WO2020067443A1 - Procédé de formation de feuille de cellules somatiques - Google Patents

Procédé de formation de feuille de cellules somatiques Download PDF

Info

Publication number
WO2020067443A1
WO2020067443A1 PCT/JP2019/038197 JP2019038197W WO2020067443A1 WO 2020067443 A1 WO2020067443 A1 WO 2020067443A1 JP 2019038197 W JP2019038197 W JP 2019038197W WO 2020067443 A1 WO2020067443 A1 WO 2020067443A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
culture
sheet
present disclosure
Prior art date
Application number
PCT/JP2019/038197
Other languages
English (en)
Japanese (ja)
Inventor
賢二 大山
文哉 大橋
Original Assignee
テルモ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by テルモ株式会社 filed Critical テルモ株式会社
Priority to JP2020527125A priority Critical patent/JPWO2020067443A1/ja
Publication of WO2020067443A1 publication Critical patent/WO2020067443A1/fr

Links

Images

Classifications

    • 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
    • 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 disclosure relates to a method for producing an implant containing various biological cells, for example, myoblasts, a sheet-like cell culture produced using the method, and a method for treating a disease using the sheet-like cell culture. And so on.
  • Non-Patent Document 1 fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, iPS cells, etc. for repairing myocardial tissue damaged by ischemic heart diseases such as angina pectoris and myocardial infarction.
  • Patent Document 1 a cell structure formed using a scaffold and a sheet-shaped cell culture in which cells are formed in a sheet shape have been developed (Patent Document 1, Non-Patent Document 2).
  • sheet-shaped cell culture to treatment use of cultured epidermis sheet for skin damage due to burns, use of corneal epithelial sheet-shaped cell culture for corneal injury, oral mucosal sheet for endoscopic resection of esophageal cancer
  • Studies on the use of cell cultures are underway, and some of them are in the stage of clinical application.
  • the present disclosure relates to a method for producing a high-quality sheet-shaped cell culture, including various living organism-derived cells, for example, myoblasts while maintaining the function of the cells at a high level, and a sheet-shaped cell produced using the method. It is an object to provide a cell culture, a method for treating a disease using the sheet-shaped cell culture, and the like.
  • Such an implant When preparing a cell-containing graft, it must be prepared in a xeno-free environment. Therefore, such an implant is usually prepared using human-derived, preferably recipient-derived serum, instead of xenogenic serum such as fetal bovine serum, so as to not contain impurities derived from the manufacturing process.
  • human-derived, preferably recipient-derived serum instead of xenogenic serum such as fetal bovine serum, so as to not contain impurities derived from the manufacturing process.
  • xenogenic serum such as fetal bovine serum
  • the present inventors have attempted to prepare a sheet-shaped cell culture for living body transplantation using myoblasts having sufficient functions even when prepared in a xeno-free environment. Even when lysate was used, a new finding was found that a sheet-shaped cell culture could be produced which was comparable to the case where serum was used. Further research based on such findings and the use of a platelet lysate can produce high-quality grafts that can withstand clinical applications in the preparation of grafts containing various biological cells. This led to the completion of the present invention.
  • the present invention relates to the following: [1] A method for producing a graft containing adherent cells; (A) inoculating a cell population containing the adherent cells on a culture substrate, and (B) incubating the seeded cell population with a medium containing platelet lysate;
  • the above method comprising: [2] The method of [1], wherein the adherent cells are cells into which no gene has been introduced. [3] The method of [1] or [2], wherein the medium does not contain serum different from the species from which the cells contained in the graft are derived. [4] The method of [1] to [3], wherein the medium further contains a cell adhesive component.
  • a graft such as a sheet-shaped cell culture can be produced with high quality and high efficiency from a cell population containing various biological cells, for example, myoblasts.
  • grafts such as sheet-shaped myoblast cell cultures, which exhibit an effect when transplanted into a living body
  • a graft having desired properties such as cytokine production at a high level. It is possible to provide a graft or the like very suitably.
  • FIG. 1 is a photograph showing the results of a test for forming a sheet-shaped skeletal myoblast culture.
  • A is a sheet-shaped skeletal myoblast cell culture formed in an FBS-containing medium
  • BD are sheet-shaped skeletal myoblast cell cultures formed in a PL-containing medium.
  • B is for UltraGro
  • C is for UltraGRO @ PURE
  • D is for UltraGRO @ ADVANCE.
  • 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 sheet-shaped cell culture of the present disclosure preferably does not include a scaffold (support). 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 sheet-shaped cell culture of the present disclosure can maintain its 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 sheet-shaped cell culture, and does not include 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).
  • the cells constituting the graft of the present disclosure are not particularly limited as long as they can form the graft, and include, for example, adherent cells (adherent cells).
  • adherent cells adherent cells
  • the cells constituting the graft are cells into which no gene has been introduced.
  • Adherent 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, embryonic stem cells, mesenchymal stem cells, etc.). Somatic cells may be those differentiated from stem cells.
  • 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.
  • stem cells eg, tissue stem cells such as myoblasts, cardiac stem cells
  • Non-limiting examples of cells constituting the graft include, for example, myoblasts (eg, skeletal myoblasts), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, muscle tissue, Endometrial, placenta, cord blood-derived cells), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells, retinal pigment epithelial cells, Nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, liver parenchymal cells), pancreatic cells (eg, pancreatic islet cells), kidney cells, adrenal cells, periodontal ligament cells, gingiva Cells, periosteal cells, skin cells and the like.
  • myoblasts eg, skeletal myoblasts
  • Non-limiting examples of stem cell-derived adherent cells include stem cell-derived cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, and 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, at least 50%, preferably at least 60%, more preferably at least 70% at the end of the formation of the sheet cell culture. %, More preferably 75% or more.
  • the culture substrate is not particularly limited as long as cells can form an implant thereon, and includes, for example, containers of various materials and / or shapes, solid or semi-solid surfaces in the containers, 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 substrate coated on the surface with a hydrophilic compound such as a collagen gel or a hydrophilic polymer, further, collagen
  • a hydrophilic compound such as a collagen gel or a hydrophilic polymer, further, collagen
  • examples include extracellular matrices such as fibronectin, laminin, vitronectin, proteoglycan, and glycosaminoglycan, and substrates coated on the surface with cell adhesion factors such as cadherin family, selectin family, and integrin family.
  • 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.
  • pluripotent stem cells is a term well known in the art and has the ability to differentiate into cells of all lineages belonging to the three germ layers, i.e., endoderm, mesoderm and ectoderm. Means cell.
  • pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), and the like.
  • ES cells embryonic stem cells
  • ntES cells nuclear transfer embryonic stem cells
  • a pluripotent stem cell of the present disclosure particularly means a pluripotent stem cell into which no gene has been introduced.
  • a suspension culture of the pluripotent stem cell is carried out to form an aggregate of any of the above three germ layers, and then a cell forming an aggregate 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 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 pluripotent stem cells can be performed using any known technique. For example, induction of differentiation from pluripotent stem cells into 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.
  • myoblast means a progenitor cell of striated muscle cell, and includes skeletal myoblast and cardioblast.
  • skeletal myoblasts means myoblasts present in skeletal muscle. Skeletal myoblasts are well known in the art, and can be prepared from skeletal muscle by any known method (eg, the method described in JP-A-2007-89442), or commercially available. It is also available (eg, Lonza, Cat # CC-2580).
  • Skeletal myoblasts include, but are not limited to, markers such as CD56, ⁇ 7 integrin, myosin heavy chain IIa, myosin heavy chain IIb, myosin heavy chain IId (IIx), MyoD, Myf5, Myf6, myogenin, desmin, PAX3, and the like. Can be identified by In certain embodiments, the skeletal myoblasts are CD56 positive. In a more particular embodiment, the skeletal myoblasts are CD56 positive and desmin positive.
  • Skeletal myoblasts include any organism having skeletal muscle, including, but not limited to, humans, non-human primates, rodents (such as mice, rats, hamsters, guinea pigs), rabbits, dogs, cats, pigs, It may be derived from mammals such as horses, cows, goats and sheep.
  • the skeletal myoblast is a mammalian skeletal myoblast.
  • the skeletal myoblast is a human skeletal myoblast.
  • Skeletal myoblasts can be collected from any skeletal muscle.
  • the skeletal myoblasts of the present disclosure are skeletal myoblasts from the thigh, neck, and abdomen.
  • One aspect of the present disclosure relates to a method of producing a high-quality implant comprising adherent cells, particularly non-transgenic adherent cells.
  • the method of the present disclosure includes the following steps (a) and (b): (A) inoculating a cell population containing the adherent cells on a culture substrate, and (B) incubating the seeded cell population with a medium containing platelet lysate.
  • a cell into which a gene has not been introduced means a cell into which a foreign gene has not been introduced in the process of obtaining the cell.
  • the cell is a cell derived from a stem cell
  • gene transfer has not been performed in any of the steps of stem cell isolation and differentiation induction.
  • a primary cell obtained from a living tissue a subcultured cell obtained by subculturing the primary cell, a cell obtained by differentiating a precursor cell of the somatic cell obtained from the living tissue, or the like can be used.
  • the seeding on the culture substrate may be performed, for example, by injecting a cell suspension in which cells are suspended in a 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 set at a density at which a graft can be formed, and such a density may vary depending on the type of the graft and the desired cells. However, those skilled in the art can use an appropriate method based on techniques known in the art. Density can be selected. For example, in the case of a sheet-shaped cell culture containing skeletal myoblasts, it may be 2.0 ⁇ 10 5 cells / cm 2 or more, for example, but may be seeded at higher density.
  • 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 The density at which contact inhibition occurs, or the 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
  • 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
  • these modifications such as laminin 511 (a modification of laminin), VTN-N (a modification of vitronectin), and RetroNectin (R) (a modification of fibronectin ) may be used.
  • step (b) the seeded cells are incubated for graft formation.
  • graft formation when the graft is a sheet-shaped cell culture is particularly referred to as “sheeting”
  • sheeting culture incubation for forming the seeded cells into a sheet
  • Incubation of the seeded cells for graft formation can be performed by any known technique 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.
  • graft formation such as sheeting of cells is achieved by cells adhering to each other via an intercellular adhesion mechanism such as an adhesion molecule or an extracellular matrix. Therefore, it is considered that the step of forming a graft (for example, forming a sheet) of the seeded cells can be achieved 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 . Incubation can be performed under normal pressure (atmospheric pressure, non-pressurized).
  • Incubation can be performed in containers of any size and shape.
  • the size and shape of the transplant can be adjusted by adjusting the size and shape of the culture vessel, or by placing a mold of the desired size and shape in the culture vessel and culturing cells inside it. It can be adjusted arbitrarily.
  • Incubation time for graft formation can vary depending on the type of seeded cells and cell density.
  • the sheet may be formed by disseminating the skeletal myoblast at a density of, for example, about 3.0 ⁇ 10 6 cells / cm 2 and incubating for 2 hours or more.
  • the seeding density reaches a confluent density, that is, when seeding is performed at a higher density, the period of sheet culture can be shortened, and the culture time may be 2 to 12 hours, more preferably 2 to 6 hours. .
  • the medium (sometimes simply referred to as a medium) used for the graft formation is not particularly limited as long as it enables formation of cell-cell adhesion.
  • physiological saline various physiological buffers (for example, PBS, HBSS, etc.) and those based on various basal media for cell culture may be used.
  • basal media include, but are not limited to, DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12 and the like. Many of these basal media are commercially available and their compositions are also known.
  • 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 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 have been added, removed, increased or reduced in weight.
  • the sheeting medium may contain additives such as normal serum (eg, bovine serum such as fetal calf serum, horse serum, human serum, etc.) and various growth factors (eg, FGF, EGF, VEGF, HGF, etc.).
  • the transplant when the transplant is produced under xeno-free conditions, it is preferable that the transplant does not contain 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 an incubation for forming a graft is performed in a medium containing platelet lysate instead of or in addition to serum or growth factors.
  • the vehicle contains 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 made it possible to form a graft as in the case of using serum by including a platelet lysate in a medium. It has been found for the first time that piece formation can be easily achieved at low cost.
  • 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 medium may be any level commonly used in the art, and may be, for example, 1%, 2.5%, 5%, 10%, 15%, 20%, and the like. .
  • the platelet lysate is contained in the 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 medium may be replaced as appropriate during the incubation period. Further, the composition of the medium may be changed in accordance with the progress of the graft formation.
  • the medium may further include a cell adhesive component.
  • the cell adhesive component is as described in detail above.
  • the culture substrate may or may not be further coated with a cell adhesive component or a platelet lysate.
  • the cell adhesive component contained in the medium may be the same as 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 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 the sheet is formed using cells having low viability, that is, cells having weak activity, the content of the cell adhesive component is preferably small.
  • the concentration of the cell adhesive component contained in the medium is about 0.1%, about 0.5% based on the concentration (100%) used when the same cell adhesive component is used as a coating agent for the culture substrate. , 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 adhesive component contained in the medium is 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. % 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 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%, about 5% to about 10%, and the like.
  • myoblasts used in the method of the present disclosure may be directly obtained from a living body or may be derived from other cells, but are preferably obtained directly from a living body. It is a thing.
  • the skeletal myoblasts of the present disclosure are skeletal myoblasts from the thigh, neck, and abdomen.
  • the other cells to be induced include pluripotent stem cells capable of differentiating into any cells.
  • Another aspect of the present disclosure treats a disease in a subject in need thereof, comprising applying an effective amount of a graft, preferably a sheet of cell culture, produced by the method of the present disclosure to the subject in need thereof. On how to do it.
  • 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.
  • 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 further include, prior to the step of producing the implant, obtaining a cell or a tissue that is a source of the cells for producing the implant from the subject.
  • 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 and recurrence of a disease, reducing symptoms, or delaying or stopping the progress (eg, size, weight, number of grafts, etc.), 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.
  • a transplant using adherent cells particularly cells that have not been transfected, such as cells collected from a transplant target
  • high-quality transplantation that is comparable to conventional cells even in a xeno-free environment You can get a piece. Therefore, in the production of a xeno-free implant used for clinical use, a high-quality implant can be easily formed.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Transplantation (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

Le but de la présente invention est de fournir un procédé de production d'une greffe de haute qualité comprenant des cellules adhérentes telles que des cellules de muscle squelettique, tout en conservant les fonctions de celui-ci à des niveaux élevés ; une greffe produite à l'aide dudit procédé ; et un procédé de traitement de maladie ou similaire à l'aide de ladite greffe. Ce procédé de production d'une greffe comprend (a) une étape de placage d'une population de cellules comprenant des cellules adhérentes sur un substrat de culture ; et (b) une étape de culture de la population de cellules plaquées à l'aide d'un milieu contenant un lysat de plaquettes.
PCT/JP2019/038197 2018-09-27 2019-09-27 Procédé de formation de feuille de cellules somatiques WO2020067443A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2020527125A JPWO2020067443A1 (ja) 2018-09-27 2019-09-27 体細胞のシート化方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018182449 2018-09-27
JP2018-182449 2018-09-27

Publications (1)

Publication Number Publication Date
WO2020067443A1 true WO2020067443A1 (fr) 2020-04-02

Family

ID=69950779

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/038197 WO2020067443A1 (fr) 2018-09-27 2019-09-27 Procédé de formation de feuille de cellules somatiques

Country Status (2)

Country Link
JP (1) JPWO2020067443A1 (fr)
WO (1) WO2020067443A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022050282A1 (fr) * 2020-09-01 2022-03-10 株式会社セルファイバ Échafaudage, procédé de production d'échafaudage, culture cellulaire, procédé de culture cellulaire

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011110368A (ja) * 2009-11-30 2011-06-09 Terumo Corp シート状細胞培養物の製造方法
JP2011115058A (ja) * 2009-12-01 2011-06-16 Terumo Corp 単離されたシート状細胞培養物の製造方法
JP2014143971A (ja) * 2013-01-30 2014-08-14 Terumo Corp シート状細胞培養物の製造方法
JP2014533715A (ja) * 2011-11-23 2014-12-15 セル セラピー リミテッドＣｅｌｌ Ｔｈｅｒａｐｙ Ｌｉｍｉｔｅｄ 血小板溶解物ゲル
JP2017501740A (ja) * 2013-11-04 2017-01-19 アイソポジェン ピーティーワイ リミテッド 細胞培養方法
JP2018506986A (ja) * 2015-03-04 2018-03-15 メゾブラスト・インターナショナル・エスアーエールエル 間葉系幹細胞の細胞培養法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP5407345B2 (ja) * 2009-01-13 2014-02-05 大日本印刷株式会社 生体組織の作製方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011110368A (ja) * 2009-11-30 2011-06-09 Terumo Corp シート状細胞培養物の製造方法
JP2011115058A (ja) * 2009-12-01 2011-06-16 Terumo Corp 単離されたシート状細胞培養物の製造方法
JP2014533715A (ja) * 2011-11-23 2014-12-15 セル セラピー リミテッドＣｅｌｌ Ｔｈｅｒａｐｙ Ｌｉｍｉｔｅｄ 血小板溶解物ゲル
JP2014143971A (ja) * 2013-01-30 2014-08-14 Terumo Corp シート状細胞培養物の製造方法
JP2017501740A (ja) * 2013-11-04 2017-01-19 アイソポジェン ピーティーワイ リミテッド 細胞培養方法
JP2018506986A (ja) * 2015-03-04 2018-03-15 メゾブラスト・インターナショナル・エスアーエールエル 間葉系幹細胞の細胞培養法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HEMEDA, H. ET AL.: "Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells", CYTOTHERAPY, vol. 16, 2014, pages 170 - 180, XP055202535, ISSN: 1465-3249, DOI: 10.1016/j.jcyt.2013.11.004 *
RANZATO, E. ET AL.: "Scratch wound closure of C2C12 mouse myoblasts is enhanced by human platelet lysate", CELL BIOLOGY INTERNATIONAL, vol. 33, 2009, pages 911 - 917, XP026519735, ISSN: 1065-6995, DOI: 10.1016/j.cellbi.2009.06.017 *
TIAN, B-M. ET AL.: "Human platelet lysate supports the formation of robust human periodontal ligament cell sheets", J. TISSUE ENG. REGEN. MED., vol. 12, 17 July 2017 (2017-07-17), pages 961 - 972, XP055701079, ISSN: 1932-6254 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022050282A1 (fr) * 2020-09-01 2022-03-10 株式会社セルファイバ Échafaudage, procédé de production d'échafaudage, culture cellulaire, procédé de culture cellulaire

Also Published As

Publication number Publication date
JPWO2020067443A1 (ja) 2021-08-30

Similar Documents

Publication Publication Date Title
JP7256818B2 (ja) 心筋細胞のシート化方法
JP2023175787A (ja) 多能性幹細胞由来細胞のシート化方法
WO2020067443A1 (fr) Procédé de formation de feuille de cellules somatiques
US11395863B2 (en) Modification method for sheet-shaped cell culture
WO2021065984A1 (fr) Procédé de formation de feuille de cardiomyocytes
US20200109368A1 (en) Method for preparing differentiation-induced cells
WO2020067436A1 (fr) Procédé de formation d'un greffon de cellules dérivées de cellules souches pluripotentes
WO2021065986A1 (fr) Récipient pour séparer des cellules vivantes
WO2019177135A1 (fr) Procédé de production de culture cellulaire stratiforme
JP7550778B2 (ja) Ｃｄ５６陽性細胞の比率を高めるための方法
WO2021065976A1 (fr) Procédé d'amélioration de l'activité dans un greffon
US20200392466A1 (en) Sheet-forming method for pluripotent stem cell-derived cells
JP7471069B2 (ja) 移植片の活性を高める方法
JP2019154363A (ja) 医療用細胞培養物
TWI745655B (zh) 片狀細胞培養物的製造方法
WO2023210609A1 (fr) Procédé de production de feuillet cellulaire
WO2021065971A1 (fr) Solution tampon de dilution pour cellules cryoconservées
WO2020067434A1 (fr) Méthode de cryoconservation de cellules
WO2020067442A1 (fr) Méthode de cryoconservation des cellules
KR20210015911A (ko) 망막 색소 상피 세포 막 및 그의 제조 방법
JP2021052658A (ja) 生体由来組織からの生細胞の分離方法
Ka et al. STEM CELLS–THE ULTIMATE BODY REPAIR KIT
Soong Development of a novel technology to engineer heart muscle for contractile and paracrine support in heart failure
JP2019154362A (ja) シート状細胞培養物の製造方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19864196

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2020527125

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19864196

Country of ref document: EP

Kind code of ref document: A1