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WO2020067439A1 - Procédé de gainage pour cardiomyocytes - Google Patents

Procédé de gainage pour cardiomyocytes Download PDF

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Publication number
WO2020067439A1
WO2020067439A1 PCT/JP2019/038192 JP2019038192W WO2020067439A1 WO 2020067439 A1 WO2020067439 A1 WO 2020067439A1 JP 2019038192 W JP2019038192 W JP 2019038192W WO 2020067439 A1 WO2020067439 A1 WO 2020067439A1
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WIPO (PCT)
Prior art keywords
cells
cell
culture
graft
cardiomyocytes
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Ceased
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PCT/JP2019/038192
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English (en)
Japanese (ja)
Inventor
芳樹 澤
繁 宮川
賢二 大山
文哉 大橋
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Terumo Corp
University of Osaka NUC
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Terumo Corp
Osaka University NUC
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Priority to JP2020549444A priority Critical patent/JP7256818B2/ja
Publication of WO2020067439A1 publication Critical patent/WO2020067439A1/fr
Anticipated expiration legal-status Critical
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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
    • A61L27/36—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
    • 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
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a method, a graft manufactured using the method, a method for treating a disease using the graft, and the like.
  • Non-Patent Document 1 As a source of cardiomyocytes used for preparing such grafts, cardiomyocytes derived from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have recently been receiving attention. Production of sheet-shaped cell cultures containing such pluripotent stem cell-derived cardiomyocytes and treatment experiments on animals have been attempted (Non-patent Documents 2 and 3). However, the development of sheet-like cell cultures containing cardiomyocytes derived from pluripotent stem cells has only just begun, and there are still many unknowns regarding their functional properties and factors affecting them.
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • the present disclosure relates to a method for producing a transplant such as a high-quality sheet-shaped cell culture, which includes iPS cell-derived differentiation-inducing cells, particularly cardiomyocytes, while maintaining the function of the cells at a high level, and using the method. It is an object of the present invention to provide a graft manufactured by the method, a method for treating a disease using the graft, and the like.
  • a graft containing cardiomyocytes to be used for transplantation When preparing a graft containing cardiomyocytes to be used for transplantation, it must be prepared in a xeno-free environment. In addition, when using fetal bovine serum (FBS) or a serum-free medium, a problem such as insufficient pulsation of cardiomyocytes has been found.
  • FBS fetal bovine serum
  • the present inventors have attempted to prepare a sheet-shaped cell culture for living body transplantation using iPS cell-derived cardiomyocytes having a sufficient function even if prepared in a xenofree environment, Using a platelet (lysate), a new finding was found that a stronger pulsation could be observed in a shorter time than when serum was used. Based on such findings, further research has been carried out to demonstrate that it is possible to produce high-quality transplants that can withstand clinical applications in the preparation of transplants containing various somatic cells differentiated from iPS cells. As a result, the present invention has been completed.
  • the present invention relates to the following: [1] A method for producing a transplant containing cells induced to differentiate from pluripotent stem cells; (A) a step of inoculating a cell population containing the cells on a culture substrate, and (b) a step of explanting and culturing the inoculated cell population with an explant forming medium containing a platelet lysate;
  • the above method comprising: [2] The method of [1], wherein the pluripotent stem cells are iPS cells. [3] The method of [1] or [2], wherein the transplant is a sheet-shaped cell culture.
  • a method for accelerating the onset of pulsation of cardiomyocytes in a graft containing cardiomyocytes differentiated from iPS cells, wherein the cell population containing cardiomyocytes is transplanted with a graft-forming medium containing platelet lysate comprising culturing a piece.
  • a transplant such as a sheet-shaped cell culture, having a higher quality than before can be produced with high efficiency from a cell population including differentiation-inducing cells, particularly cardiomyocytes, that have been induced to differentiate from iPS cells.
  • differentiation-inducing cells particularly cardiomyocytes
  • cardiomyocytes differentiated from iPS cells it is possible to prepare grafts retaining desired properties at a high level. It is possible to provide a graft which is very suitable for transplantation.
  • FIG. 1 is a table showing a comparison of the results when sheet culture was performed using the serum-free medium and the PL-containing medium in Example 2 respectively.
  • the PL-containing medium was used, sheets were obtained in all lots after two days of culture, but when the serum-free medium was used, sheets could not be formed due to breakage in half of the lots.
  • FIG. 2 is a graph comparing the state of pulsation of a sheet-shaped cardiomyocyte culture prepared using an FBS-containing medium and using a PL-containing medium. A stronger pulsation was observed earlier in the medium prepared with the PL-containing medium than in the medium prepared with the FBS-containing medium.
  • the present disclosure provides a method for producing an implant comprising cells induced to differentiate from pluripotent stem cells; (A) inoculating a cell population containing the cells on a culture substrate, and (b) explanting and culturing the inoculated cell population in a medium containing platelet lysate;
  • the method relates to:
  • the term “graft” refers to a structure for transplantation into a living body, and particularly refers to a structure for transplantation containing cells as a component.
  • the so-called suspension state in which at least one state in which cells are adhered to each other in a transplant to form a certain shape as a whole, and each and every cell is present separately, is referred to as the present disclosure.
  • the implant is an implantable structure that does not include structures other than cells and cell-derived substances (eg, a scaffold).
  • Examples of the graft in the present disclosure include, but are not limited to, a sheet-shaped cell culture, a spheroid, a cell aggregate, a cell suspension, a cell suspension containing fibrin gel, and a cell using a nanofiber. Cultures and the like are preferable, and a sheet cell culture or a spheroid is preferable, and a sheet cell culture is more preferable.
  • the “sheet-shaped cell culture” refers to a cell in which cells are connected to each other to form a sheet.
  • spheroid refers to a cell in which cells are connected to each other to form a substantially spherical shape.
  • the cells may be connected to each other directly (including via a cellular element such as an adhesion molecule) and / or via an intermediary substance.
  • the intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells, and examples thereof include an extracellular matrix.
  • the intervening substance is preferably derived from cells, particularly from cells constituting a sheet-shaped cell culture or spheroid.
  • the sheet-shaped cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminate (multilayer), for example, two or three layers, Four layers, five layers, six layers, etc.). Further, the sheet-shaped cell culture may have a three-dimensional structure having a thickness exceeding the thickness of one cell without the cells showing a clear layer structure. For example, in the vertical cross section of the sheet-shaped cell culture, the cells may not be uniformly arranged in the horizontal direction, but may be non-uniformly arranged (for example, in a mosaic).
  • the implants of the present disclosure especially sheet cell cultures, preferably do not contain a scaffold. Scaffolds are sometimes used in the art to attach cells on and / or to their surfaces and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride ( Although PVDF) membranes and the like are known, the implants of the present disclosure can maintain their physical integrity without such a scaffold.
  • the sheet-shaped cell culture of the present disclosure preferably includes only a substance derived from the cells constituting the graft, and does not include any other substances.
  • the cell may be a cell derived from a different species or a cell derived from the same species.
  • heterologous cell means a cell derived from an organism of a different species from the recipient when a sheet-shaped cell culture is used for transplantation.
  • cells derived from monkeys and pigs correspond to xenogeneic cells.
  • Allogeneic cell means a cell derived from an organism of the same species as the recipient.
  • human cells correspond to cells derived from the same species.
  • Allogeneic cells include autologous cells (also called autologous cells or autologous cells), that is, cells derived from the recipient and allogeneic non-autologous cells (also called allogeneic cells). Autologous cells are preferred in the present disclosure because rejection does not occur even when transplanted. However, it is also possible to use xenogeneic cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be necessary to suppress rejection.
  • cells other than autologous cells that is, non-autologous cells of the same species as cells of xenogeneic origin may be collectively referred to as non-autologous cells.
  • the cells are autologous cells or allogeneic cells. In one aspect of the present disclosure, the cells are autologous cells (including autologous iPS cells). In another aspect of the present disclosure, the cells are allogeneic cells (including allogeneic iPS cells).
  • Cells constituting the graft of the present disclosure are cells that have been induced to differentiate from pluripotent stem cells, and are not particularly limited as long as they can form a graft such as a sheet-shaped cell culture.
  • Cells include, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg, tissue stem cells such as myoblasts, cardiac stem cells, mesenchymal stem cells, etc.).
  • adherent somatic cells eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin
  • the somatic cells may be stem cells, particularly those differentiated from iPS cells (iPS cell-derived adherent cells).
  • iPS cell-derived adherent cells include, for example, iPS cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells Gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes and the like.
  • the cells constituting the graft can be derived from any organism that can be treated with the graft. Such organisms include, without limitation, 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 graft is not particularly limited, and may be composed of only one type of cell, or may be a type using 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% or more at the end of the graft formation. More preferably, it can be 75% or more.
  • the culture substrate is not particularly limited as long as cells can form a cell culture thereon, and includes, for example, containers of various materials and / or shapes, a solid or semi-solid surface in the container, and the like.
  • the container is preferably made of a structure / material that does not allow the passage of a liquid such as a culture solution. Examples of such a material include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, and polydimethyl.
  • Acrylamide, metal for example, iron, stainless steel, aluminum, copper, brass
  • metal for example, iron, stainless steel, aluminum, copper, brass
  • the container preferably has at least one flat surface.
  • a culture container having a bottom surface formed of a culture substrate capable of forming a cell culture and a liquid impermeable side surface.
  • culture vessels include, but are not limited to, cell culture dishes, cell culture bottles, and the like.
  • the bottom of the container may be transparent or opaque. If the bottom surface of the container is transparent, observation and counting of cells can be performed from the back side of the container.
  • the container may have a solid or semi-solid surface inside. Examples of the solid surface include plates and containers made of various materials as described above, and examples of the semi-solid surface include a gel and a soft polymer matrix.
  • the culture substrate may be prepared using the above materials, or a commercially available substrate may be used.
  • Preferred culture substrates include, but are not limited to, for example, a substrate having an adhesive surface suitable for forming a sheet-shaped cell culture, and a substrate having a low adhesive surface suitable for forming a spheroid. And / or a substrate having a uniform well-like structure.
  • a hydrophilic compound such as a collagen gel or a hydrophilic polymer
  • collagen And extracellular matrices such as fibronectin, laminin, vitronectin, proteoglycan, and glycosaminoglycan
  • substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, and integrin family.
  • such substrates are commercially available (e.g., Corning (R) TC-Treated Culture Dish, Corning , etc.).
  • temperature-responsive gel obtained by crosslinking soft agar, poly (N-isopropylacrylamide) (PIPAAm) with polyethylene glycol (PEG), polyhydroxyethyl methacrylate (A substrate coated with a non-cell-adhesive compound such as a hydrogel such as poly (HEMA) or 2-methacryloyloxyethylphosphorhoscholine (MPC) polymer and / or a substrate having a uniform uneven structure on the surface.
  • a non-cell-adhesive compound such as a hydrogel such as poly (HEMA) or 2-methacryloyloxyethylphosphorhoscholine (MPC) polymer and / or a substrate having a uniform uneven structure on the surface.
  • HEMA poly (HEMA) or 2-methacryloyloxyethylphosphorhoscholine
  • MPC 2-methacryloyloxyethylphosphorhoscholine
  • the culture substrate may be entirely or partially transparent or opaque.
  • the culture substrate may be coated on its surface with a material whose properties change in response to a stimulus, for example, temperature or light.
  • 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, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfuryl methacryl Amide), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-e
  • a predetermined stimulus By applying a predetermined stimulus to these materials, their physical properties, for example, hydrophilicity or hydrophobicity, can be changed, and the detachment of the cell culture adhered on the materials can be promoted.
  • Culture dishes coated with a temperature-responsive materials are commercially available (e.g., UpCell of CellSeed Inc. (R)), they can be used in the production method of the present disclosure.
  • the culture substrate may be in various shapes.
  • the area 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 , and the like.
  • a circular culture dish having a diameter of 10 cm is used as a culture substrate.
  • the area is 56.7 cm 2 .
  • the culture surface may be flat or may have an uneven structure. In the case of having an uneven structure, it is preferable to have a uniform uneven structure.
  • inducible pluripotent stem cells or “iPS cells” are terms well known in the art, and by introducing multiple genes into somatic cells, three germ layers, that is, endoderm, mesoderm and A cell that has acquired the ability to differentiate into all lineage cells belonging to the ectoderm. That is, induced pluripotent stem cells or iPS cells are cells having pluripotency and self-renewal ability induced by introducing a gene.
  • the iPS cells are first cultured in suspension to form aggregates of any of the above three germ layers, and then the cells that form the aggregates are identified. Cells are induced to differentiate.
  • the iPS cells can be any mammalian cells, but are preferably iPS cells derived from human cells.
  • differentiation-inducing cells derived from iPS cells means any cells that have been subjected to differentiation-inducing treatment so as to differentiate from iPS cells into cells of a specific type.
  • Non-limiting examples of differentiation-inducing cells include muscular cells such as cardiomyocytes and skeletal myoblasts, neuronal cells such as neuronal cells, oligodendrocytes and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells.
  • hematopoietic cells such as bone marrow cells
  • immune cells such as T cells, NK cells, NKT cells, dendritic cells, B cells, cells constituting organs such as hepatocytes, pancreatic ⁇ cells, kidney cells,
  • progenitor cells and somatic stem cells that differentiate into these cells are included.
  • progenitor cells and somatic stem cells include, for example, mesenchymal stem cells in cardiomyocytes, pluripotent heart progenitor cells, unipotent heart progenitor cells, neural stem cells in nervous system cells, hematopoietic cells and immune cells.
  • hematopoietic stem cells and lymphoid stem cells.
  • Induction of differentiation of iPS cells can be performed using any known technique. For example, differentiation induction from iPS cells to cardiomyocytes can be performed based on the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, and WO 2014/185358.
  • the differentiation-inducing cell may be a cell derived from an iPS cell into which any useful gene other than a gene for reprogramming has been introduced.
  • Non-limiting examples of such cells include, for example, iPS cells into which the gene for the chimeric antigen receptor described in Themeli M. et al. Nature Biotechnology, vol. 31, vol. 10, pp. 928-933, 2013 has been introduced. And T cells derived therefrom.
  • cells into which any useful gene has been introduced after differentiation induction from iPS cells are also included in the differentiation-inducing cells of the present invention.
  • One aspect of the present disclosure relates to a method for producing a high-quality sheet-shaped cell culture containing cardiomyocytes differentiated from iPS cells.
  • the method of the present disclosure includes the following steps (a) and (b): (A) a step of seeding a cell population containing cardiomyocytes differentiated from iPS cells on a culture substrate; and (b) a sheet culture of the seeded cell population with a sheeting medium containing a platelet lysate. Process.
  • cardiomyocytes means cells having characteristics of cardiomyocytes.
  • the characteristics of the cardiomyocyte include, but are not limited to, for example, the expression of a cardiomyocyte marker, the presence of an autonomous beat, and the like.
  • Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (alias SIRPA or SHPS-1), KDR (alias CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM and the like.
  • the iPS cell-derived cardiomyocytes are c-TNT positive and / or CD172a positive.
  • the 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 container provided with the culture substrate.
  • a device suitable for the operation of injecting the cell suspension such as a dropper or pipette, can be used.
  • the seeding density of the cells is determined at a density capable of forming a sheet-shaped cell culture, and the density may vary depending on the desired cells. However, those skilled in the art can select an appropriate density from techniques known in the art. can do.
  • a sheet-shaped cell culture containing cardiomyocytes it may 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, densities that reach confluence, i.e., densities at which cells are expected to cover the entire adhesive surface of the culture vessel upon seeding, e.g., upon seeding, cells contact each other , A density at which contact inhibition occurs, or a density at which cell growth is substantially stopped by contact inhibition.
  • the upper limit of the seeding density is not particularly limited. However, if the seeding density is excessively high, the number of dead cells increases, resulting in inefficiency.
  • the seeding density is, for example, from about 1.0 ⁇ 10 6 / cm 2 to about 1.0 ⁇ 10 7 / cm 2 , from about 1.0 ⁇ 10 6 / cm 2 to about 5 0.0 ⁇ 10 6 / cm 2 , about 1.0 ⁇ 10 6 / cm 2 to about 3.0 ⁇ 10 6 / cm 2 , about 1.5 ⁇ 10 6 / cm 2 to about 1.0 ⁇ 10 7 pieces / cm 2 , about 1.5 ⁇ 10 6 pieces / cm 2 to about 5.0 ⁇ 10 6 pieces / cm 2 , about 1.5 ⁇ 10 6 pieces / cm 2 to about 3.0 ⁇ 10 6 pieces / cm 2 , about 2.0 ⁇ 10 6 pieces / cm 2 to about 1.0 ⁇ 10 7 pieces / cm 2 , about 2.0 ⁇ 10 6 pieces / cm 2 to about 5.0 ⁇ 10 6 pieces / Cm 2 , about 2.0 ⁇ 10 6 / cm 2 to about 3.0 ⁇ 10 6 / cm 2 , and the like.
  • the seeding density is, for
  • the culture substrate to be seeded is as described in detail above, but in a preferred embodiment, is a culture substrate having a surface coated with a cell adhesive component such as an extracellular matrix or a cell adhesion factor.
  • Cell adhesion components include, but are not limited to, for example, extracellular matrices such as collagen, fibronectin, laminin, vitronectin, proteoglycans, glycosaminoglycans, cadherin family, selectin family, cell adhesion factors such as integrin family And modifications thereof, for example, laminin 511 (a modification of laminin), VTN-N (a modification of vitronectin), and RetroNectin (R) (a modification of fibronectin).
  • the seeded cell population may contain other cells as long as it contains cardiomyocytes differentiated from iPS cells.
  • a cell population obtained by inducing differentiation from iPS cells into cardiomyocytes may include, for example, fibroblasts and vascular endothelial cells in addition to cardiomyocytes.
  • a cell population obtained by inducing differentiation from iPS cells using the method described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 or WO 2014/185358, or the like is used as it is. It may be used, or may be used after carrying out cryopreservation or pre-culture.
  • the cell population to be seeded is induced to differentiate from iPS cells, and then inoculated on a culture substrate (preferably on a planar culture substrate) to perform adherent culture. is there. Cryopreservation and thawing may be performed before or after such adherent culture. By performing the adherent culture, it is possible to achieve the formation of a high-quality graft at a high probability in the subsequent formation of the graft.
  • the formed graft may include vascular endothelial cells, cell walls, fibroblasts in addition to cardiomyocytes.
  • the percentage of cells contained in the graft of the present disclosure may be, for example, about 30-70% of cardiomyocytes, 0.1% to about 20% of vascular endothelial cells, and about 1% to about 40% of mural cells. Good.
  • the culture conditions and the like may be in accordance with the conditions for performing ordinary adhesion culture.
  • the culture may be performed at 37 ° C. under 5% CO 2 using a commercially available culture vessel for adhesion culture.
  • the seeding density of the cells may be any density as long as the density does not prevent adhesion between cells and / or formation of adhesion between the cells and the culture substrate, for example, a subconfluent density, The density may be confluent or higher.
  • the cultivation time may be a time period such that adhesion between cells and / or adhesion between cells and a culture substrate is formed, and specifically, for example, 2 to 168 hours, 2 to 144 hours, 2 to 120 hours It may be about 2 to 96 hours, 2 to 72 hours, 2 to 48 hours, 2 to 24 hours, 2 to 12 hours, 2 to 6 hours, 2 to 4 hours.
  • the seeded cells are cultured on a sheet.
  • a culture for forming the seeded cells as a graft is referred to as a “graft formation culture”, and the graft is a sheet-shaped cell culture, and the culture for forming the seeded cells into a sheet is performed.
  • sheet culture it is referred to as “sheet culture”. Sheeting of the seeded cells can be performed by any known method and conditions. Non-limiting examples of such techniques are described in, for example, JP-A-2010-081829, JP-A-2010-226991, JP-A-2011-110368, JP-A-2011-172925, WO 2014/185517, and the like.
  • the step of forming the seeded cells into a sheet can be achieved, for example, by culturing the cells under conditions that form intercellular adhesion.
  • 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, culture at about 37 ° C., 5% CO 2 .
  • the cultivation can be performed under normal pressure (atmospheric pressure, non-pressurized).
  • the culture can be performed in a container of any size and shape.
  • the size and shape of the sheet-shaped cell culture can be adjusted by adjusting the size and shape of the cell attachment surface of the culture container, or by installing a mold of the desired size and shape on the cell attachment surface of the culture container, It can be adjusted arbitrarily by culturing the cells therein.
  • the time of sheeting culture may vary depending on the type of seeded cells and cell density.
  • the sheets may be seeded at a density of, for example, about 2.1 ⁇ 10 5 cells / cm 2 and cultured for 4 days or more to form sheets.
  • the seeding density reaches a confluent density, that is, when the seeding is performed at a higher density, the period of sheet culture can be shortened, and the culture time may be 2 to 4 days, more preferably 2 to 3 days. .
  • a medium used for graft formation for example, sheeting
  • a graft forming medium sometimes referred to as a sheeting medium particularly when the graft formation is a sheeting
  • a medium capable of forming a cell graft is used.
  • physiological saline various physiological buffers (for example, PBS, HBSS, etc.), or those based on various cell culture base media.
  • basal medium examples 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 and the like.
  • MCDB MCDB102, 104, 107, 120, 131, 153, 199, etc.
  • L15 SkBM, RITC80-7
  • DMEM / F12 examples 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 and the like.
  • the basal medium may be used with its standard composition (for example, as it is commercially available), or its composition may be appropriately changed depending on the cell type and cell conditions. Therefore, the
  • the graft forming medium may contain additives such as normal serum (eg, bovine serum such as fetal bovine serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).
  • normal serum eg, bovine serum such as fetal bovine serum, horse serum, human serum, etc.
  • various growth factors eg, FGF, EGF, VEGF, HGF, etc.
  • the transplant contains no serum different from the species from which the cells contained in the transplant are derived, such as bovine serum and horse serum.
  • the present disclosure is characterized in that a graft forming culture is performed using a graft forming medium containing a platelet lysate instead of or in addition to serum or a growth factor.
  • the graft forming medium comprises platelet lysate but does not contain serum.
  • platelet lysate (PL) refers to a composition rich in growth factors and the like, obtained by repeatedly freezing and thawing platelets.
  • platelet lysates have been known to promote the growth of mesenchymal stem cells.
  • the present inventors have added a platelet lysate to a sheeting medium, thereby achieving a strong autonomic pulsation in a shorter culture period than before. Was observed for the first time.
  • Platelet lysates are commercially available as media additives for cell culture and are known in the art.
  • the platelet lysate can be prepared, for example, by the method described in JP-T-2014-533715, Bieback et al., STEM CELLS, 2009; 27: 2331-2341.
  • it can be prepared by, for example, dissolving a platelet population and removing contaminants such as platelet particles and a membrane therefrom to obtain a supernatant. Lysis of platelets can be achieved through steps such as chemical means (eg, using CaCl 2 ), osmotic means (eg, using distilled water), freeze-thaw means, mechanical disruption means, and the like. Removal of contaminants can be achieved by a method such as centrifugation or filtration.
  • the concentration of the platelet lysate contained in the graft forming medium may be a level generally used in the art, such as 1%, 2.5%, 5%, 10%, 15%, 20%, and the like. May be.
  • the platelet lysate is contained in the graft forming medium in an amount of about 1% to 20%, more preferably about 2% to 10%, and still more preferably about 2.5% to 10%.
  • the graft forming medium may be appropriately replaced during the graft forming culture. Further, the composition of the medium may be changed in accordance with the progress of the graft formation.
  • the present inventors use a sheeting medium to which a Rho kinase (ROCK) inhibitor is added as a medium on the first day of sheeting culture in the production of a sheet-shaped cell culture containing cardiomyocytes derived from iPS cells. As a result, it was newly found that a sheet-shaped cell culture was effectively formed.
  • the sheeting medium used for day 1 sheeting culture 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 sheeting medium may further contain a cell adhesive component.
  • the cell adhesive component is as described in detail above.
  • the culture substrate may or may not be coated with the cell adhesive component.
  • the cell adhesive component contained in the sheeting medium may be the same as or different from the cell adhesive component coating the culture substrate. However, they are preferably the same cell adhesive component.
  • the concentration of the cell-adhesive component contained in the graft-forming medium may vary depending on the type of the cell-adhesive component contained, the state of the cells forming the graft, and the like. For example, when cells having low viability, that is, cells having weak activity, are used, the content of the cell adhesive component is preferably small.
  • the concentration of the cell adhesive component contained in the sheeting medium is about 0.1% and about 0.1% based on the concentration (100%) used when the same cell adhesive component is used as a coating agent for the culture substrate. It may be 5%, about 1%, about 5%, about 10%, about 20%, about 25%, about 50%, about 75%, about 100%, etc.
  • the concentration range of the cell adhesive component contained in the sheeting medium is about 0% based on the concentration (100%) used when the same cell adhesive component is used as a coating agent for the culture substrate. 0.1% 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% 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%, About 5% to about 20%, May the like 5% to about 10%.
  • Another aspect of the present disclosure is a method of hastening the onset of pulsation of cardiomyocytes in a graft containing cardiomyocytes differentiated from iPS cells, comprising:
  • the present invention relates to the above method, comprising culturing the explant in a formation medium.
  • the present inventors compared platelet formation with a serum-free medium by adding a platelet lysate to a graft formation medium when forming a graft containing cardiomyocytes differentiated from iPS cells. It was found that the onset of pulsation of cardiomyocytes was accelerated. This makes it possible to easily form a high-quality implant in a xeno-free environment.
  • Another aspect of the present disclosure relates to a method of treating a disease in a subject, comprising applying an effective amount of an implant produced by the method of the present disclosure to the subject in need thereof.
  • the disease to be treated is as described above.
  • treatment is intended to include all types of medically acceptable prophylactic and / or therapeutic interventions, such as for the cure, temporary remission or prevention of disease.
  • treatment includes medically acceptable treatments for a variety of purposes, including slowing or stopping the progression of a disease associated with tissue abnormalities, regressing or eliminating lesions, preventing the onset of the disease or preventing its recurrence, and the like. Involve interventions.
  • a component that enhances the survival, engraftment, and / or function of a graft, and other active components that are useful for treating a target disease are used in combination with the graft or the like of the present disclosure. be able to.
  • the treatment method of the present disclosure may further include a step of manufacturing the implant of the present disclosure according to the manufacturing method of the present disclosure.
  • the method of treatment of the present disclosure may comprise, prior to the step of producing the graft, cells (eg, somatic cells for inducing iPS cells, such as skin cells, blood cells, etc.) or cells for producing the graft from the subject.
  • the method may further include a step of collecting a tissue (for example, a tissue containing a somatic cell for inducing iPS cells, such as skin tissue or blood) serving as a source of the protein.
  • a tissue for example, a tissue containing a somatic cell for inducing iPS cells, such as skin tissue or blood
  • the subject from whom the cells or tissue from which the cells are to be sourced is harvested is the same individual as the subject to whom a cell culture, composition, or explant is administered.
  • the subject from whom the cells or tissue from which the cells are to be sourced is harvested is a homologous distinct body from the subject to be administered, such as a cell culture, composition, or implant.
  • the subject from which the cells or the tissue from which the cells are sourced is harvested is an individual that is heterogeneous to the subject receiving the administration, such as a graft.
  • an effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing symptoms, or delaying or stopping the progress (eg, size, weight, number, etc. of a graft), Preferably, it is an amount that prevents the onset and recurrence of the disease or cures the disease. Also preferred is an amount that does not cause adverse effects beyond the benefit of administration. Such an amount can be appropriately determined, for example, by a test in a laboratory 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 a tissue lesion to be treated can be an important index for determining an effective amount.
  • the administration method examples 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 administrations are possible if the desired effect is not obtained.
  • the cell culture, the composition, the sheet-shaped cell culture, or the like of the present invention may be fixed to a target tissue by a locking means such as a suture or staple.
  • human iPS cells for clinical use established at Kyoto University iPS Cell Research Institute were used as pluripotent stem cells.
  • Human iPS cells were maintained by a feeder-free method with reference to M. Nakagawa et al., Scientific Reports, 4: 3594 (2014).
  • human iPS cells were induced to differentiate into cardiomyocytes by referring to Miki et al., Cell Stem Cell 16, 16, 699-711, June 4, 2015 and WO 2014/185358 and WO 2017/038562 to induce embryoid bodies.
  • the cells were cultured in a culture solution containing the agent (Dorsomorphin) and a TGF ⁇ inhibitor (SB431542), and then cultured in a culture solution containing VEGF and bFGF to obtain iPS cell-derived human cardiomyocytes.
  • the percentage of cardiomyocytes in the resulting cell population was between 50% and 90%.
  • Example 1 Comparison between FBS-containing medium and PL-containing medium Using the cell population containing cardiomyocytes differentiated from human iPS cells obtained above, sheet-forming culture conditions were examined.
  • a sheet medium was prepared by adding 20% FBS or 5% human platelet lysate to DMEM / F12 medium.
  • laminin iMatrix-5111 was further added as a cell adhesive component at 0.1 ⁇ g / mL, 0.25 ⁇ g / mL or 0.5 ⁇ g / mL, respectively.
  • the Rho kinase inhibitor Y27632 was added to the sheeting medium.
  • Cell population comprising cardiomyocytes, temperature responsive culture dishes at a density of 1.5 ⁇ 10 6 cells / cm 2 (UpCell (R) , Cellseed) seeded, 37 ° C., 3 days of culture in the 5% CO 2 environment did.
  • the temperature-responsive culture dish used was the same as the culture solution (excluding Y27632), and was incubated at 37 ° C. overnight and precoated. After the culture, the sheet-shaped cell culture containing cardiomyocytes was detached from the culture dish.
  • Example 2 Similar to Comparative Example 1 between serum-free medium and PL-containing medium , DMEM / F12 medium supplemented with 0.1 ⁇ g / mL laminin (iMatrix-511), and further supplemented with 5% platelet lysate Were used as sheeting media to perform sheeting culture. The results are shown in FIG. Comparing the sheeting success rate on the second day of sheeting culture, the PL group was 100% successful, whereas the serum-free medium was 50%.
  • the state of the pulsation was photographed using the cell motion imaging system SI8000 (Sony), and the pulsation acceleration (acceleration) and pulsation deformation distance ( contration deformation distance) was calculated and compared between the case where the FBS-containing medium of Example 1 was used and the case where the PL-containing medium of Example 2 was used.
  • the results are shown in FIG. It was confirmed that the group in which the sheet-shaped cell culture was formed using the PL-containing medium had a stronger pulsation than the group using the FBS-containing medium.
  • a high-quality sheet-shaped cell culture can be obtained when a sheet-shaped cell culture is formed using cells or the like that have been induced to differentiate from pluripotent stem cells.
  • a high-quality sheet cell culture can be easily formed.

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Abstract

Le but de la présente invention est de fournir un procédé de production d'un greffon de haute qualité, tel qu'une culture cellulaire en forme de feuille, comprenant une variété de cellules dérivées de cellules SPi, tels que des cardiomyocytes, tout en possédant les fonctions des cellules à un degré élevé; un greffon produit à l'aide dudit procédé; et un procédé de traitement d'une maladie à l'aide du greffon. La solution selon l'invention consiste à fournir un procédé de production d'un greffon, ledit procédé comprenant : (a) une étape d'inoculation d'une population de cellules qui comprend des cellules induites par différenciation à partir d'une cellule SPi sur un substrat de culture, et (b) une étape pour effectuer une culture de formation de greffon de la population de cellules inoculées sur un milieu de formation de greffon qui comprend un lysat plaquettaire.
PCT/JP2019/038192 2018-09-27 2019-09-27 Procédé de gainage pour cardiomyocytes Ceased WO2020067439A1 (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
WO2022080455A1 (fr) * 2020-10-14 2022-04-21 ダ・ヴィンチ ユニバーサル株式会社 Procédé de production de cellules souches/précurseurs de muscle cardiaque et procédé d'inhibition de la fibrose myocardique
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