3), etc.

Patent No. 4943844 JP 2016-52272 Publication

Haraguchi et al., Stem Cells Trans1 Med. 2012 Feb;1(2):136-41 Sawa et al., Surg Today. 2012 Jan;42(2):181-4 Ilya Y. Shadrin et al., Nature Communications 8: 1825, 2017

The present invention relates to a method for producing a sheet containing differentiation-induced cells derived from pluripotent stem cells, a sheet produced by the method, a kit for producing the sheet, and a sheet. The purpose was to provide a method for applying objects.

When producing a sheet-shaped cell culture, cells are frozen and thawed, but these operations may reduce the adhesion ability of cells. In addition to these, cell adhesion ability may decrease for unknown reasons. When cells with low adhesion ability are used in the conventional method of producing sheet-like cell cultures, the cells tend to form aggregates or detach, resulting in the cells not being evenly distributed between cells. Cell-cell adhesion is not formed and sheet-like cell cultures cannot be obtained.
The present inventors discovered that a cell-containing sheet can be produced even from cells with low adhesion ability, and based on this knowledge, as a result of further research, the present invention was completed. I ended up letting it happen.

That is, the present invention relates to the following.
[1] A method for producing a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, comprising the following steps:
disposing the cells on the substrate with a gap therebetween;
filling the gaps between the cells with gel to form a sheet;
The method described above.
[2] The method according to [1], further comprising the step of adhering the cells to the substrate.
[3] The method according to [1] or [2], wherein the cells are in the form of cell clusters.
[4] The method according to any one of [1] to [3], wherein the pluripotent stem cells are human iPS cells.
[5] The method according to any one of [1] to [4], wherein the gel contains fibrin gel, gelatin, or collagen.
[6] The method according to any one of [1] to [5], wherein the gel is gelled by mixing two liquids.
[7] The method according to any one of [1] to [6], wherein the substrate is a substrate containing a stimulus-responsive material.
[8] A sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, produced by the method described in any one of [1] to [7].
[9] A kit for producing a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells according to [8],
The above-described kit includes differentiation-induced cells derived from pluripotent stem cells, a substrate for placing the cells, and a gel for forming the cells into a sheet.
[10] A method for treating a disease that is improved by applying a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, the method comprising: The method described above includes applying the produced sheet-like product to a subject in need of it.

According to the method of the present invention, a sheet-like material can be obtained even from cells with low adhesive ability. In Cardiopatch described in Non-Patent Document 2, cells are embedded inside the patch, but in the sheet-like product according to the present invention, cells are placed on a base material and then a sheet is formed with gel. As a result, cells are localized on one side of the sheet-like material and exposed on that side. Therefore, in the sheet-like material according to the present invention, the cells can take in oxygen, nutrients, etc. from the exposed surface, and the cytokines produced by those cells are not hindered by the gel, and the cells can take in oxygen and nutrients from the exposed surface. It can act directly and/or indirectly on cells surrounding the site of application.
In addition, unlike conventional sheet-like cell cultures, the sheet-like material according to the present invention does not shrink after peeling, so a sheet-like material of the same size as the substrate used for production can be obtained, and a sheet-like material of the same size can be obtained. Larger sheets are obtained than sheet-like cell cultures made with substrates.

FIG. 1 is a photograph of the sheet-like material 1. FIG. 2 is a photograph of a laminate of fibrin gel and sheet-like cell culture. FIG. 3 is a cross-sectional view of the sheet-like material 1. As shown in FIG. Cells are localized in the area surrounded by the broken line, and gel is present in the area surrounded by the dashed-dotted line. FIG. 4 is a cross-sectional view of the sheet-like material 2. As shown in FIG.

The present invention will be explained in detail below.
One aspect of the present invention is a method for producing a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, which includes a step of arranging cells on a substrate with gaps between the cells. The present invention relates to a method including the step of filling gaps with gel to form a sheet.

In the present invention, the term "sheet-like material" refers to a material in which cells are formed into a sheet-like shape. In one embodiment of the present invention, the sheet-like material contains cells and gel, and preferably does not contain any scaffold other than gel. Scaffolds may be used in the art to attach cells onto and/or within their surfaces and maintain the physical integrity of the sheet, such as polyvinylidene difluoride (PVDF). Membranes made from the above are known. The sheet-like article of the present invention can maintain its physical integrity even without such a scaffold.

In the present invention, "arranging cells on a substrate with gaps between them" refers to a state in which the cells are in contact with or adhered to the bottom surface of the substrate with gaps between the cells. refers to doing. It is only necessary that the cells have a gap in some part, and this does not mean that the cells are not in contact with each other. In the present invention, the state in which cells are arranged on a substrate with gaps between them means, for example, that a plurality of island-shaped cell populations exist on the substrate, and there are gaps between the island-shaped cell populations. It also includes the state of being. In the present invention, a state where the entire surface of the base material is continuously covered with cells does not include a state where the cells are arranged on the base material with gaps between them.
In one embodiment, the cells are placed on the substrate by allowing the cells to settle naturally after seeding the cells. Natural sedimentation is carried out, for example, by allowing the mixture to stand for 1 minute to 24 hours, preferably for 5 minutes to 6 hours, and more preferably for 5 minutes to 1 hour.
In one embodiment, the cells are placed on the substrate by centrifugation after seeding the cells. Centrifugation is performed using a centrifugal separator by appropriately setting centrifugal force, centrifugation time, and internal temperature of the centrifugal separator. The centrifugal force is set to, for example, 10G to 1000G, preferably 50G to 1000G, more preferably 50G to 400G. The time for centrifugation is set to, for example, 10 seconds to 2 hours, preferably 1 minute to 60 minutes, more preferably 1 to 15 minutes, and still more preferably 1 minute to 10 minutes. The temperature inside the machine is set to, for example, 0°C to 42°C, preferably 4°C to 37°C.

In one embodiment, the disposed cells are in contact with the substrate. In the present invention, the state in which the cells are in contact with the base material refers to the state in which the cells are in contact with the base material, but the cells and the base material are bonded via the extracellular matrix produced by the cells. Refers to the state where it is not done.
In the present invention, the contact between the cells and the substrate depends on various conditions, for example, after seeding the cells, for 5 minutes to 24 hours, for example, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, Approximately 40 minutes, approximately 50 minutes, approximately 1 hour, approximately 2 hours, approximately 3 hours, approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 This occurs by culturing for about 12 hours, about 18 hours, or about 24 hours.
In another embodiment, the disposed cells are adherent to the substrate. In the present invention, the state in which cells are adhered to a substrate refers to a state in which the cells and the substrate are bonded via an extracellular matrix produced by the cells.
The method of the invention, in one embodiment, further includes the step of adhering the cells to the substrate.
In the present invention, adhesion between cells and the substrate depends on various conditions, but for example, after seeding the cells, for 24 hours or more, for example, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, It is produced by culturing for 60 hours, 66 hours, or 72 hours.

The medium for use in culture may be any known medium used for culturing cells. Examples of such a medium 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.

In embodiments of the invention, the cells are in the form of cell clusters. In the present invention, the term "cell cluster" refers to two or more cells closely adhered to form a cluster. In the present invention, the size of the cell cluster is, for example, 10 μm to 1000 μm in diameter. Preferably, the diameter is 50 μm to 500 μm, more preferably 100 μm to 300 μm. In one embodiment, the size of the cell clusters may not be uniform. In the present invention, when the size of the cell cluster is uniform, for example, the relative standard deviation of the individual size of the cell cluster measured by a cell analyzer or the like is less than 40%, preferably less than 30%, more preferably is less than 20%, and the expression that the size of the cell cluster is not uniform means that the relative standard deviation is 60% or more, preferably 50% or more, and more preferably 45% or more. Cell clusters are also called spheroids.
In one aspect of the present invention, there is provided a method for producing a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, comprising: a step of arranging a cell mass on a base material with a gap therebetween; filling the gaps between cell clusters with gel to form a sheet;
The method includes:
In one embodiment, the method according to the present invention further includes the step of culturing the cells to form a cell mass before the step of disposing the cell mass on the substrate with a gap therebetween.
Any known method can be used to form cell clusters, but is not limited thereto. For example, the inner surface is coated with a cell adhesion inhibitor and a plurality of depressions are formed on the surface of the base material. There is a method (Japanese Patent Application No. 2012-533946) in which culture is performed using a substrate having an uneven surface between the depressions.
When cell clusters are seeded, the thickness of the cell portion seen from the side varies from a minimum of 10 μm to a maximum of 100 μm, but this difference is filled by the gel and formed into a sheet. Therefore, the thickness of the sheet itself is uniform regardless of the thickness of the cell portion, and even when formed from cell aggregates, the sheet-like material can be easily handled.

In the present invention, the cells contained in the sheet-like material are not particularly limited as long as they are cells induced to differentiate from pluripotent stem cells and can form a sheet-like material. cells). Adherent cells include, for example, adherent somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal gland cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (for example, myoblasts, tissue stem cells such as cardiac stem cells, mesenchymal stem cells, etc.). The somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells). Non-limiting examples of cells that can form sheet-like materials include iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal gland cells, periodontal ligament cells, Examples include gingival cells, periosteal cells, skin cells, synovial cells, and cartilage cells. Among these, cardiomyocytes, whose recovery rate and survival rate can directly affect the quality of the graft, are preferred.

In the present invention, "pluripotent stem cell" is a term well known in the art and has the ability to differentiate into cells of all lineages belonging to the three germ layers: endoderm, mesoderm and ectoderm. means cell. Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Induced pluripotent stem cells (iPS cells) are cells induced by introducing genes. Normally, when inducing differentiation of pluripotent stem cells into specific cells, the pluripotent stem cells are first cultured in suspension to form aggregates of cells from any of the three germ layers, and then the cells that form the aggregates are induce differentiation into specific cells of interest.

In the present invention, a "differentiation-induced cell derived from a pluripotent stem cell" means any cell that has been subjected to differentiation-inducing treatment so that it differentiates into a specific type of cell from a pluripotent stem cell. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous system cells such as neuron cells, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. cells, hematopoietic cells such as 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 kidney cells; In addition to chondrocytes and reproductive cells, it includes progenitor 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 cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in nervous system cells, hematopoietic system cells, and immune cells. Related cells include hematopoietic stem cells and lymphoid stem cells. Induction of differentiation of pluripotent stem cells can be performed using any known method. When using differentiation-induced cells derived from pluripotent stem cells such as iPS-derived cardiomyocytes as the desired cells, undifferentiated cells may be removed after differentiation induction. Treatment for removing undifferentiated cells is known in the art, and methods described in WO2017/038562, WO2016/072519, WO2007/088874, etc. can be used, for example.

Further, the differentiation-induced cells may be cells derived from iPS cells into which any useful genes other than genes for reprogramming have 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. Furthermore, cells into which any useful gene has been introduced after being induced to differentiate from pluripotent stem cells are also included in the differentiation-induced cells of the present invention.
In one embodiment, the differentiation-induced cells derived from pluripotent stem cells are cardiomyocytes derived from iPS cells. iPS cell-derived cardiomyocytes can be obtained by inducing cardiomyocytes from iPS cells, that is, by subjecting iPS cells to cardiomyocyte induction treatment. Various techniques for inducing cardiomyocytes from iPS cells are known (eg, Burridge et al., Cell Stem Cell. 2012 Jan 6;10(1):16-28). Non-limiting examples of such induction methods include, for example, a method using embryoid body formation, a method using monolayer differentiation culture, and a method using forced aggregation. In one embodiment, the sheet material containing cardiomyocytes derived from iPS cells of the present invention may contain vascular endothelial cells, parietal cells, fibroblasts, etc. in addition to cardiomyocytes. The composition ratio of cells contained in the sheet-like material of the present invention is, for example, about 30 to 70% myocardial cells, 0.1% to about 20% vascular endothelial cells, about 1% to about 40% parietal cells, and fibroblasts. It may be about 1% to about 40% of the cells. Cardiomyocytes derived from iPS cells can be purified after induction to increase purity. Purification methods include various separation methods using markers specific to cardiomyocytes (e.g., cell surface markers), such as magnetic cell separation (MACS), flow cytometry, affinity separation, and specific Examples include a method of expressing a selectable marker (for example, an antibiotic resistance gene, etc.) using a promoter, and a combination of these methods (see, for example, Burridge et al. mentioned above). Examples of cell surface markers specific to cardiomyocytes include CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM, and the like. Furthermore, examples of promoters specific to cardiomyocytes include NKX2-5, MYH6, MLC2V, and ISL1.

The cells constituting the sheet-like material can be derived from any organism, including, but not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, Includes sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, and the like. Further, the number of types of cells contained in the sheet-like material is not particularly limited, and the sheet may contain only one type of cells, or may contain two or more types of cells. When there are two or more types of cells contained in the sheet-like article, the content ratio (purity) of the largest number of cells is 60% or more, preferably 70% or more, more preferably 75% at the end of sheet-like article production. That's all.

The cells may be of xenogeneic origin or allogeneic origin. Here, "xeno-derived cells" means cells derived from a different species of organism than the recipient when the cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. Moreover, "cells derived from the same species" means cells derived from the same species of organism as the recipient. For example, when the recipient is a human, human cells correspond to allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), that is, cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferable from the viewpoint of clinical application because they do not cause rejection reactions even when transplanted. However, it is also possible to utilize cells of xenogeneic origin or allogeneic non-autologous cells. When using xenogeneic cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection reactions. In this specification, cells other than autologous cells, that is, xenogeneic cells and allogeneic non-autologous cells may be collectively referred to as non-autologous cells. In one embodiment, the cells are autologous or allogeneic. In one embodiment, the cells are autologous cells. In another embodiment, the cell is an allogeneic cell.

In one embodiment, the seeding to obtain the cells provided in the present invention is about 1.0×10 ⁵ cells/cm ² to about 5.0×10 ⁷ cells/cm ² , about 3.0×10 ⁵ cells/cm 2 . /cm ² to about 5.0×10 ⁷ pieces/cm ² , about 3.5×10 ⁵ pieces/cm ² to about 5.0×10 ⁷ pieces/cm ² , about 1.0×10 ⁶ pieces/cm ² to about 5.0×10 ⁷ pieces/cm ² , about 1.0×10 ⁵ pieces/cm ² to about 3.4×10 ⁶ pieces/cm ² , about 3.0×10 ⁵ pieces/cm ² to Approximately 3.4×10 ⁶ pieces/cm ² , about 3.5×10 ⁵ pieces/cm ² to about 3.4×10 ⁶ pieces/cm ² , about 1.0×10 ⁶ pieces/cm ² to about 3 .4×10 ⁶ pieces/cm ² , about 3.0×10 ⁵ pieces/cm ² to about 1.7×10 ⁶ pieces/cm ² , about 3.5×10 ⁵ pieces/cm ² to about 1.7 It can be carried out at a density of x10 ⁶ pieces/cm ² , about 1.0 x 10 ⁶ pieces/cm ² to about 1.7 x 10 ⁶ pieces/cm ² .

In the present invention, any gel can be used to form the sheet-like material as long as it does not have an adverse effect on living organisms. Examples of gels that do not adversely affect living bodies include, but are not limited to, fibrin gel, gelatin, collagen, and sodium alginate. In one embodiment, the gel is a biodegradable gel that is degraded in vivo, absorbed into the body, metabolized, and excreted.
In one embodiment, the gel is one in which gelation occurs by mixing two liquids. In one embodiment, the gel is in a solidified state near room temperature. In one embodiment, the gel does not exhibit temperature reversibility. Examples of such gels include, but are not limited to, fibrin gel made by mixing fibrinogen solution and thrombin solution, adhesion prevention agents made by mixing NHS-modified CM dextrin and trehalose aqueous solution, and sodium carbonate, sodium bicarbonate aqueous solution, etc. can be mentioned. These are commercially available as, for example, Volheal (registered trademark) for tissue adhesion (manufactured by Teijin Pharma), Veriplast (registered trademark) for tissue adhesion (manufactured by CSL Behring), and Adspray (registered trademark) (manufactured by Terumo Corporation). possible and can be used in the present invention. In the present invention, the gel is preferably fibrin gel, gelatin or collagen, more preferably fibrin gel.
In the present invention, the gel may serve as a support to prevent damage to the sheet when the sheet is peeled off from the substrate and when the sheet is moved for application.

In the present invention, the thickness of the sheet-like material is, for example, 10 μm to 2000 μm. Preferably, it is 10 μm to 500 μm, more preferably 50 μm to 200 μm.

In the present invention, the step of filling the intercellular spaces with gel to form a sheet is performed, for example, by gently adding the gel onto the substrate on which the cells are disposed. Although the amount added may be arbitrary, it is preferably an amount that gives the sheet-like material the above-mentioned thickness. In one embodiment, the gel is gelled by mixing two liquids, and any known method can be used for gelling using such a gel. Examples of such methods include a method in which fibrinogen solution and thrombin solution are added simultaneously, and a method in which a fibrinogen solution is dropped onto cells and then a thrombin solution is sprayed to form a fibrin gel (Japanese Patent No. 6495603). be able to.
In this step, the gaps (pores) that existed at the time of placement are filled with gel, and on one side of the sheet where cells are localized, the cells exist through the gel and continue to exist over the entire surface. Not yet.

In the present invention, the "substrate" is not particularly limited as long as cells can form a cell culture thereon, and includes, for example, containers made of various materials, solid or semi-solid surfaces in containers, etc. include. The container preferably has a structure and material that do not allow liquid such as culture solution to pass through. Such materials include, without limitation, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicone, polystyrene, glass, polyacrylamide, polydimethyl Examples include acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass), and the like. It is also preferred that the container has at least one flat surface. Examples of such containers include, without limitation, cell culture dishes, cell culture bottles, and the like. The container may also have a solid or semi-solid surface within it. Examples of solid surfaces include plates and containers made of the various materials listed above, and examples of semi-solid surfaces include gels, soft polymer matrices, and the like. The base material may be produced using the above-mentioned materials, or a commercially available material may be used. Preferred substrates include, without limitation, for example, substrates with adhesive surfaces suitable for forming sheet-like articles. Specifically, substrates having a hydrophilic surface, such as polystyrene treated with corona discharge, substrates coated with a hydrophilic compound such as collagen gel or a hydrophilic polymer, and furthermore, collagen, fibronectin, laminin, etc. Examples include substrates whose surfaces are coated with extracellular matrices such as vitronectin, proteoglycans, and glycosaminoglycans, and cell adhesion factors such as the cadherin family, selectin family, and integrin family. Additionally, such substrates are commercially available (eg, Corning™ TC-Treated Culture Dish, Corning, etc.).

The surface of the base material may be coated with a material whose physical properties change in response to a stimulus, such as temperature or light. Such materials include, but are not limited to, (meth)acrylamide compounds, N-alkyl-substituted (meth)acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide amide, etc.), N,N-dialkyl-substituted (meth)acrylamide derivatives (e.g., N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, N,N-diethylacrylamide, etc.), having a cyclic group ( meth)acrylamide derivatives (e.g. 1-(1-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) temperature-responsive materials consisting of homopolymers or copolymers of vinyl ether derivatives (e.g., methyl vinyl ether), light-absorbing polymers having azobenzene groups, vinyl derivatives of triphenylmethane leucohydroxide, and acrylamide monomers. Known materials such as photoresponsive materials such as copolymers with spirobenzopyran and N-isopropylacrylamide gels containing spirobenzopyran can be used (for example, see JP-A-2-211865 and JP-A-2003-33177). ). By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed and the detachment of cell cultures attached to the materials can be promoted. Culture dishes coated with temperature-responsive materials are commercially available (eg, UpCell® from Cellseed Corporation and Cepallet® from DIC Corporation) and can be used in the present invention.

The base material may have various shapes, but is preferably flat. Although the area is not particularly limited, it is typically about 1 cm ² to about 200 cm ² , preferably about 2 cm ² to about 100 cm ² , and more preferably about 3 cm ² to about 50 cm ² .

The substrate may be coated with serum. "Coated with serum" means that serum components are attached to the surface of the base material. Such a state can be obtained, for example, without limitation, by treating the substrate with serum. Treatment with serum includes contacting the substrate with serum and optionally incubating for a predetermined period of time.

As the serum, heterologous serum and homologous serum can be used. Heterologous serum means serum derived from an organism of a different species than the recipient when the sheet-like material is used for transplantation. For example, when the recipient is a human, serum derived from a cow or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), or horse serum (HS), corresponds to the xenogeneic serum. Moreover, "allogeneic serum" means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum (also referred to as autologous serum), that is, serum derived from the recipient, and allogeneic allogeneic serum derived from a conspecific individual other than the recipient. Note that herein, serum other than autologous serum, that is, xenogeneic serum and allogeneic allogeneic serum may be collectively referred to as non-autologous serum.

Serum for coating the substrate is commercially available, or can be prepared by conventional methods from blood collected from the desired organism. Specifically, for example, there is a method in which collected blood is allowed to stand at room temperature for about 20 minutes to about 60 minutes to coagulate, and then centrifuged at about 1000 G to about 1200 G to collect the supernatant. .

When incubating on the substrate, the serum may be used neat or diluted. Dilution can be performed in any medium such as, without limitation, water, saline, various buffers (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DME, RPMI1640, This can be done with MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM/F12, etc.). The dilution concentration is not particularly limited as long as the serum component can be deposited on the substrate, for example, about 0.5% to about 100% (v/v), preferably about 1% to about 60% (v/v). v), more preferably about 5% to about 40% (v/v).

The incubation time is also not particularly limited as long as the serum component can adhere to the substrate, for example, about 1 hour to about 72 hours, preferably about 4 hours to about 48 hours, more preferably about 5 hours to about 24 hours. time, more preferably about 6 hours to about 12 hours. The incubation temperature is also not particularly limited as long as the serum components can adhere to the substrate, for example, about 0°C to about 60°C, preferably about 4°C to about 45°C, more preferably room temperature to about 40°C. be.

The serum may be discarded after incubation. As a method for disposing of serum, conventional liquid disposal methods such as suction with a pipette or decantation can be used. In a preferred embodiment of the invention, after serum disposal, the substrate may be washed with a serum-free washing solution. The serum-free cleaning solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not have an adverse effect on serum components attached to the substrate, such as, without limitation, water, physiological saline, and various buffer solutions. (e.g., PBS, HBSS, etc.), various liquid media (e.g., DMEM, MEM, F12, DMEM/F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, etc.). The cleaning method may be a conventional substrate cleaning method, such as, but not limited to, a method in which a serum-free cleaning solution is added onto the substrate, stirred for a predetermined period of time (for example, about 5 seconds to about 60 seconds), and then discarded. Can be used.

In the present invention, the substrate may be coated with a growth factor. Here, "growth factor" means any substance that promotes cell proliferation compared to its absence, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor, etc. Contains cell growth factors (FGF), etc. The method of coating a substrate with a growth factor, the method of discarding the method, and the method of washing are such 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. It is basically the same as serum except that it is more preferably about 0.001 μg/mL to about 0.01 μg/mL.

In the present invention, the substrate may be coated with a steroid agent. The term "steroidal agent" as used herein refers to compounds having a steroid core that can have adverse effects on living bodies, such as adrenal cortex insufficiency and Cushing's syndrome. Such compounds include, without limitation, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone, and the like. The method of coating, discarding, and washing the substrate with a steroid agent is such 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 100 μg/mL, as dexamethasone. It is basically the same as serum except that it is 40 μg/mL, more preferably about 1 μg/mL to about 10 μg/mL.

The substrate may be coated with any one of serum, growth factors, and steroids, or any combination thereof, i.e., serum and growth factors, serum and steroids, serum, growth factors, and steroids, or , may be coated with a combination of growth factors and steroids. When coating with multiple components, these components may be mixed and coated simultaneously or may be coated in separate steps.

The substrate may be coated with serum or the like and then immediately seeded with cells, or it may be stored after coating and then seeded with cells. The coated substrate can be stored for a long period of time by keeping it at, for example, about 4°C or less, preferably about -20°C or less, more preferably about -80°C or less.

Cells can be seeded onto the substrate using any known method and conditions. The cells may be seeded onto the substrate, for example, by injecting a cell suspension in which cells are suspended in a culture solution into the substrate (culture container). For injecting the cell suspension, an instrument suitable for injecting the cell suspension, such as a dropper or pipette, can be used.

In one embodiment, the medium may be removed before adding the gel to the cells placed on the substrate. In further embodiments, after removing the medium, the cells adhered to the substrate may be washed with a washing solution. In one embodiment, any washing solution that does not disrupt adhesion between cells and substrate may be used. Examples of washing solutions that do not disrupt cell-substrate adhesion include, but are not limited to, Hank's balanced salt buffer, HEPES, DMEM, RPMI, and Ham's F-12.
In one aspect, the method for producing a sheet-like article according to the present invention can further include any known step for culturing cells. Examples of such steps include, but are not limited to, thawing frozen cells, adding a wash solution to the thawed cells, and centrifuging the cells with the wash solution. It will be done.

Another aspect of the present invention is a pluripotent stem cell-derived cell prepared by a method comprising the steps of: arranging cells on a substrate with gaps therebetween; and filling the gaps between the cells with a gel to form a sheet. The present invention relates to a sheet-like material containing differentiation-induced cells. In the present invention, the gel for forming the sheet-like material is preferably fibrin gel, gelatin, and collagen, and more preferably fibrin gel. In the present invention, the substrate on which the cells are placed is preferably a substrate comprising a stimuli-responsive material, more preferably a substrate comprising a temperature-responsive material. In one aspect of the invention, the cells are in the form of cell clusters.

Another aspect of the present invention is a sheet-like article containing differentiation-induced cells derived from pluripotent stem cells, which includes cells, a substrate for arranging the cells, and a gel for forming the cells into a sheet-like form. This invention relates to a kit for producing.
The kit of the present invention may further include, but is not limited to, a medium, a washing solution, a buffer solution, a tube, an instrument used for cell culture, a transportation container, instructions regarding how to use the kit, and the like.

Examples of instruments used for cell culture include pipettes, cell strainers, cell scrapers, and the like.
Examples of instructions regarding how to use include a usage manual, a medium on which information regarding the manufacturing method and usage method is recorded, such as a flexible disk, CD, DVD, Blu-ray disc, memory card, and USB memory.

Yet another aspect of the present invention is a method for treating a disease that is improved by applying a sheet-like material containing differentiation-induced cells derived from pluripotent stem cells, the sheet produced using the method according to the present invention. The present invention relates to a method comprising applying a substance to a subject in need thereof.

In the following examples, clinical human iPS cells established at Kyoto University iPS Cell Research Institute (CiRA) were used as pluripotent stem cells. Human iPS cells were maintained using a feeder-free method with reference to M. Nakagawa et al., Scientific Reports, 4:3594 (2014). Next, human iPS cells were induced to differentiate into cardiomyocytes to form embryoid bodies, with reference to the descriptions in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014/185358, and WO2017/038562. I got it. Specifically, human iPS cells maintained and cultured in a culture solution containing no feeder cells were cultured in StemFit AK03 medium containing 10 μM Y27632 (Wako Pure Chemical Industries, Ltd.) on EZSPERE (registered trademark) (manufactured by AGC Techno Glass). The embryoid bodies obtained were cultured in a culture medium containing activin A, bone morphogenetic protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further treated with Wnt inhibition. Human cardiomyocytes derived from iPS cells are cultured in a culture solution containing a drug (IWP3), a BMP4 inhibitor (Dorsomorphin), and a TGFβ inhibitor (SB431542), and then cultured in a culture solution containing VEGF and bFGF. I got it. The percentage of cardiomyocytes in the obtained cells was 50% to 90%.

Example 1. Preparation of sheet-like material Human iPS cell-derived cardiomyocytes cryopreserved in cell preservation solution (MCDB medium containing 10% DMSO) were thawed at 37°C, diluted with 10% FBS/DMEM, centrifuged, and then After removing the supernatant, the cells were suspended at a density of 2 x 10 ⁶ cells/cm ² in 10 mL of DMEM medium containing 20% human serum (manufactured by Gibco) and placed on a temperature-responsive substrate (UpCell (registered)) with a diameter of 6 cm. Trademark), 6 cm dish, CS3006, manufactured by Cell Seed Co., Ltd.). After seeding, the cell population was cultured for 72 hours in an incubator (BNA-121D, manufactured by ESPEC) set at 37° C. and 5% CO ₂ . After culturing, the substrate was removed from the incubator, it was confirmed that the cells were attached to the substrate, and the medium was discarded. It was observed that the seeded cells did not form a complete sheet-like cell culture, but had gaps (pores).
After discarding the medium, transfer the contents of vial 1 (fibrinogen lyophilized powder) of 100 μl of fibrinogen solution (Beriplast® for tissue adhesion (CSL Behring)) to the contents of vial 2 (fibrinogen dissolution). The contents of vial 3 (freeze-dried thrombin powder) of 100 μl of thrombin solution (Veriplast® tissue adhesion (manufactured by CSL Behring)) were dissolved in vial 4 (freeze-dried thrombin powder). The contents (thrombin dissolution solution) (thrombin concentration: 300 units/mL) were added dropwise and allowed to stand for about 5 minutes to form a fibrin gel.
After the fibrin gel was formed, 1 mL of Hank's balanced salt solution (HBSS (+), Cat No. 14025, manufactured by Life Technologies) was added and washed three times to remove unreacted fibrinogen and thrombin. Thereafter, the temperature-responsive material was allowed to stand for 5 to 30 minutes at room temperature (20 to 25°C) for temperature treatment, and the sheet-like material was peeled off from the base material by pipetting, and the sheet-like material was recovered (sheet-like Item 1).
Example 2. Preparation of sheet-like material (comparative example)
As a comparative example, sheet-like material 2 was prepared by forming a sheet-like material from fibrin gel in the same manner as sheet-like material 1 described above immediately after seeding without disposing cells on the base material.

Example 3. Preparation of a laminate of fibrin gel and sheet-shaped cell culture Human iPS cell-derived cardiomyocytes cryopreserved in a storage solution for cell preservation (MCDB medium containing 10% DMSO) were thawed at 37°C, and 0.5% serum It was washed twice using a physiological buffer containing albumin (manufactured by Gibco). 6.0 × 10 ⁷ washed cells were suspended in 10 mL of DMEM medium containing 20% human serum (manufactured by Gibco), and suspended in a substrate with a diameter of 10 cm (UpCell (registered trademark), 10 cm dish, CS3005, CellSeed). The seeds were sown in After seeding, cells were cultured for 1 day in an incubator set at 37°C and 5% _CO2 . After culturing, remove the substrate from the incubator and leave it at room temperature (20-25°C) for 5-30 minutes for temperature treatment of the temperature-responsive material, then peel the sheet-like cell culture from the substrate by pipetting. and collected it.

Next, the contents of vial 1 (fibrinogen lyophilized powder) of 100 μl of fibrinogen solution (Beriplast® tissue adhesion (manufactured by CSL Behring)) were added to the collected sheet-like cell culture. The contents of vial 3 (thrombin lyophilized powder) of 100 μl of thrombin solution (Beriplast® tissue adhesion (manufactured by CSL Behring)) were added to vial 4. (Thrombin concentration: 300 units/mL) was added dropwise to the solution and left to stand for about 5 minutes to form a fibrin gel. After the fibrin gel was formed, it was washed three times with 1 mL of Hank's balanced salt solution (HBSS (+), Cat No. 14025, manufactured by Life Technologies) to remove unreacted fibrinogen and thrombin, and the fibrin gel and sheet A laminate with a cell culture was obtained.

Example 4. Evaluation of sheet materials
(1) Evaluation of size of sheet-like material 1 and laminate The sheet-like material 1 produced in Example 1 and the laminate produced in Example 3 were each transferred to a 10 cm Petri dish and compared in size.
FIG. 1 shows a photograph of the sheet-like material 1, and FIG. 2 shows a photograph of the laminate. The sheet-like material 1 did not shrink and its size was 6 cm in diameter, which was the same as the size of the prepared Petri dish. The laminate shrank after peeling, and the size of the laminate was 2.5 cm in diameter.

(2) Evaluation of cross-sectional views of sheet-like material 1 and sheet-like material 2 Sheet-like material 1 produced in Example 1 and sheet-like material 2 produced in Example 2 were soaked in 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd.), respectively. , overnight fixation, and then each sheet was soaked in 30% sucrose/PBS. Thereafter, the sheet-like materials were each coated with Tissue Tech (registered trademark) O. C. T. It was immersed in a compound (Sakura Finetech Japan) and subjected to freezing treatment to prepare frozen sections. The prepared frozen sections were stained with hematoxylin and eosin to stain cell nuclei in bluish purple with hematoxylin and other structures in various shades of red with eosin, and observed under a microscope (100x magnification).
FIG. 3 shows a cross-sectional view of the sheet-like material 1, and FIG. 4 shows a cross-sectional view of the sheet-like material 2. In sheet-like material 1, the gaps (pores) that existed when the cells were placed are filled with gel, and the cells are localized on one side (the area surrounded by the dashed line), and the cells are localized on one side (the area surrounded by the dashed line), and the cells are localized on one side (the area surrounded by the dashed line). It existed separately from the other parts. On the other hand, in sheet-like material 2, cells were evenly scattered throughout the sheet-like material.

Claims (11)

A method for producing a sheet-like material containing human iPS cell-derived cardiomyocytes, the method comprising:
arranging the cell clusters in an island shape with gaps on the substrate;
Filling the gaps between the cell clusters with fibrin gel to form a sheet,
The method described above, wherein the gel serves as a support to prevent damage to the sheet when it is peeled off from the substrate or when the sheet is moved for application. A method according to claim 1, wherein the thickness of the gel is in the range of 10 μm to 500 μm . Prior to the step of arranging the cell clusters in islands with gaps on the substrate:
(i) Seeding human iPS cell-derived cardiomyocytes onto a culture substrate and allowing them to adhere;
(ii) A step capable of forming a sheet-like cell culture with gaps between cell clusters rather than a complete sheet-like cell culture
3. The method of claim 1 or 2, further comprising: The method according to any one of claims 1 to 3 , wherein the gel is in a solidified state at around room temperature. The method according to any one of claims 1 to 4 , wherein the gel is formed from a fibrinogen lysate with a fibrinogen concentration of 80 mg/mL and a thrombin concentration of 300 units/mL. A sheet-like material containing human iPS cell-derived cardiomyocytes and fibrin gel ,
The cell clusters are arranged in an island shape with gaps on the bottom surface of the sheet, and
The gaps between the cell clusters are filled with fibrin gel to form a sheet shape,
Here, the fibrin gel serves as a support to prevent breakage of the sheet when the sheet is moved for application. 7. The sheet-like material according to claim 6, wherein the sheet-like material is made of a lump-like cell mass and a fibrin gel that fills a gap between the cell mass. A kit for producing a sheet containing the human iPS cell-derived cardiomyocytes according to claim 6 or 7 ,
The kit comprising human iPS cell-derived cardiomyocytes, a substrate for placing the cells, and the gel for forming the cells into a sheet. The sheet-like material according to claim 6 or 7, which is used to treat a disease that is improved by application of the sheet-like material containing cardiomyocytes derived from human iPS cells. A method for producing a sheet-like material containing human iPS cell-derived cardiomyocytes, the method comprising:
placing cells on a substrate, where the cells placed on the substrate do not form a complete sheet-like cell culture, but have gaps (pores);
A step of producing a sheet-like object by molding the cells placed on the base material into a sheet-like form using fibrin gel,
Here, cells are localized on one side of the sheet-like material, exposed on one side, and
The method described above, wherein the gel serves as a support to prevent damage to the sheet when it is peeled off from the substrate or when the sheet is moved for application. A sheet-like material containing human iPS cell-derived cardiomyocytes and fibrin gel that does not contract immediately after its preparation until it is applied ,
Cells exist on one side of the sheet-like material via fibrin gel, but they do not exist continuously over the entire surface,
The fibrin gel acts as a support to prevent the sheet from breaking, and maintains the physical integrity of the sheet.
The sheet- like material.