WO2021065984A1 - Procédé de formation de feuille de cardiomyocytes - Google Patents

Procédé de formation de feuille de cardiomyocytes Download PDF

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WO2021065984A1
WO2021065984A1 PCT/JP2020/037084 JP2020037084W WO2021065984A1 WO 2021065984 A1 WO2021065984 A1 WO 2021065984A1 JP 2020037084 W JP2020037084 W JP 2020037084W WO 2021065984 A1 WO2021065984 A1 WO 2021065984A1
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cells
cell
culture
sheet
component
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PCT/JP2020/037084
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Japanese (ja)
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芳樹 澤
繁 宮川
賢二 大山
文哉 大橋
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国立大学法人大阪大学
テルモ株式会社
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Priority to JP2021551353A priority Critical patent/JP7575392B2/ja
Publication of WO2021065984A1 publication Critical patent/WO2021065984A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • This disclosure is a commissioned research project of the Japan Medical Research and Development Organization, Regenerative Medicine Realization Center Network Program, Practical Application Research Center for Diseases and Tissues (Center A), "Center for the Creation of Myocardial Regenerative Therapy Using iPS Cells" in FY2018. It is a patent application to which Article 17 of the Development and Industrial Technology Enhancement Law applies, and transplants of various cells such as sheet-shaped cell cultures containing myocardial cells, especially pluripotent stem cell-derived myocardial cells.
  • the present invention relates to a method for producing, a transplanted piece manufactured by using the method, a method for treating a disease using the transplanted piece, and the like.
  • Non-Patent Document 1 a graft containing cardiomyocytes prepared by a cell engineering technique into an affected area.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • Non-Patent Documents 2 to 3 There are attempts to prepare sheet-like cell cultures containing such pluripotent stem cell-derived cardiomyocytes and therapeutic experiments in animals.
  • Non-Patent Documents 2 to 3 the development of sheet-like cell cultures containing pluripotent stem cell-derived cardiomyocytes has just begun, and there are still many unclear points about their functional characteristics and factors that influence them.
  • the graft-forming medium contains as little heterologous components other than humans as possible, that is, a so-called xeno-free state.
  • a sheet-shaped cell culture in a xeno-free state is often costly and laborious, and depending on the cells used, it is possible to form a sheet-shaped cell culture while maintaining the desired properties of the cells. It can be difficult.
  • the present disclosure is a method for producing a high-quality implant, which comprises various cells such as cardiomyocytes, particularly cardiomyocytes derived from pluripotent stem cells, while preserving the functions of the cells, using the method. It is an object of the present invention to provide a manufactured implant, a method for treating a disease using the implant, and the like.
  • a graft containing cardiomyocytes to be used for transplantation it is necessary to prepare with a xenofree graft-forming medium.
  • FBS fetal bovine serum
  • problems such as cardiomyocytes not beating sufficiently and grafts not being formed well have been found. ..
  • the present inventors are studying the preparation of sheet-shaped cell cultures for living body transplantation using cardiomyocytes derived from pluripotent stem cells, which have sufficient functions even when prepared with a Xenofree implant-forming medium.
  • a serum-free medium can be used to form a sheet. I found it. Based on this finding, further research was carried out, and it was found that it is possible to produce a high-quality graft that can withstand clinical application in the preparation of a graft containing various cells, and to complete the present invention. I arrived.
  • the present invention relates to the following: [1] A method for producing a graft containing somatic cells; (A) a step of seeding the cell population containing the somatic cells on a culture substrate, and (b) a step of seeding the seeded cell population in a transplantation piece-forming medium containing a cell adhesion component. Included, said method. [2] The method according to [1], wherein the culture substrate is further coated with a cell adhesion component and / or a masking component. [3] The method according to [1] or [2], wherein the content of the cell adhesion component in the graft-forming medium is 1/10 to 1/100 of the amount used for coating the culture substrate. ..
  • a method for producing a sheet-like cell culture containing cardiomyocytes (A) A step of seeding myocardial cells at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component, and (B) sheet-forming culture using a sheet-forming medium, and sheet-like cell culture.
  • a method for producing a sheet-shaped cell culture which comprises a step of forming a substance.
  • the sheet formation medium contains or does not contain a cell adhesion component.
  • [9] A method for evaluating the surface of a culture medium for producing a sheet-shaped cell culture.
  • the step of coating the surface of the culture substrate with the cell adhesion component thereby improving the adhesion of the cells to the surface of the culture substrate.
  • a step of coating the surface of the culture substrate with a masking component thereby reducing the adhesion of cells to the surface of the culture substrate.
  • the above-mentioned step including (iv) a step of forming a sheet-like cell culture by sheet-forming culture in a sheet-forming medium, and (v) a step of evaluating a peeling state of the sheet-like cell culture from the surface of a culture substrate.
  • transplants such as sheet-like cell cultures of higher quality than before can be efficiently produced from a cell population containing various cells, for example, cardiomyocytes, particularly cardiomyocytes induced to differentiate from pluripotent stem cells.
  • a cell population containing various cells for example, cardiomyocytes, particularly cardiomyocytes induced to differentiate from pluripotent stem cells.
  • cardiomyocytes particularly cardiomyocytes induced to differentiate from pluripotent stem cells.
  • the graft-forming medium does not use serum, that is, it does not contain impurities derived from the manufacturing process. It is particularly useful in regenerative medicine because it enables graft formation even when the graft is formed and cultured under the conditions.
  • a platelet lysate to the graft-forming medium, it is possible to prepare a graft having a desired property at a high level, and particularly to provide a very suitable graft for living-donor transplantation into humans. It becomes possible.
  • FIG. 1 is a photograph showing a state of sheet formation when laminin (iMatrix-511), vitronectin (VTN-N), and fibronectin (RetroNectin (R)) are used as cell adhesion components.
  • the upper row (A and B) is for laminin
  • the middle row (C and D) is for vitronectin
  • the lower row (E and F) is for fibronectin
  • the left column (A, C and E) is for use as a coating agent.
  • the right column (B, D and F) shows the result when used at 1/10 of the recommended concentration. In any case, it was confirmed that the sheet was formed.
  • FIG. 1 is a photograph showing a state of sheet formation when laminin (iMatrix-511), vitronectin (VTN-N), and fibronectin (RetroNectin (R)) are used as cell adhesion components.
  • the upper row (A and B) is for laminin
  • FIG. 2 shows a state in which the sheet-shaped cell culture is peeled off after sheeting with iMatrix-511 as a cell adhesion component and FBS as a masking component on a culture substrate coated with various concentrations. It is a photograph showing. By coating with FBS together with iMatrix-511, it was confirmed that peeling was possible even when iMatrix-511 with a wide concentration was used.
  • the present disclosure is a method of producing a graft containing the desired cells; (A) a step of seeding the cell population containing the cells on a culture substrate, and (b) a step of forming and culturing the seeded cell population in a medium containing a cell adhesion component.
  • the present invention relates to the above-mentioned method.
  • the "graft” means a structure for transplantation into a living body, and particularly means a structure for transplantation containing cells as a constituent component.
  • the implant is a structure for transplantation that is free of cells and structures other than cell-derived substances (eg, scaffolds, etc.).
  • the implants in the present disclosure include, but are not limited to, sheet-like cell cultures, spheroids, cell aggregates, and the like, preferably sheet-like cell cultures or spheroids, and more preferably sheet-like. It is a cell culture.
  • sheet-shaped cell culture refers to cells connected to each other to form a sheet.
  • the cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances.
  • the intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix.
  • the mediator is preferably derived from cells, particularly from the cells that make up the sheet-like cell culture.
  • the cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected.
  • the sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminate (multilayer), for example, two layers, three layers, etc. It may be 4 layers, 5 layers, 6 layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without showing a clear layered structure of the cells. For example, in the vertical cross section of a sheet-shaped cell culture, cells may be present in a non-uniformly (for example, mosaic-like) arrangement without being uniformly aligned in the horizontal direction.
  • a non-uniformly for example, mosaic-like
  • the grafts of the present disclosure preferably do not contain scaffolds (supports). Scaffolds may be used in the art to attach cells to and / or inside the scaffold to maintain the physical integrity of the implant, such as a sheet cell culture, eg, poly. Although membranes made of vinylidene fluoride (PVDF) and the like are known, the implants of the present disclosure can maintain their physical integrity without such scaffolds. In addition, the implants of the present disclosure preferably consist only of cell-derived substances constituting the implants and do not contain any other substances.
  • scaffolds supports
  • Scaffolds may be used in the art to attach cells to and / or inside the scaffold to maintain the physical integrity of the implant, such as a sheet cell culture, eg, poly.
  • PVDF vinylidene fluoride
  • the implants of the present disclosure can maintain their physical integrity without such scaffolds.
  • the implants of the present disclosure preferably consist only of cell-derived substances constituting the implants and do not contain any other substances.
  • the cell may be a heterologous cell or an allogeneic cell.
  • heterologous cell means a cell derived from an organism of a species different from the recipient when the graft is used for transplantation.
  • cells derived from monkeys and pigs correspond to heterologous cells.
  • homoogeneous cell means a cell derived from an organism of the same species as the recipient.
  • the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells).
  • Autologous cells are preferred in the present disclosure because they do not cause rejection when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection.
  • cells other than autologous cells that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells.
  • the cell is an autologous cell or an allogeneic cell.
  • the cell is an autologous cell (including an autologous iPS cell).
  • the cell is an allogeneic cell (including an allogeneic iPS cell).
  • the cells constituting the implant of the present disclosure are not particularly limited as long as they can form the implant, and include, for example, adherent cells (adhesive cells).
  • Adhesive cells include, for example, adherent somatic cells (eg, myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, root membrane cells, gingival cells, bone membrane cells, skin.
  • iPS cells induced pluripotent stem cells
  • Somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells).
  • Non-limiting examples of cells constituting the transplant include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair roots, muscle tissue, etc.) Endometrial, placenta, umbilical cord blood, etc.), myocardial cells, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigments) Epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells, etc.), hepatocytes (eg, hepatic parenchymal cells, etc.), pancreatic cells (eg, pancreatic islet cells, etc.), kidney cells, adrenal cells, root membrane Examples include cells, gingival cells, bone membrane cells, skin cells
  • Non-limiting examples of iPS cell-derived adherent cells include iPS cell-derived myocardial cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, renal cells, adrenal cells, root membrane cells, gingival cells, and bone membrane cells. , Skin cells, synovial cells, cartilage cells and the like.
  • the cells that make up the implant can be derived from any organism that can be treated with the implant. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included.
  • the number of types of cells constituting the transplant is not particularly limited, and may be composed of only one type of cells, or may be composed of two or more types of cells.
  • the content ratio (purity) of the most abundant cells is, for example, 50% or more, preferably 60% or more, more preferably 70 at the end of formation of the sheet-like cell culture. % Or more, more preferably 75% or more.
  • the culture substrate is not particularly limited as long as the cells can form a cell culture on the cells, and includes, for example, containers of various materials and / or shapes, solid or semi-solid surfaces in the containers, and the like. ..
  • the container preferably has a structure / material that does not allow a liquid such as a culture solution to permeate.
  • Such materials include, without limitation, for example, polyethylene, polypropylene, Teflon®, polyethylene terephthalate, polymethylmethacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl.
  • Acrylamide, metals (eg, iron, stainless steel, aluminum, copper, brass) and the like can be mentioned.
  • the container preferably has at least one flat surface.
  • a culture container having a bottom surface made of a culture substrate capable of forming a cell culture and a liquid-impermeable side surface.
  • a culture vessel include, but are not limited to, a cell culture dish, a cell culture bottle, and the like.
  • the bottom surface of the container may be transparent or opaque. If the bottom surface of the container is transparent, cells can be observed and counted from the back side of the container.
  • the container may have a solid or semi-solid surface inside the container. Examples of the solid surface include plates and containers of various materials as described above, and examples of the semi-solid surface include gels and soft polymer matrices.
  • the culture substrate may be prepared using the above-mentioned materials, or a commercially available one may be used.
  • Preferred culture substrates include, without limitation, for example, a substrate having an adhesive surface suitable for forming a sheet-like cell culture, and a substrate having a low adhesive surface suitable for forming spheroids. And / or a substrate having a uniform well-like structure and the like.
  • a substrate coated with a hydrophilic compound such as corona discharge-treated polystyrene, collagen gel or hydrophilic polymer on the surface thereof, and further, collagen.
  • Fibronectin Fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan and other extracellular matrix, and base materials coated with cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface.
  • cell adhesion factors such as cadoherin family, selectin family and integrin family on the surface.
  • substrates are commercially available (eg, Corning (R) TC-Treated Culture Dish, Corning, etc.).
  • spheroid formation for example, soft agar, temperature-responsive gel obtained by cross-linking poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG), polyhydroxyethyl methacrylate (commercially available name: mebiol gel), polyhydroxyethyl methacrylate ( Examples include a base material coated with a non-cell adhesive compound such as a hydrogel such as poly-HEMA) and 2-methacryloyloxyethyl phosphorischoline (MPC) polymer and / or a base material having a uniform uneven structure on the surface. Be done. Such substrates are also commercially available (eg, EZSPHERE (R), etc.).
  • the culture medium may be transparent or opaque in whole or in part.
  • the surface of the culture substrate may be coated with a material whose physical properties change in response to irritation, for example, temperature or light.
  • a material whose physical properties change in response to irritation, for example, temperature or light.
  • Such materials include, but are not limited to, for example, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (eg, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, etc.
  • Known materials such as a copolymer with a body and a photoresponsive material such as N-isopropylacrylamide gel containing spirobenzopyran can be used (see, for example, JP-A-2-21186 and JP-A-2003-33177). ). By giving a predetermined stimulus to these materials, their physical characteristics, for example, hydrophilicity and hydrophobicity can be changed, and the exfoliation of the cell culture adhering on the material can be promoted. Culture dishes coated with a temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell (R) ) and can be used in the production methods of the present disclosure.
  • a temperature-responsive material are commercially available (eg, CellSeed Inc.'s UpCell (R) ) and can be used in the production methods of the present disclosure.
  • the culture medium may have various shapes.
  • the area thereof is not particularly limited, but may be, for example, about 1 cm 2 to about 200 cm 2 , about 2 cm 2 to about 100 cm 2 , about 3 cm 2 to about 50 cm 2 .
  • a circular culture dish having a diameter of 10 cm can be mentioned. In this case, the area is 56.7 cm 2 .
  • the culture surface may be flat or may have an uneven structure. When it has a concavo-convex structure, it is preferable that it has a uniform concavo-convex structure.
  • pluripotent stem cell is a well-known term in the art and has the ability to differentiate into three germ layers, i.e. cells of all lineages belonging to endoderm, mesoderm and ectoderm. Means cell.
  • pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear-transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like.
  • ES cells embryonic stem cells
  • ntES cells nuclear-transplanted embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • pluripotent stem cells are first suspended-cultured to form aggregates of any of the above three germ layers, and then cells that form aggregates. Induce differentiation into specific cells of interest.
  • pluripotent stem cell-derived differentiation-inducing cell means any cell that has been subjected to differentiation-inducing treatment so as to differentiate from a pluripotent stem cell into a specific type of cell.
  • differentiation-inducing cells include muscular cells such as myocardial cells and skeletal myoblasts, neural cells such as neuron cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells.
  • Hematopoietic cells such as cells and bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells and B cells, cells constituting organs such as hepatocytes, pancreatic ⁇ cells and renal cells, In addition to cartilage cells and germ cells, it also includes precursor cells and somatic stem cells that differentiate into these cells.
  • progenitor cells and somatic stem cells include mesenchymal stem cells in myocardial cells, pluripotent cardiac progenitor cells, monopoly cardiac progenitor cells, neural stem cells in neural cells, hematopoietic cells and immunity. Examples include hematopoietic stem cells and lymphoid stem cells in related cells.
  • Induction of differentiation of pluripotent stem cells can be performed using any known method.
  • the induction of differentiation of pluripotent stem cells into cardiomyocytes can be performed based on the methods described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 and WO2014 / 185358.
  • a differentiation-inducing cell derived from a pluripotent stem cell such as an iPS-derived cardiomyocyte
  • the undifferentiated cell may be removed after the differentiation induction.
  • the treatment for removing undifferentiated cells is known in the art, and the methods described in, for example, WO2017 / 038562, WO2016 / 072519, WO2007 / 088744, etc. can be used.
  • the differentiation-inducing cell may be a cell derived from an iPS cell into which any useful gene other than the gene for reprogramming has been introduced.
  • 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.
  • cells into which any useful gene has been introduced after induction of differentiation from pluripotent stem cells are also included in the differentiation-inducing cells of the present invention.
  • cell adhesion component means an isolated polypeptide having the property of adhering to cells, including extracellular matrix and cell adhesion factors described in the above description of the culture medium. To do. Such cell-adhesive components are generally used to coat the surface of the culture medium. Examples of cell-adhesive components include, but are not limited to, extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, and cells such as cadoherin family, selectin family, and integrin family.
  • these variants or functional equivalents such as laminin variants such as laminin 511 and laminin 221 and vitronectin variants such as VTN-N, Kimizuka et al., J. Biochem.
  • laminin variants such as laminin 511 and laminin 221
  • vitronectin variants such as VTN-N, Kimizuka et al., J. Biochem.
  • fibronectin such as retronectin (R) described in (1991), 110, pp.284-291 and the like.
  • R retronectin
  • the cell adhesion components of the present disclosure do not contain blood-derived components other than cell adhesion factors and / or their functional equivalents.
  • the present invention will be described in detail below, taking as an example the case where the desired cell is a cardiomyocyte and the implant is a sheet-like cell culture.
  • One aspect of the disclosure relates to a method for producing a high quality sheet cell culture containing cardiomyocytes.
  • the method of the present disclosure includes the following steps (a) and (b): (A) A step of seeding a cell population containing myocardial cells on a culture substrate, and (b) a step of sheeting and culturing the seeded cell population in a sheeting medium containing a cell adhesion component.
  • cardiomyocyte means a cell having the characteristics of cardiomyocyte.
  • the characteristics of cardiomyocytes include, but are not limited to, expression of cardiomyocyte markers, presence of autonomous pulsation, and the like.
  • Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like. ..
  • cardiomyocytes derived from pluripotent stem cells are c-TNT positive and / or CD172a positive.
  • the cardiomyocytes used in the method of the present disclosure may be directly obtained from a living body or derived from other cells, but are preferably derived from other cells. ..
  • Examples of the induction into cardiomyocytes include a method of introducing a myocardial inducing factor into fibroblasts and the like, and a method of inducing differentiation of cells having the property of differentiating into cardiomyocytes such as pluripotent stem cells and cardiac progenitor cells. ..
  • seeding on the culture substrate may be performed, for example, by injecting a cell suspension in which cells are suspended in a sheeting medium into a culture vessel provided with the culture substrate.
  • a cell suspension in which cells are suspended in a sheeting medium into a culture vessel provided with the culture substrate.
  • an instrument suitable for the injection operation of the cell suspension such as a dropper or a pipette, can be used.
  • the seeding density of cells is set to a density capable of forming a sheet-like cell culture, and the density may vary depending on the desired cells, but those skilled in the art select an appropriate density from methods known in the art. can do.
  • a sheet-shaped cell culture containing cardiomyocytes it can be, for example, 2.0 ⁇ 10 5 cells / cm 2 or more, but may be seeded at a higher density.
  • Examples of higher densities include, for example, a density that reaches confluence, that is, a density at which cells are expected to cover the entire adhesive surface of the culture vessel when seeded, for example, cells come into contact with each other when seeded. It can be as dense as expected, the density at which contact inhibition occurs, or the density at which cell proliferation is substantially stopped by contact inhibition or higher.
  • the upper limit of the seeding density is not particularly limited, but if the seeding density is excessively high, more cells will die, resulting in inefficiency. In one aspect of the present disclosure, the seeding density is, for example, about 1.0 ⁇ 10 6 / cm 2 to about 1.0 ⁇ 10 7 / cm 2 , about 1.0 ⁇ 10 6 / cm 2 to about 5.
  • the sowing density is from about 1.76 ⁇ 10 6 / cm 2 to about 2.33 ⁇ 10 6 / cm 2 .
  • the seeded cell population may contain other cells as long as they contain the desired cells (eg, myocardial cells), and when the desired cells are myocardial cells, for example, fibroblasts, vascular endothelial cells. And / or wall cells and the like may be further included.
  • the cell population the cell population collected from the tissue may be used as it is, or by using, for example, the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 or WO 2014/185358.
  • the cell population obtained by inducing differentiation from iPS cells may be used as it is, or may be used after cryopreservation, pre-culture, removal of undifferentiated cells, or the like.
  • the seeded cell population is a cell population that is induced to differentiate from iPS cells, seeded on a culture substrate (preferably on a flat culture substrate), adherently cultured, and then recovered. is there. Cryopreservation and thawing may be performed before or after such adhesive culture.
  • the culture conditions and the like may be the same as those for normal adhesive culture.
  • a commercially available culture container for adhesive culture may be used for culturing under 37 ° C. and 5% CO 2 conditions.
  • the seeding density of the cells may be any density as long as it does not interfere with the adhesion between the cells and / or the formation of the adhesion between the cells and the culture substrate, and may be, for example, a subconfluent density. It may be at or above a density that reaches confluence.
  • the culturing time may be such that adhesion between cells and / or adhesion between cells and the culture substrate is formed, and specifically, for example, 2 to 24 hours, 2 to 12 hours, and 2 to 6 hours. It may be about 2 to 4 hours.
  • the cell population does not include undifferentiated cells.
  • a cell population obtained by collecting from a living body or inducing differentiation from pluripotent stem cells may be subjected to an undifferentiated cell removal operation.
  • the "undifferentiated cell removal operation” refers to undifferentiated cells having tumorigenicity from a cell population, typically a cell population containing differentiation-inducing cells obtained by inducing differentiation of pluripotent stem cells. It means a removal operation and can be performed using any known method.
  • Non-limiting examples of such a method include various separation methods using markers specific to undifferentiated cells (for example, cell surface markers), for example, magnetic cell separation method (MACS), flow cytometry method, affinity separation.
  • markers specific to undifferentiated cells for example, cell surface markers
  • MCS magnetic cell separation method
  • Methods, methods for expressing selective markers (eg, antibiotic resistance genes, etc.) by specific promoters, factors necessary for the survival of undifferentiated cells nutrient sources (nutrient sources such as methionine, maintenance of undifferentiated state such as bFGF)
  • a method of exterminating undifferentiated cells by culturing in a medium excluding (factors, etc.) a method of culturing in the presence of factors that promote differentiation (VEGF, BMP, activin, etc.) and promoting undifferentiated cells, not yet Examples thereof include a method of treating the surface antigen of differentiated cells with a drug targeting the surface antigen.
  • WO2016 / 072519, WO2013 / 10080 examples thereof include the method described in JP-A-2016-093178 and the method using the heat treatment described in WO2017 / 038526.
  • the operation for removing undifferentiated cells is performed by culturing in a sugar-free medium as described in WO2007 / 088874.
  • the culture base material to be seeded is as described in detail above, but in a preferred embodiment, it is a culture base material whose surface is coated with a cell adhesion component such as an extracellular matrix or a cell adhesion factor.
  • the cell-adhesive component is not limited to this, for example, extracellular matrix such as collagen, fibronectin, laminin, vitronectin, proteoglycan, glycosaminoglycan, and cell adhesion factors such as cadoherin family, selectin family, and integrin family.
  • these variants may be, for example, laminin 511 (a variant of laminin), VTN-N (a variant of vitronectin), retronectin (R) (a variant of fibronectin).
  • Coating of the cell adhesion component can be achieved by contacting and incubating a medium containing the cell adhesion component with the culture substrate.
  • the amount of the cell adhesion component that can be contained in such a medium may vary depending on the area of the substrate to be coated, the cell adhesion component used, the cell type to be cultured, and the like.
  • a person skilled in the art can set the optimum amount or concentration according to the product protocol, etc., and the amount or concentration can be set when coating with only the adhesive component without using the masking component, for example, laminin 511 or VTN-.
  • N it is about 0.1 to 1.0 ⁇ g / cm 2
  • retronectin (R) it is about 4 to 20 ⁇ g / cm 2 .
  • a culture substrate in which the surface is coated with a masking component such as a blood-derived component in addition to a cell adhesion component such as an extracellular matrix or a cell adhesion factor.
  • the "masking component” means a component capable of reducing the cell adhesion on the surface of the culture medium coated with the cell adhesion component.
  • Such components are typically blood-derived components, such as normal serum (eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.), as well as albumin and platelet lysates contained in normal serum. , Skim milk, albumin substitutes such as polyvinyl alcohol, and the like.
  • Coating of the medium containing the masking component may be performed simultaneously with or separately from the coating of the cell adhesion component, and is achieved by contacting and incubating the medium containing the cell adhesion component and / or the masking component with the culture substrate. obtain.
  • concentration of the masking component that can be contained in such a medium may vary depending on the concentration of the cell adhesion component or the cell adhesion component used, the area of the substrate to be coated, the cell type to be cultured, and the like.
  • the concentration of the masking component is 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.16%, 0.2% in the coating medium. , 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0% 2.25%, 2.5%, 2.75%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 7.5% , 9%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 30%, 40% or more, and the upper limit is not particularly limited, but 45.
  • the amount or concentration of laminin 511 is 0.01 ⁇ g / cm 2 to 100 ⁇ g / cm 2 with respect to 0.1% to 30% FBS, laminin 511 0.
  • the incubation time is not particularly limited as long as the masking component can adhere to the culture substrate, for example, 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, still more preferably 6 to 12 hours. It's time.
  • the incubation temperature is also not particularly limited as long as the masking component can adhere to the culture substrate, and is, for example, 0 to 60 ° C., preferably 4 to 45 ° C., more preferably room temperature to 40 ° C.
  • the seeded cells are sheet-cultured.
  • the culture for forming the seeded cells as a transplant is referred to as "transplant formation culture”
  • the transplant is a sheet-like cell culture
  • the culture for forming the seeded cells into a sheet is referred to as a culture.
  • sheet culture it is referred to as "sheet culture”. Sheeting of seeded cells can be performed by any known method and condition. Non-limiting examples of such a method are described in, for example, Japanese Patent Application Laid-Open No. 2010-081829, Japanese Patent Application Laid-Open No. 2010-226991, Japanese Patent Application Laid-Open No. 2011-110368, Japanese Patent Application Laid-Open No.
  • Cell graft formation (eg, sheet formation) is believed to be achieved by the cells adhering to each other via adhesion molecules or intercellular adhesion mechanisms such as extracellular matrix.
  • the step of forming a graft of seeded cells can be accomplished, for example, by culturing the cells under conditions that form cell-cell adhesions. Such conditions may be any as long as they can form cell-cell adhesion, but usually, cell-cell adhesion can be formed under the same conditions as general cell culture conditions. Such conditions include, for example, culturing at about 37 ° C. and 5% CO 2.
  • the culture can be carried out under normal pressure (atmospheric pressure, non-pressurization).
  • the graft-forming culture sheet-forming culture
  • the graft-forming culture sheeted culture
  • Culturing can be carried out in containers of any size and shape. For the size and shape of the sheet-shaped cell culture, adjust the size and shape of the cell adhesion surface of the culture vessel, or install a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing cells inside the cell.
  • the time for sheeting culture can vary depending on the type of cells to be seeded and the cell density.
  • the sheet may be formed by seeding at a density of, for example, about 2.1 ⁇ 10 5 cells / cm 2 and culturing for 4 days or more.
  • the seeding density reaches the confluence, that is, when the seeding is performed at a higher density, the period of sheeting culture can be shortened, and the culture time may be 2 to 4 days, more preferably 2 to 3 days. ..
  • the medium used for graft formation (for example, sheet formation) (the graft-forming medium, which may be referred to as a sheet-forming medium when the graft formation is sheet-forming) contains a cell adhesion component and transplants cells. It is not particularly limited as long as it enables fragment formation, and for example, physiological saline, various physiological buffer solutions (for example, PBS, WBSS, etc.), various basal medium-based liquids for cell culture, and the like can be used. You may use it.
  • Such basal medium is not limited to, for example, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM. / F12 and the like are included.
  • Many of these basal media are commercially available, and their compositions are also known.
  • the basal medium may be used as it has a standard composition (for example, as it is on the market), or the composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the basal medium used in the present invention is not limited to those having a known composition, and includes those in which one or more components are added, removed, increased or decreased.
  • Transplantation media may also include additives such as normal serum (eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).
  • normal serum eg, bovine serum such as bovine fetal serum, horse serum, human serum, etc.
  • various growth factors eg, FGF, EGF, VEGF, HGF, etc.
  • the graft-forming medium is serum-free.
  • concentration of the cell adhesive component contained in the sheeting medium is not particularly limited as long as it does not adversely affect the cells, but may be equal to or less than the concentration used in the above coating.
  • concentration is, for example, about 0.01 to 10 ⁇ g / mL for laminin 511 or VTN-N, preferably about 0.1 to 1 ⁇ g / mL, and about 0.2 to 100 ⁇ g / mL for retronectin (R). , Preferably about 2-10 ⁇ g / mL and the like.
  • the sheeting medium further comprises a platelet lysate.
  • platelet lysate refers to a composition rich in growth factors and the like, which is obtained by repeatedly freezing and thawing platelets.
  • Such a composition is commercially available as a medium additive for cell culture, is known in the art, and can be prepared by, for example, the method described in Bieback et al., STEM CELLS, 2009; 27: 2331-2341. Is. In recent years, it is known to promote the proliferation of mesenchymal stem cells. In the production of sheet-shaped cell cultures containing cardiomyocytes, the present inventors have observed that by incorporating platelet lysates into the sheeting medium, strong autonomous pulsation is observed in a faster culture time than before. I found it for the first time.
  • the concentration of platelet lysate contained in the graft-forming medium may be such that it is usually used in the art, for example, 1%, 2.5%, 5%, 10%, 15%, 20% and the like. It may be there.
  • the platelet lysate is contained in the graft-forming medium in an amount of 1% to 20%, more preferably 2% to 10%, still more preferably 2.5% to 10%.
  • the graft-forming medium may be appropriately replaced during the graft-forming culture.
  • the composition of the medium may be changed according to the progress of graft formation.
  • the present inventors use a sheeted medium to which a Rho-kinase (ROCK) inhibitor is added as a medium for the first day of sheeted culture.
  • a Rho-kinase (ROCK) inhibitor is added as a medium for the first day of sheeted culture.
  • the sheeting medium used for the sheeting culture on day 1 comprises a Rho-kinase inhibitor.
  • the sheeting medium after the second day may or may not contain a Rho kinase inhibitor, but preferably does not contain a Rho kinase inhibitor.
  • the culture substrate may or may not be coated with a cell adhesive component.
  • the cell adhesion component coating the culture substrate may be the same as or different from the cell adhesion component contained in the sheeting medium. It may be, but preferably the same cell adhesion component.
  • the culture substrate is coated with only the cell adhesive component, preferably only the cell adhesive component contained in the sheeting medium. Therefore, in such an embodiment, the culture medium does not contain components other than the cell adhesive component, such as serum.
  • the culture substrate may be coated with another component in place of or in addition to the cell adhesion component. Examples of such other components include coating components exemplified in the above description of the culture medium, such as temperature-responsive materials.
  • the culture medium of the present disclosure is coated with serum, FBS or albumin in addition to the cell adhesive components.
  • the concentration of the cell adhesion component contained in the graft-forming medium may differ depending on the type of the cell adhesion component contained and the state of the cells forming the graft. For example, when cells with low viability, that is, weak activity are used, the content of the cell adhesion component should be low.
  • the concentration of the cell adhesion component contained in the sheeting medium is about 0.1%, about 0.%, based on the concentration used when the same cell adhesion component is used as the coating agent for the culture substrate (100%). It may be 5%, about 1%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, and the like.
  • the concentration range of the cell adhesion component contained in the sheeting medium is about 0 based on the concentration used when the same cell adhesion component is used as the coating agent for the culture substrate (100%). .1% to about 100%, about 0.1% to about 100%, about 0.1% to about 50%, about 0.1% to about 25%, about 0.1% to about 20%, about 0 .1% to about 10%, about 1% to about 100%, about 1% to about 100%, about 0.5% to about 100%, about 0.5% to about 100%, about 0.5% to About 50%, about 0.5% to about 25%, about 0.5% to about 20%, about 0.5% to about 10%, about 1% to about 50%, about 1% to about 25%, About 1% to about 20%, about 1% to about 10%, about 0.5% to about 100%, about 5% to about 100%, about 5% to about 50%, about 5% to about 25%, It may be about 5% to about 20%, about 5% to about 10%, and the like.
  • the cardiomyocytes used in the method of the present disclosure may be directly obtained from a living body or derived from other cells, but are preferably derived from other cells. It is a thing.
  • other cells to be induced include fibroblasts such as myocardial fibroblasts, progenitor cells that differentiate into myocardial cells such as cardiac progenitor cells, and pluripotent stem cells that can differentiate into arbitrary cells, which are preferable.
  • pluripotent stem cells more preferably iPS cells, and even more preferably human iPS cells.
  • Another aspect of the disclosure relates to a method of producing a sheet-like cell culture containing desired somatic cells, such as cardiomyocytes.
  • a method of producing a sheet-like cell culture containing desired somatic cells includes the following steps: (A) A step of seeding a cell population containing desired somatic cells (hereinafter, may be referred to as sheet-forming cells) at a density reaching confluence on a culture substrate coated with a cell-adhesive component and a masking component; And (B) a step of forming a sheet-like cell culture by sheet-forming and culturing the seeded cell population in a sheet-forming medium.
  • step (A) a cell population containing the desired somatic cells (sheet-forming cells) for forming a sheet-like cell culture is seeded on a culture substrate coated with a cell adhesion component.
  • the seeding on the culture substrate is as described in detail in the above-mentioned method for producing the implant.
  • the sheet-forming cells the cells described in detail in the cells constituting the implant can be used.
  • the culture medium is as described in detail above.
  • the method and concentration of coating with the cell adhesion component and the masking component are as detailed in the coating of the medium containing the masking component.
  • a cell population containing sheet-forming cells seeded on a culture substrate is sheet-cultured in a sheet-forming medium.
  • the sheet-forming culture is as described in detail in the above-mentioned graft-forming culture.
  • the sheeting medium in the method of this aspect is as detailed above, except that it may or may not contain cell adhesion components.
  • the sheeting medium comprises a cell adhesive component. In another embodiment, the sheeting medium is free of cell adhesive components.
  • Another aspect of the present disclosure relates to a method of evaluating the surface of a culture medium for producing a sheet-like cell culture.
  • Such a method includes the following steps: (I) The step of coating the surface of the culture substrate with the cell adhesion component, thereby improving the adhesion of the cells to the surface of the culture substrate. (Ii) Further, a step of coating the surface of the culture substrate with a masking component, thereby reducing the adhesion of cells to the surface of the culture substrate, (Iii) A step of seeding cardiomyocytes at a density reaching confluence on a culture substrate coated with a cell adhesion component and a masking component. (Iv) A step of forming a sheet-like cell culture by sheet-forming culture in a sheet-forming medium, and (v) a step of evaluating a peeling state of the sheet-like cell culture from the surface of the culture substrate.
  • evaluating the surface of the culture substrate means that the sheet-like cell culture is cultured in a sheet using a culture medium coated with a medium containing a cell adhesive component and a masking component. It means that it is evaluated whether or not it has an appropriate substrate surface as a sheeted culture substrate through the evaluation of the peeled state. Since the cells used for producing the sheet-shaped cell culture have different peeling states from the culture base material for each lot, it is necessary to evaluate the surface of the culture base material for each lot of cells. According to the method of this aspect, the surface of the culture medium can be evaluated easily and accurately.
  • step (i) the surface of the culture substrate is coated with a cell adhesive component.
  • the cell adhesion component and coating are as described in detail in the above-mentioned method for producing a graft.
  • step (ii) the surface of the cell substrate coated in step (i) is further coated with a masking component.
  • Steps (iii) and (iv) are as described in detail in the method for producing a sheet-shaped cell culture.
  • step (v) the exfoliation state of the sheet-shaped cell culture from the surface of the culture substrate is evaluated.
  • evaluation the exfoliation state means evaluating the presence or absence of exfoliation of the sheet-like cell culture from the culture substrate and the type of exfoliation when sheet-forming culture and / or exfoliation work is performed using the coated culture substrate. Means to do. By evaluating such a peeled state, the surface of the culture medium can be evaluated.
  • exfoliation mainly include spontaneous exfoliation and exfoliation by exfoliation work, and those that can be exfoliated by exfoliation work are more likely to cause shrinkage of the sheet-like cell culture due to the intercellular binding force. It can be evaluated as a preferable surface of the culture substrate. For the evaluation, a person skilled in the art can select any method such as visual inspection or image analysis.
  • the sheet cell culture produced by the production method of the present disclosure comprises cardiomyocytes.
  • the sheet cell culture produced by the production method of the present disclosure comprises cardiomyocytes, fibroblasts, vascular endothelial cells and / or parietal cells.
  • the sheet cell cultures of the present disclosure are useful in treating diseases that are ameliorated by the application of sheet cell cultures, such as various diseases associated with tissue abnormalities. Therefore, in one aspect, the sheet cell cultures of the present disclosure are intended for use in the treatment of diseases that are ameliorated by the application of sheet cell cultures, particularly those associated with tissue abnormalities.
  • the sheet-shaped cell cultures of the present disclosure have similar properties peculiar to constituent cells except that they have higher mechanical strength than conventional sheet-shaped cell cultures, and therefore at least conventional myoblasts. It can be applied to tissues and diseases that can be treated with sheet-like cell cultures containing cells or fibroblasts.
  • the tissues to be treated include, for example, myocardium, cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear, bone marrow, stomach, etc.
  • Examples include the gastrointestinal tract such as the small intestine, duodenum, and large intestine.
  • the diseases to be treated are not limited, for example, heart diseases (for example, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.), corneal diseases (for example, corneal epithelial stem cell exhaustion, corneal membrane).
  • heart diseases for example, myocardial injury (myocardial infarction, cardiac trauma), cardiomyopathy, etc.
  • corneal diseases for example, corneal epithelial stem cell exhaustion, corneal membrane.
  • Eye / chemical corrosion corneal ulcer, corneal opacification, corneal perforation, corneal scar, Stevens Johnson syndrome, ocular herbitis, etc.
  • retinal disease eg, retinal pigment degeneration, age-related yellow spot degeneration, etc.
  • Esophageal disease eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)
  • skin disease eg, skin injury (trauma, burn), etc.
  • joint disease eg, osteoarthritis
  • Cartilage disease eg, cartilage damage
  • Liver disease eg, chronic liver disease
  • Pancreatic disease eg, diabetes
  • Dental disease eg, periodontal disease, etc.
  • Kidney disease eg, renal failure
  • thyroid disease eg, hypothyroidism
  • muscle disease eg, muscle injury, myitis, etc.
  • Patent Document 1 Non-Patent Document 1 Tanaka et al., J Gastroenterol. 2013; 48 (9): 1081-9.
  • the sheet-like cell cultures of the present disclosure can also be fragmented to an injectable size and injected at the site requiring treatment for greater efficacy than injection with a single cell suspension (Wang et. al., Cardiovasc Res. 2008; 77 (3): 515-24). Therefore, such a use is also possible for the sheet-shaped cell culture of the present disclosure.
  • Another aspect of the present disclosure relates to a method of treating a disease in a subject in need thereof, including applying an effective amount of the implant produced by the method of the present disclosure to a subject in need thereof.
  • the diseases to be treated are as described above.
  • the term “treatment” shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at the cure, temporary remission or prevention of disease, etc.
  • the term “treatment” is medically acceptable for a variety of purposes, including delaying or stopping the progression of a disease associated with a tissue abnormality, regressing or eliminating a lesion, preventing the onset or recurrence of the disease, and the like. Including interventions to be performed.
  • an ingredient that enhances the viability, engraftment and / or function of the implant, other active ingredients useful for treating the target disease, etc. are used in combination with the implant of the present disclosure. be able to.
  • the treatment method of the present disclosure may further include the step of producing the implant of the present disclosure according to the production method of the present disclosure.
  • the treatment method of the present disclosure serves as a cell (for example, skin cells, blood cells, etc. when using iPS cells) or a source of cells for producing a graft from a subject before the step of producing the graft. It may further include the step of collecting tissue (for example, skin tissue, blood, etc. when using iPS cells).
  • the subject from which the cell or tissue from which the cell is source is collected is the same individual as the subject to whom the cell culture, composition, implant, or the like is administered.
  • the subject from which the cell or tissue from which the cell is sourced is harvested is a separate entity of the same species as the subject receiving the administration, such as a cell culture, composition, or implant.
  • the subject from which the cell or tissue that is the source of the cell is collected is an individual different from the subject to which the implant or the like is administered.
  • the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progression (for example, size, weight, number of sheet-like cell cultures, etc.).
  • the amount is preferably an amount that prevents the onset and recurrence of the disease or cures the disease.
  • an amount that does not cause an adverse effect exceeding the benefit of administration is preferable.
  • Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art.
  • the size of the tissue lesion to be treated can be an important index for determining the effective amount.
  • Examples of the administration method include intravenous administration, intramuscular administration, intraosseous administration, intrathecal administration, and direct application to tissues.
  • the frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained.
  • the cell culture, composition, sheet-like cell culture or the like of the present invention may be fixed to the target tissue by a locking means such as a suture or a staple.
  • human iPS cells maintained and cultured in a culture medium containing no feeder cells are cultured on EZSphere (Asahi Glass) for one day in StemFit AK03 medium (Ajinomoto) containing 10 ⁇ M Y27632 (Wako Pure Chemical Industries). Then, the obtained embryo-like body was cultured in a culture medium containing Actibin A, bone-forming protein (BMP) 4, and basic fibroblast growth factor (bFGF), and further, Wnt inhibitor (IWP3) and BMP4 inhibition were performed.
  • EZSphere Asahi Glass
  • StemFit AK03 medium Ajinomoto
  • bFGF basic fibroblast growth factor
  • Human myocardial cells derived from iPS cells were obtained by culturing in a culture medium containing a drug (Dorsomorphin) and a TGF ⁇ inhibitor (SB431542) and then in a culture medium containing VEGF and bFGF.
  • the proportion of cardiomyocytes in the resulting cell population ranged from 50% to 90%.
  • Example 1 Comparison between FBS-containing medium and PL-containing medium Using the cell population obtained above containing cardiomyocytes induced to differentiate from human iPS cells, the sheeting culture conditions were examined. 20% FBS or 5% human platelet lysate was added to DMEM / F12 medium as a sheeting medium. Laminin (iMatrix-511) as a cell adhesion component was further added to the sheeting medium containing the platelet lysate at 0.1 ⁇ g / mL, 0.25 ⁇ g / mL or 0.5 ⁇ g / mL, respectively. In addition, the Rho-kinase inhibitor Y27632 was added to the sheeting medium only on the first day of the sheeting culture.
  • the cell population containing cardiomyocytes was seeded in a temperature-responsive culture dish (UpCell (R) , CellSeed) at a density of 1.5 ⁇ 10 6 cells / cm 2 and placed in an environment of 37 ° C. and 5% CO 2 for 3 days. It was cultured. As the temperature-responsive culture dish, the same one as the culture solution (however, Y27632 was not included) was put therein, and the culture dish was incubated overnight at 37 ° C. and coated. After culturing, the sheet-shaped cell culture containing cardiomyocytes was exfoliated from the culture dish.
  • UpCell (R) UpCell (R) , CellSeed
  • Example 2 Sheeting in serum-free medium Similar to Test Example 1, 0.1 ⁇ g / mL of standard medium with 20% FBS added to DMEM / F12 medium and laminin (iMatrix-511) without adding blood-derived components was added. Sheet culture was performed using each of the medium (E6) as a sheet medium. The results are shown in the table below.
  • Example 3 Examination of cell adhesion components
  • laminin iMatrix-5111
  • vitronectin VTN
  • Sheeting culture was performed using -N) and fibronectin ( R) (Takarabio).
  • R fibronectin
  • the recommended concentrations are 0.5 ⁇ g / cm 2 , 0.5 ⁇ g / cm 2 and 8 ⁇ g / cm 2 , respectively.
  • sheet-forming culture was carried out using the recommended concentration and a concentration of 1/10 of the recommended concentration.
  • Example 4 Examination of cell condition and optimum concentration We investigated how the cell adhesion component affects the step of removing undifferentiated cells that can affect the activity of cells. In order to confirm whether the optimum concentration of the cell adhesion component that can be used for sheeting culture changes depending on the state of the cells, three types of cells were used for examination. As the highly active cells, a cell population containing myocardial cells induced to differentiate from the human iPS cells obtained above (no step of freezing and thawing the cells, no step of removing undifferentiated cells) was used as the above-mentioned medium-active cells.
  • the cell population containing the myocardial cells induced to differentiate from the human iPS cells obtained in 1 above was once frozen and thawed, and the cells containing the myocardial cells induced to differentiate from the human iPS cells obtained above were regarded as low-activity cells. Undifferentiated cells were removed from the population, then frozen and thawed.
  • the treatment for removing undifferentiated cells was carried out in order by using the heat treatment described in WO2017 / 038562, the sugar-free medium culture method described in WO2007 / 0888874, and the anti-CD30 antibody-binding drug treatment described in WO2016 / 072519.
  • Example 5 Examination of coating with cell adhesion component and masking component DMEM / F12 investigated how the peeling state of sheet-shaped cell culture changes when the cell culture dish is coated with a masking component such as FBS together with the cell adhesion component. 0%, 0.31%, 0.63%, 1.25%, 2.5%, 5%, 10%, 20% FBS and 0.076 ⁇ g / cm 2 , 0.76 ⁇ g / cm 2 , 7 A total of 40 coating solutions were prepared with .6 ⁇ g / cm 2 and 76 ⁇ g / cm 2 of iMatrix-511, which were added to each well of a temperature responsive culture dish and incubated overnight at 37 ° C. for coating.
  • a masking component such as FBS together with the cell adhesion component.
  • HBSS manufactured by Invitrogen
  • the culture solution was removed, HBSS (+) was added, and the mixture was allowed to stand at room temperature for 1 hour and 30 minutes for peeling work.
  • the exfoliated state of the sheet-shaped cell culture is shown in Table 3 and FIG. “Peeling” of the colorless cells in Table 3 indicates spontaneous peeling, and “peeling” of the gray cells indicates peeling by the peeling operation. X indicates that the material was not peeled off.
  • the peeling state of the sheet-shaped cell culture can be controlled so that the natural peeling can be suppressed by adjusting the concentrations of the masking component and the cell adhesion component, and the peeling can be performed only by the peeling operation.
  • the present invention it is possible to obtain a high-quality sheet-shaped cell culture when forming a sheet-shaped cell culture using cells or the like induced to differentiate from pluripotent stem cells.
  • a high-quality sheet-shaped cell culture even in the production of a xeno-free sheet-shaped cell culture used for clinical use, it becomes possible to easily form a high-quality sheet-shaped cell culture.

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Abstract

La présente invention aborde le problème de la fourniture : d'un procédé de production d'une culture cellulaire de type feuille de haute qualité contenant une variété de cellules telles que des cardiomyocytes, en particulier, des cardiomyocytes dérivés de cellules souches pluripotentes, les cellules conservant fortement leur fonction ; d'une culture cellulaire de type feuille produite à l'aide du procédé ; et d'un procédé de traitement d'une maladie à l'aide de la culture cellulaire de type feuille. Le problème est résolu par un procédé de production de culture cellulaire de type feuille comprenant : (a) une étape d'étalement d'une population de cellules comprenant des cardiomyocytes sur un substrat de culture ; et (b) une étape de culture de la population de cellules étalée avec un milieu de formation de feuille comprenant un constituant d'adhérence cellulaire permettant de former une feuille.
PCT/JP2020/037084 2019-09-30 2020-09-30 Procédé de formation de feuille de cardiomyocytes WO2021065984A1 (fr)

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CN112272699A (zh) * 2018-07-06 2021-01-26 株式会社迈傲锐治 细胞片的制造方法、心肌细胞片和用于制造心肌细胞片的试剂盒

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WO2016076368A1 (fr) * 2014-11-12 2016-05-19 テルモ株式会社 Feuillet de cellules myocardiques

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WO2016076368A1 (fr) * 2014-11-12 2016-05-19 テルモ株式会社 Feuillet de cellules myocardiques

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Title
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112272699A (zh) * 2018-07-06 2021-01-26 株式会社迈傲锐治 细胞片的制造方法、心肌细胞片和用于制造心肌细胞片的试剂盒

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