WO2015025957A1 - Procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage d'un composé candidat médicamenteux - Google Patents

Procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage d'un composé candidat médicamenteux Download PDF

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WO2015025957A1
WO2015025957A1 PCT/JP2014/072029 JP2014072029W WO2015025957A1 WO 2015025957 A1 WO2015025957 A1 WO 2015025957A1 JP 2014072029 W JP2014072029 W JP 2014072029W WO 2015025957 A1 WO2015025957 A1 WO 2015025957A1
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myocardial
cells
myocardial tissue
tissue
cardiomyocytes
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PCT/JP2014/072029
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English (en)
Japanese (ja)
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明石満
松▲崎▼典弥
澤芳樹
宮川繁
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国立大学法人大阪大学
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Priority to JP2015532917A priority Critical patent/JP6608281B2/ja
Publication of WO2015025957A1 publication Critical patent/WO2015025957A1/fr

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    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • C12N2513/00—3D culture

Definitions

  • the present disclosure relates to a method for producing a myocardial tissue chip used for screening a drug candidate compound, a myocardial tissue chip, and a method for screening a drug candidate compound using the myocardial tissue chip.
  • a myocardial tissue chip that can be used for drug screening in which a three-dimensional myocardial tissue is formed, a method for manufacturing the myocardial tissue chip, and a drug screening method using the myocardial tissue chip I will provide a.
  • the present disclosure relates to a method for producing a myocardial tissue chip used for drug screening, in which a coated cell whose surface is coated with a coating containing an extracellular matrix component is disposed on a substrate. And forming a myocardial three-dimensional tissue body on the substrate by repeatedly arranging the coated cells, wherein the cells include cardiomyocytes derived from induced pluripotent stem cells
  • the present invention relates to a chip manufacturing method.
  • the present disclosure is a myocardial tissue chip used for drug screening, which includes a base material and a myocardial three-dimensional tissue body formed on the base material, and the myocardial three-dimensional tissue
  • the body relates to a myocardial tissue chip comprising cardiomyocytes derived from induced pluripotent stem cells and an extracellular matrix component, wherein the cardiomyocytes are stacked via the extracellular matrix component.
  • the present disclosure is a method for screening a drug candidate compound using the myocardial tissue chip of the present disclosure, wherein the drug candidate compound is brought into contact with the myocardial tissue chip, and the myocardial tissue of the candidate compound
  • the present invention relates to a screening method including observing the influence of a chip on a myocardial three-dimensional tissue and evaluating a candidate compound based on the observation result.
  • the present disclosure includes a cardiomyocyte derived from an induced pluripotent stem cell, an extracellular matrix component, and a fibroblast, and the cardiomyocyte and the fibroblast are arranged in three dimensions. It is related with the cultured myocardial tissue obtained by culture.
  • a myocardial three-dimensional tissue is formed, and a myocardial tissue chip that can be used for drug screening, a manufacturing method thereof, and a drug screening method using the myocardial tissue chip are provided. it can.
  • FIG. 1 is an example of a micrograph of a myocardial three-dimensional tissue body of a reference example.
  • FIG. 2 is an example of a graph showing changes in the number of pulsations accompanying drug administration of the myocardial three-dimensional tissue body of the reference example.
  • FIG. 3 is an example of a micrograph of a myocardial three-dimensional tissue body in the myocardial tissue chip produced in Example 1.
  • FIG. 4 is an example of a graph showing changes in the number of pulsations accompanying drug administration of a myocardial three-dimensional tissue body in the myocardial tissue chip produced in Example 2.
  • FIG. 1 is an example of a micrograph of a myocardial three-dimensional tissue body of a reference example.
  • FIG. 2 is an example of a graph showing changes in the number of pulsations accompanying drug administration of the myocardial three-dimensional tissue body of the reference example.
  • FIG. 3 is an example of a micrograph of a myocardial three-dimensional
  • FIG. 5 is a tissue section image (HE-stained image) of a myocardial three-dimensional tissue body (after 7 days of culture) in the myocardial tissue chip produced in Example 3.
  • 6 is a phase contrast micrograph of a myocardial three-dimensional tissue body (after 7 days of culture) in the myocardial tissue chip produced in Example 3.
  • FIG. 7 is a confocal laser micrograph of a myocardial three-dimensional tissue body (after 6 days of culture) having a vascular network prepared in Example 8
  • FIG. 7A is a myocardial three-dimensional tissue body prepared in Example 8-1.
  • the “myocardial tissue chip” includes a base material and a myocardial three-dimensional tissue body formed on the base material.
  • the “cardiac myocardial three-dimensional tissue body” includes at least cardiomyocytes derived from induced pluripotent stem cells and an extracellular matrix component, and a cell structure in which cardiomyocytes are stacked via the extracellular matrix component In one or a plurality of embodiments, it is preferable to imitate a myocardial structure in a living body.
  • the induced pluripotent stem cells include disease-specific iPS cells and normal iPS cells in one or a plurality of embodiments.
  • the origin of iPS cells is not particularly limited, and examples include humans and mice, but human-derived iPS cells are preferable from the viewpoint of providing a myocardial tissue chip capable of more accurately evaluating human cardiotoxicity.
  • commercially available ones or self-prepared ones may be used as the cardiomyocytes derived from iPS cells in one or a plurality of embodiments.
  • the myocardial three-dimensional tissue in the present disclosure includes cells and organs (and / or organs) other than cardiomyocytes capable of constituting the myocardium in addition to the myocardial cells derived from iPS cells and extracellular matrix components. Constituent cells) and the like.
  • Examples of cells other than cardiomyocytes include, in one or more embodiments, vascular endothelial cells, myoblasts, fibroblasts, mesenchymal stem cells, hematopoietic stem cells, nerve cells, cancer cells and the like.
  • Examples of the organ include blood vessels, lymphatic vessels, nerves, and the like in one or a plurality of embodiments.
  • Examples of the cells constituting the organ include vascular endothelial cells, myoblasts, fibroblasts, mesenchymal stem cells, hematopoietic stem cells, nerve cells, and cancer cells in one or a plurality of embodiments.
  • the “coated cell” refers to a cell containing a coating containing an extracellular matrix component and a cell, and the cell surface of which is covered with the coating.
  • the cells to be coated include the cells described above.
  • the coated cells can be prepared by the methods and examples described later, and the method disclosed in JP2012-115254A.
  • the “extracellular matrix component” refers to a substance that fills the space outside the cell in a living body and performs a function such as a skeletal role, a role of providing a scaffold, and a role of holding a biological factor.
  • the extracellular matrix component may further contain a substance that can perform functions such as a skeletal role, a role of providing a scaffold, and a role of retaining a biological factor in in vitro cell culture.
  • the present disclosure relates to a method for producing a myocardial tissue chip used for drug screening, wherein a coated cell having a cell surface coated with a coating containing an extracellular matrix component is disposed on a substrate; Cardiomyocytes derived from induced pluripotent stem cells (hereinafter also referred to as “iPS cell-derived cardiomyocytes”), comprising forming a myocardial three-dimensional tissue on the substrate by repeatedly arranging
  • the present invention relates to a method for producing a myocardial tissue chip including the following (hereinafter, also referred to as “production method of the present disclosure”).
  • a myocardial three-dimensional tissue body in which the number of layers and the structure are uniformly controlled is formed in commercially available microwells of various sizes used for drug screening.
  • An improved myocardial tissue chip can be provided.
  • a myocardial three-dimensional tissue can be formed in the microwell within one day at the latest.
  • a myocardial three-dimensional tissue body of at least five layers or more can be provided inside the microwell.
  • the myocardial tissue chip obtained by the production method of the present disclosure can be used for testing / inspection related to safety and pharmacokinetics in the fields of pharmaceuticals, pharmaceuticals, cosmetics, foods, and the environment.
  • the production method of the present disclosure forms a myocardial three-dimensional tissue body on a base material by repeatedly placing the coated cells, the surface of which is coated with a coating containing an extracellular matrix component, on the base material.
  • a three-dimensional myocardial tissue on the base material by culturing.
  • the culture temperature is 4 to 60 ° C., 20 to 40 ° C., or 30 to 37 ° C.
  • the culture time is not particularly limited, and in one or more embodiments, it is 1 to 168 hours, 3 to 24 hours, or 3 to 12 hours.
  • the medium is not particularly limited and can be appropriately determined depending on the cell.
  • the medium may be a medium supplemented with serum or a serum-free medium.
  • the production method of the present disclosure includes disposing fibroblasts on a substrate in one or a plurality of embodiments from the viewpoint of forming a tissue body closer to a living myocardial tissue.
  • the fibroblasts may be mixed with the coated cells containing iPS cell-derived cardiomyocytes or may be disposed separately from the coated cells.
  • the fibroblasts are arranged so that the ratio of cardiomyocytes (cardiomyocytes: fibroblasts, number of cells) is 99: 1 to 1:99.
  • the ratio of cardiomyocytes to fibroblasts is 80:20 to 30:70, 80: from the viewpoint of generating action potentials in cardiomyocytes and realizing more regular pulsations. 20 to 45:55 or 75:25 to 50:50 is preferred.
  • the fibroblast is preferably a cardiac fibroblast in one or more embodiments.
  • the fibroblast may be a cell derived from a human or a cell derived from other than a human.
  • the fibroblast may be a cell derived from an embryonic stem cell, an induced pluripotent stem cell, or the like in one or a plurality of embodiments. Fibroblasts may or may not be coated with a coating containing extracellular matrix components.
  • the manufacturing method according to the present disclosure places a vascular endothelial cell on a base material from the viewpoint that a vascular network is formed in a three-dimensional tissue body to make the tissue body closer to a living body's myocardial tissue.
  • the vascular endothelial cells may be mixed with the coated cells containing the cardiomyocytes derived from iPS cells or may be disposed separately from the coated cells.
  • the vascular endothelial cell is preferably a cardiac microvascular endothelial cell in one or more embodiments.
  • the vascular endothelial cell may be a cell derived from a human or a cell derived from other than a human. In one or a plurality of embodiments, the vascular endothelial cell may be a cell derived from embryonic stem cells, induced pluripotent stem cells and the like in one or a plurality of embodiments. Vascular endothelial cells may or may not be coated with a coating containing an extracellular matrix component.
  • the density of the coated cells at the time of myocardial three-dimensional tissue formation is the size and thickness of the target myocardial three-dimensional tissue, the size of the container to be cultured, the number of cells to be stacked, etc. 1 ⁇ 10 2 pieces / cm 3 to 1 ⁇ 10 9 pieces / cm 3 , 1 ⁇ 10 4 pieces / cm 3 to 1 ⁇ 10 8 pieces / cm 3 in one or a plurality of embodiments. Or 1 ⁇ 10 5 pieces / cm 3 to 1 ⁇ 10 7 pieces / cm 3 .
  • the production method of the present disclosure includes arranging the coated cells such that at least four layers of iPS cell-derived cardiomyocytes are laminated.
  • the number of cell layers to be stacked is not particularly limited, and in one or a plurality of embodiments, 5, 6, 7, 8, 9, or 10 layers or more may be mentioned.
  • the arrangement of the coated cells improves the reproducibility of the myocardial three-dimensional tissue to be formed and improves the production efficiency, and enables faster production and easier control of the number of layers and the structure. In one or a plurality of embodiments, this is preferably performed by discharging the coated cells onto the substrate using a liquid discharge nozzle, and more preferably using an injection device such as an ink jet discharge device.
  • the ejection device is a device capable of ejecting one coated cell per ejection to a predetermined region of the base material, and a piezoelectric drive ink jet ejection device can be preferably employed.
  • discharge of coated cells is preferably performed by discharging one coated cell per discharge.
  • a myocardial tissue pattern by discharging one coated cell per discharge, it is possible to form a myocardial three-dimensional tissue that is more similar to a myocardial tissue in a living body.
  • the arrangement of the coated cells in the production method of the present disclosure is based on a preset pattern, so that the coated cells coated with predetermined cells are arranged at predetermined positions. It is preferable to carry out while moving the nozzle.
  • the manufacturing method of the present disclosure uses coated cells coated with a coating containing an extracellular matrix component, in one or a plurality of embodiments, it is possible to reduce stress on the cells during the placement of the cells by ejection, and Cells can be stacked efficiently.
  • the area of the myocardial three-dimensional tissue body is 1 mm 2 or more, 5 mm 2 or more, 10 mm 2 or more, or 20 mm 2 or more, and 1000 mm 2 or less, 700 mm 2 in one or more embodiments.
  • it is 600 mm 2 or less, or 500 mm 2 or less.
  • a multiwell plate is mentioned in one or some embodiment.
  • the substrate may be a multi-well plate in which a membrane filter is arranged in each well in one or a plurality of embodiments from the viewpoint of easy handling, and preferably includes a housing part and a base part.
  • a multi-well plate in which a container whose base is a membrane filter is arranged.
  • the number of wells in the multi-well plate is not particularly limited, and examples thereof include 24, 96, 384, and 1536 in one or more embodiments.
  • the pore size of the membrane filter is not particularly limited as long as the cultured cells can be retained on the membrane filter, and in one or a plurality of embodiments, it is 0.1 ⁇ m to 2 ⁇ m, or 0.4 ⁇ m to 1.0 ⁇ m. .
  • the material of the membrane includes, for example, polyethylene terephthalate (PET), polycarbonate, or polytetrafluoroethylene (PTFE).
  • the present disclosure is a myocardial tissue chip used for drug screening, including at least a cardiomyocyte derived from an induced pluripotent stem cell and an extracellular matrix component, and the induced pluripotency
  • the present invention relates to a myocardial tissue chip (hereinafter also referred to as “myocardial tissue chip of the present disclosure”), which is a myocardial three-dimensional tissue structure in which stem cells are laminated via the extracellular matrix component.
  • the myocardial tissue chip of the present disclosure can be manufactured by the manufacturing method of the present disclosure.
  • the myocardial tissue chip of the present disclosure in one or a plurality of embodiments, it is possible to perform a test / inspection regarding safety and pharmacokinetics in the fields of medicine, pharmaceuticals, cosmetics, foods, and the environment.
  • the substrate, the extracellular matrix, and the cardiomyocytes are as described above.
  • the iPS cell is preferably a human-derived iPS cell because in one or a plurality of embodiments, human cardiotoxicity evaluation difficult in animal experiments can be more accurately performed.
  • the myocardial three-dimensional tissue body further includes fibroblasts in one or a plurality of embodiments from the viewpoint that the myocardial three-dimensional tissue is a tissue close to a living myocardial tissue.
  • the fibroblast is preferably a cardiac fibroblast in one or more embodiments.
  • the fibroblast may be a cell derived from a human or a cell derived from other than a human.
  • the fibroblast may be a cell derived from an embryonic stem cell, an induced pluripotent stem cell, or the like in one or a plurality of embodiments.
  • the ratio of cardiomyocytes to fibroblasts (cardiomyocytes: fibroblasts, number of cells) in the three-dimensional myocardial tissue is 99: 1 to 1:99 in one or more embodiments. In one or a plurality of embodiments, the ratio of cardiomyocytes to fibroblasts is 80:20 to 30:70, 80: from the viewpoint of generating action potentials in cardiomyocytes and realizing more regular pulsations. 20 to 45:55 or 75:25 to 50:50 is preferred.
  • the myocardial three-dimensional tissue body further includes vascular endothelial cells in one or a plurality of embodiments.
  • the myocardial three-dimensional tissue body preferably includes a vascular network formed of vascular endothelial cells in order to stably maintain the function of the myocardial three-dimensional tissue for a longer period of time.
  • the vascular endothelial cell is preferably a cardiac microvascular endothelial cell in one or more embodiments.
  • the vascular endothelial cell may be a cell derived from a human or a cell derived from other than a human.
  • the vascular endothelial cell may be a cell derived from embryonic stem cells, induced pluripotent stem cells and the like in one or a plurality of embodiments.
  • the myocardial three-dimensional tissue structure preferably includes four or more layers of cardiomyocytes, and more preferably five or more layers of cardiomyocytes. Are stacked with 6, 7, 8, 9, or 10 or more layers.
  • the myocardial three-dimensional tissue is formed in each well of the multi-well plate.
  • the area of the myocardial three-dimensional tissue body in the myocardial tissue chip of the present disclosure is 1 mm 2 or more, 5 mm 2 or more, 10 mm 2 or more, or 20 mm 2 or more, and 1000 mm 2 or less, 700 mm 2. Hereinafter, it is 600 mm 2 or less, or 500 mm 2 or less.
  • the density of cells in the myocardial three-dimensional tissue body is 1 ⁇ 10 5 cells / mm 3 or more, 5 ⁇ 10 5 cells / mm 3 or more, 1 ⁇ 10 6 cells / mm 3 or more, 5 ⁇ 10 6 pieces / mm 3 or more, or 1 ⁇ 10 7 pieces / mm 3 or more, and 1 ⁇ 10 10 pieces / mm 3 or less, 5 ⁇ 10 9 pieces / mm 3 or less, or 1 ⁇ 10 9 pieces / Mm 3 or less.
  • the present disclosure is a method for screening a drug candidate compound using the myocardial tissue chip of the present disclosure, wherein the drug candidate compound is brought into contact with the myocardial tissue chip, and the myocardial tissue of the candidate compound
  • a screening method including observing the effect on the myocardial three-dimensional tissue in the chip and evaluating a candidate compound based on the observation result hereinafter also referred to as “screening method of the present disclosure”.
  • screening method of the present disclosure in one or a plurality of embodiments, it is possible to perform a test / inspection regarding safety and pharmacokinetics in the fields of medicine, pharmaceuticals, cosmetics, foods, and the environment.
  • Examples of drug candidate compounds to be screened in the screening method of the present disclosure include therapeutic agents for heart diseases and the like in one or a plurality of embodiments.
  • the present disclosure is a kit for screening a drug candidate compound, which includes a myocardial tissue chip of the present disclosure, a culture medium, and an instruction manual (hereinafter, “kit of the present disclosure”). Also called).
  • kit of the present disclosure includes the above-mentioned medium.
  • the coated cell includes a cell and an extracellular matrix component, and the surface of the cell is coated with an extracellular matrix component coating.
  • the coating containing the extracellular matrix component preferably includes a membrane containing the substance A and a membrane containing the substance B that interacts with the substance A.
  • a protein or polymer having an RGD sequence hereinafter also referred to as “substance having an RGD sequence” and a protein or polymer having the RGD sequence are used.
  • a combination with a protein or polymer that interacts with the protein hereinafter also referred to as “substance having interaction”
  • a protein or polymer that has a positive charge hereinafter also referred to as “substance with a positive charge”.
  • a protein or polymer having a negative charge hereinafter also referred to as “substance having a negative charge”.
  • the thickness of the coating containing the extracellular matrix component is preferably 1 nm to 1 ⁇ 10 3 nm, or 2 nm to 1 ⁇ 10 2 nm, and the myocardial three-dimensional structure in which the coated cells are stacked more densely. From the reason that a tissue body can be obtained, 3 nm to 1 ⁇ 10 2 nm is more preferable.
  • the thickness of the coating containing the extracellular matrix component can be appropriately controlled by, for example, the number of membranes constituting the coating.
  • the coating containing the extracellular matrix component is not particularly limited, and may be a single layer. In one or a plurality of embodiments, for example, 3, 5, 7, 9, 11, 13, 15 layers or more It may be a multilayer.
  • the production method of the present disclosure may include a step of preparing a coated cell.
  • Coated cells can be prepared by alternately bringing a solution containing substance A and a solution containing substance B into contact with cells such as cardiomyocytes.
  • the combination of the substance A and the substance B includes a combination of a substance having an RGD sequence and a substance having an interaction, or a combination of a substance having a positive charge and a substance having a negative charge. It is done.
  • a substance having an RGD sequence refers to a protein or polymer having an “Arg-Gly-Asp” (RGD) sequence, which is an amino acid sequence responsible for cell adhesion activity.
  • RGD Arg-Gly-Asp
  • having an RGD sequence may originally have an RGD sequence, or may have a RGD sequence chemically bound thereto.
  • the substance having the RGD sequence is preferably biodegradable.
  • Examples of the protein having an RGD sequence include conventionally known adhesive proteins or water-soluble proteins having an RGD sequence in one or a plurality of embodiments.
  • Examples of the adhesive protein include fibronectin, vitronectin, laminin, cadherin, and collagen in one or a plurality of embodiments.
  • Examples of the water-soluble protein having an RGD sequence include, in one or more embodiments, collagen, gelatin, albumin, globulin, proteoglycan, an enzyme, an antibody, or the like to which the RGD sequence is bound.
  • Examples of the polymer having an RGD sequence include a naturally-derived polymer or a synthetic polymer in one or a plurality of embodiments.
  • Examples of the naturally-derived polymer having an RGD sequence include, in one or more embodiments, a water-soluble polypeptide, a low-molecular peptide, a polyamino acid such as ⁇ -polylysine or ⁇ -polylysine, and a sugar such as chitin or chitosan.
  • Examples of the synthetic polymer having an RGD sequence include, in one or more embodiments, a polymer or copolymer having an RGD sequence such as a linear type, graft type, comb type, dendritic type, or star type.
  • the polymer or copolymer may be polyurethane, polycarbonate, polyamide, or a copolymer thereof, polyester, poly (N-isopropylacrylamide-co-polyacrylic acid), polyamide amine dendrimer, polyethylene Examples thereof include oxide, poly ⁇ -caprolactam, polyacrylamide, or poly (methyl methacrylate- ⁇ -polyoxymethacrylate).
  • the substance having the RGD sequence is preferably fibronectin, vitronectin, laminin, cadherin, polylysine, elastin, collagen to which the RGD sequence is bound, gelatin, chitin or chitosan to which the RGD sequence is bound, and more preferably fibronectin.
  • the substance that interacts refers to a protein or polymer that interacts with a substance having an RGD sequence.
  • “interact” means, in one or more embodiments, electrostatic interaction, hydrophobic interaction, hydrogen bond, charge transfer interaction, covalent bond formation, specific interaction between proteins. , And / or a substance that interacts chemically and / or physically with a substance having an RGD sequence by van der Waals force or the like is close enough to allow bonding, adhesion, adsorption, or electron transfer.
  • the interacting substance is preferably biodegradable.
  • Examples of the protein that interacts with a substance having an RGD sequence include collagen, gelatin, proteoglycan, integrin, enzyme, or antibody in one or a plurality of embodiments.
  • Examples of the polymer that interacts with a substance having an RGD sequence include a naturally-derived polymer or a synthetic polymer in one or a plurality of embodiments.
  • the naturally-derived polymer that interacts with a substance having an RGD sequence includes, in one or more embodiments, a water-soluble polypeptide, a low-molecular peptide, a polyamino acid, elastin, heparin, a sugar such as heparan sulfate or dextran sulfate, and Examples include hyaluronic acid.
  • the polyamino acid include, in one or more embodiments, polylysine such as ⁇ -polylysine or ⁇ -polylysine, polyglutamic acid, or polyaspartic acid.
  • the synthetic polymer that interacts with a substance having an RGD sequence include, in one or a plurality of embodiments, synthetic molecules having the RGD sequence described above.
  • the interacting substance is preferably gelatin, dextran sulfate, heparin, hyaluronic acid, globulin, albumin, polyglutamic acid, collagen, or elastin, more preferably gelatin, dextran sulfate, heparin, hyaluronic acid, or collagen, More preferred is gelatin, dextran sulfate, heparin, or hyaluronic acid.
  • the combination of the substance having the RGD sequence and the substance that interacts is not particularly limited as long as it is a combination of different substances that interact with each other, and either one is a polymer or protein containing the RGD sequence, and the other is this. Any polymer or protein that interacts with the protein may be used.
  • the combination of the substance having an RGD sequence and the substance having an interaction includes, in one or more embodiments, fibronectin and gelatin, fibronectin and ⁇ -polylysine, fibronectin and hyaluronic acid, fibronectin and dextran sulfate, fibronectin and heparin, fibronectin And collagen, laminin and gelatin, laminin and collagen, polylysine and elastin, vitronectin and collagen, RGD-bound collagen or RGD-bound gelatin and collagen or gelatin, and the like.
  • fibronectin and gelatin fibronectin and ⁇ -polylysine, fibronectin and hyaluronic acid, fibronectin and dextran sulfate, fibronectin and heparin, or laminin and gelatin are preferable, and fibronectin and gelatin are more preferable.
  • sequence, and the substance which has interaction may be one each, respectively, and may use 2 or more types together in the range which shows interaction, respectively.
  • a substance having a positive charge refers to a protein or polymer having a positive charge.
  • the protein having a positive charge is preferably a water-soluble protein in one or a plurality of embodiments.
  • the water-soluble protein include basic collagen, basic gelatin, lysozyme, cytochrome c, peroxidase, or myoglobin in one or more embodiments.
  • the polymer having a positive charge include naturally-derived polymers and synthetic polymers in one or a plurality of embodiments.
  • Examples of the naturally-derived polymer include, in one or more embodiments, a water-soluble polypeptide, a low-molecular peptide, a polyamino acid, a sugar such as chitin or chitosan, and the like.
  • Examples of the polyamino acid include polylysine such as poly ( ⁇ -lysine) and poly ( ⁇ -lysine), polyarginine, and polyhistidine in one or more embodiments.
  • Examples of the synthetic polymer include, in one or more embodiments, a polymer or copolymer such as a linear type, a graft type, a comb type, a dendritic type, or a star type.
  • the polymer or copolymer may be polyurethane, polyamide, polycarbonate, or a copolymer thereof, polyester, polydiallyldimethylammonium chloride (PDDA), polyallylamine hydrochloride, polyethyleneimine, polyvinyl. Examples thereof include amines and polyamide amine dendrimers.
  • PDDA polydiallyldimethylammonium chloride
  • polyallylamine hydrochloride polyethyleneimine
  • polyvinyl examples thereof include amines and polyamide amine dendrimers.
  • a substance having a negative charge refers to a protein or polymer having a negative charge.
  • the protein having a negative charge is preferably a water-soluble protein in one or a plurality of embodiments.
  • the water-soluble protein include acidic collagen, acidic gelatin, albumin, globulin, catalase, ⁇ -lactoglobulin, thyroglobulin, ⁇ -lactalbumin, or ovalbumin in one or more embodiments.
  • Examples of the negatively charged polymer include naturally derived polymers and synthetic polymers.
  • Examples of the naturally-derived polymer include, in one or more embodiments, water-soluble polypeptides, low-molecular peptides, polyamino acids such as poly ( ⁇ -lysine), dextran sulfate, and the like.
  • Examples of the synthetic polymer include, in one or more embodiments, a polymer or copolymer such as a linear type, a graft type, a comb type, a dendritic type, or a star type.
  • the polymer or copolymer may be polyurethane, polyamide, polycarbonate, and a copolymer thereof, polyester, polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyacrylamide methylpropane sulfonic acid. , Terminal carboxylated polyethylene glycol, polydiallyldimethylammonium salt, polyallylamine salt, polyethyleneimine, polyvinylamine, or polyamidoamine dendrimer.
  • a combination of a positively charged substance and a negatively charged substance may be ⁇ -polylysine salt and polysulfonate, ⁇ -polylysine and polysulfonate, chitosan and dextran sulfate, poly Examples include allylamine hydrochloride and polystyrene sulfonate, polydiallyldimethylammonium chloride and polystyrene sulfonate, or polydiallyldimethylammonium chloride and polyacrylate, preferably ⁇ -polylysine salt and polysulfonate, or polydiallyl. Dimethylammonium chloride and polyacrylate.
  • polysulfonate examples include sodium polysulfonate (PSS) and the like in one or more embodiments.
  • PPS sodium polysulfonate
  • the substance having a positive charge and the substance having a negative charge may each be one kind, or two or more kinds may be used in combination within a range showing an interaction.
  • a solution is first brought into contact with a solution A containing a substance having an RGD sequence, and then containing a substance having an interaction with the substance having an RGD sequence.
  • a method for preparing coated cells by contacting with B will be described by way of example. However, the present disclosure is not construed as being limited to the following embodiments.
  • the cells are brought into contact with the solution A.
  • a film containing a substance having an RGD sequence is formed on the cell surface, and the cell surface is covered with a film containing a substance having an RGD sequence.
  • the contact between the cells and the solution A is performed by applying or adding the solution A to the cells, immersing the cells in the solution A, dropping or spraying the solution A on the cells, or the like. be able to.
  • the contact condition can be appropriately determined according to a contact method, a substance having an RGD sequence and / or a cell type, a concentration of a contained liquid, and the like.
  • the contact time is preferably 30 seconds to 24 hours, 1 minute to 60 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, or 1 minute to 5 minutes.
  • the ambient temperature at the time of contact and / or the temperature of the solution A is preferably 4 to 60 ° C., 20 to 40 ° C., or 30 to 37 ° C.
  • Solution A only needs to contain a substance having an RGD sequence, and preferably contains a substance having an RGD sequence and a solvent or dispersion medium (hereinafter also simply referred to as “solvent”).
  • solvent a solvent or dispersion medium
  • the content of the substance having an RGD sequence in the solution A is 0.0001 to 1% by mass, 0.01 to 0.5% by mass, or 0.02 to 0.1% by mass. preferable.
  • the solvent include an aqueous solvent such as water, phosphate buffered saline (PBS), and a buffer solution in one or more embodiments.
  • the buffer includes Tris buffer such as Tris-HCl buffer, phosphate buffer, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer. , Britton-Robinson buffer, GTA buffer, and the like.
  • Tris buffer such as Tris-HCl buffer, phosphate buffer, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer. , Britton-Robinson buffer, GTA buffer, and the like.
  • the pH of the solvent is not particularly limited, and in one or more embodiments, 3 to 11, 6 to 8, or 7.2 to 7.4 is preferable.
  • the solution A may further contain a salt, a cell growth factor, a cytokine, a chemokine, a hormone, a bioactive peptide, a pharmaceutical composition, or the like.
  • a pharmaceutical composition include, in one or a plurality of embodiments, a therapeutic agent, preventive agent, inhibitor, antibacterial agent, or anti-inflammatory agent for diseases.
  • the salt include sodium chloride, calcium chloride, sodium bicarbonate, sodium acetate, sodium citrate, potassium chloride, sodium hydrogen phosphate, magnesium sulfate, and sodium succinate in one or more embodiments.
  • One kind of salt may be contained, or two or more kinds of salts may be contained.
  • Both the solution A and the solution B may contain a salt, or one of them may contain a salt.
  • the salt concentration in the solution A is not particularly limited, but in one or more embodiments, it is 1 ⁇ 10 ⁇ 6 M to 2M, preferably 1 ⁇ 10 ⁇ 4 M to 1M, more preferably 1 ⁇ 10 ⁇ . 4 M to 0.05 M.
  • the removal can be performed by centrifugation or filtration.
  • the removal by centrifugation can be performed by centrifuging in a state where the cells are dispersed in the solution A, and then removing the supernatant. Centrifugation conditions can be appropriately determined depending on the type of cells, the concentration of cells, and the composition of inclusions contained in the solution A.
  • washing can be performed by centrifugation or filtration.
  • washing by centrifugation can be performed by adding a solvent to the cells from which the supernatant has been removed, followed by centrifugation and removal of the supernatant.
  • the solvent used for washing is preferably the same as the solvent of the solution A.
  • the cell covered with the membrane containing the substance having the RGD sequence is brought into contact with the solution B.
  • a membrane containing an interacting substance is formed on the membrane surface containing the substance having the RGD sequence, and the cell surface covered with the membrane containing the substance having the RGD sequence is covered with the membrane containing the interacting substance.
  • the contact with the solution B can be performed in the same manner as the contact with the solution A, except that a substance that interacts instead of the substance having the RGD sequence is used.
  • the extracellular surface in which the membrane containing the substance having the RGD sequence and the membrane containing the interacting substance are alternately laminated on the entire cell surface A coating containing a matrix component can be formed.
  • the number of times that the solution A or the solution B is brought into contact with the cells can be appropriately determined according to the thickness of the coating containing the extracellular matrix component to be formed.
  • the present disclosure is also referred to as a cultured myocardial tissue containing cardiomyocytes derived from induced pluripotent stem cells, an extracellular matrix component, and fibroblasts (hereinafter referred to as “cultured myocardial tissue of the present disclosure”). ) In one or a plurality of embodiments, the cultured myocardial tissue of the present disclosure can be obtained by arranging and culturing iPS cell-derived cardiomyocytes and fibroblasts in three dimensions.
  • the cultured myocardial tissue of the present disclosure is obtained by three-dimensionally arranging and culturing iPS cell-derived cardiomyocytes and fibroblasts coated with a coating containing an extracellular matrix component. Can do.
  • cultured myocardial tissue refers to a structure or aggregate of cells including iPS cell-derived cardiomyocytes, extracellular matrix components, and fibroblasts, and iPS cell-derived cardiomyocytes arranged in three dimensions.
  • the cultured myocardial tissue of the present disclosure can be used as a graft or a therapeutic agent for treating heart failure or the like.
  • the cultured myocardial tissue of the present disclosure can be used in the practice of regenerative medicine in one or more embodiments.
  • the cultured myocardial tissue further includes fibroblasts from the viewpoint that it is a tissue close to a living myocardial tissue.
  • the fibroblast is preferably a cardiac fibroblast in one or more embodiments.
  • the fibroblast may be a cell derived from a human or a cell derived from other than a human.
  • the fibroblast may be a cell derived from an embryonic stem cell, an induced pluripotent stem cell, or the like in one or a plurality of embodiments.
  • the ratio of cardiomyocytes to fibroblasts (cardiomyocytes: fibroblasts, number of cells) in the three-dimensional myocardial tissue is 99: 1 to 1:99 in one or more embodiments. In one or a plurality of embodiments, the ratio of cardiomyocytes to fibroblasts is 80:20 to 30:70, 80: from the viewpoint of generating action potentials in cardiomyocytes and realizing more regular pulsations. 20 to 45:55 or 75:25 to 50:50 is preferred.
  • the cultured myocardial tissue further includes vascular endothelial cells in one or a plurality of embodiments.
  • the cultured myocardial tissue preferably includes a vascular network formed of vascular endothelial cells in order to stably maintain the function for a longer period of time.
  • the vascular endothelial cell is preferably a cardiac microvascular endothelial cell in one or more embodiments.
  • the vascular endothelial cell may be a cell derived from a human or a cell derived from other than a human.
  • the vascular endothelial cell may be a cell derived from embryonic stem cells, induced pluripotent stem cells and the like in one or a plurality of embodiments.
  • the vascular network can be formed by mixing and seeding vascular endothelial cells with iPS-derived cardiomyocytes and fibroblasts and culturing.
  • the area of the cultured myocardial tissue is 20 mm 2 or more, 100 mm 2 or more, 200 mm 2 or more, 500 mm 2 or more, or 1000 mm 2 or more. In one or more embodiments, the thickness of the cultured myocardial tissue is 1 ⁇ m or more, 10 ⁇ m or more, and 10000 ⁇ m or less.
  • the present disclosure may relate to one or more of the following embodiments.
  • a method for producing a myocardial tissue chip used for drug screening Arrangement of coated cells, the surface of which is coated with a coating containing an extracellular matrix component, on the substrate, and forming the myocardial three-dimensional tissue on the substrate by repeatedly arranging the coated cells.
  • Including A method for producing a myocardial tissue chip, wherein the cells include cardiomyocytes derived from induced pluripotent stem cells.
  • the production method according to [1] comprising disposing fibroblasts on the substrate.
  • the manufacturing method of description [4] The production method according to any one of [1] to [3], wherein the cells are arranged such that four or more layers of cells including the cardiomyocytes are laminated. [5] The production method according to any one of [1] to [4], wherein the placement of the coated cells is performed by discharging the coated cells onto a substrate using a liquid discharge nozzle. [6] The production method according to [5], wherein the discharge of the coated cells is performed by discharging one coated cell per discharge.
  • the myocardial tissue chip according to [9], wherein the three-dimensional myocardial tissue further includes fibroblasts.
  • [17] comprising cardiomyocytes derived from induced pluripotent stem cells, extracellular matrix components and fibroblasts, A cultured myocardial tissue obtained by culturing the cardiomyocytes and the fibroblasts in three dimensions.
  • 50 mM Tris-HCl (pH 7.4): 50 mM Tris adjusted to pH 7.4 with HCl (manufactured by Nacalai Tesque) sterilized and filtered with 0.2 ⁇ m ⁇ -raysteryl filter (manufactured by Kurashiki Boseki Co., Ltd.)
  • BFN Fibrectin from bovine plasma (manufactured by SIGMA)
  • BFN solution 0.2 mg BFN / 1 ml 50 mM Tris-HCl (pH 7.4)
  • DMEM Dulbecco's easy's medium (manufactured by SIGMA) including 10% FBS (manufactured by GIBCO)
  • Gelatin solution 0.2 mg Gelatin / 1 ml 50 mM Tris-HCl (pH 7.4) sterilized and filtered with 0.2 ⁇ m ⁇ -ray sterile filter (manufactured by Kurashiki Bose
  • Cardiomyocytes were collected from a newborn rat, and a coated cell was prepared by forming a coating containing fibronectin and gelatin on the collected cell surface.
  • the FN dipping operation and the G dipping operation were each set as a washing operation and one step, and finally, the coated cells were prepared by performing the FN dipping operation 5 times and the G dipping operation 4 times for a total of 9 steps (coating layer) Thickness: 7 nm).
  • FIG. 1 is a photomicrograph of the myocardial three-dimensional tissue after staining. As shown in FIG. 1, it was confirmed that the obtained myocardial three-dimensional tissue had a myocardial tissue structure having a troponin skeleton.
  • a myocardial three-dimensional tissue body was prepared in the same procedure as described above except that the number of coated cells seeded on the membrane filter was 10 ⁇ 10 5 (10 layers, thickness: approximately 60 ⁇ m). The density of cardiomyocytes in the obtained myocardial three-dimensional tissue was 5 ⁇ 10 7 cells / mm 2 . After confirming that the obtained myocardial three-dimensional tissue was beating, 1.0 ⁇ M isoproterenol was added and incubated for 30 minutes. After measuring the number of beats, the medium was changed to DMEM to remove isoproterenol, and after culturing for 41 hours, the number of beats was measured. The result is shown in FIG. The number of beats was measured by observation with a phase contrast microscope.
  • FIG. 2 is an example of a graph showing changes in the number of beats per minute of a three-dimensional myocardial tissue.
  • the number of pulsations of the obtained three-dimensional myocardial tissue significantly increased when isoproterenol, which is a pulsatile promoter, was added.
  • Example 1 Coated cells were prepared in the same manner as described above except that human iPS cell-derived cardiomyocytes were used in place of rat neonatal cardiomyocytes.
  • the human iPS cell used was the 253G1 strain.
  • a myocardial tissue chip containing 10 layers of myocardial 3D tissue was prepared in the same manner as in preparation of myocardial 3D tissue (Part 2).
  • the obtained myocardial three-dimensional tissue was cultured for several days, and a phase contrast micrograph is shown in FIG. It was observed that the prepared myocardial three-dimensional tissue was uniformly driven throughout the tissue even after several days of culture.
  • Example 2 A myocardial tissue chip was produced in the same manner as in Example 1 except that the number of layers in the myocardial three-dimensional tissue was changed to five. After the produced myocardial tissue chip was cultured for 1 day, 1M isoproterenol (pulsation promoter) was added, and the number of pulsations before and after the addition was measured. The result is shown in FIG. As shown in FIG. 4, it was confirmed that the number of beats increased in response to drug stimulation. That is, since the obtained myocardial three-dimensional tissue body has a myocardial function having drug responsiveness, a myocardial tissue chip providing a myocardial three-dimensional tissue body capable of drug evaluation could be produced.
  • 1M isoproterenol pulsation promoter
  • Example 3 A myocardial tissue chip including a myocardial 3D tissue was produced in the same manner as in Example 1 except that the number of layers in the myocardial 3D tissue was changed to 4.
  • FIG. 5 shows a tissue section image (HE-stained image) after culturing the prepared myocardial three-dimensional tissue for 7 days
  • FIG. 6 shows a phase contrast micrograph. The number of beats of the obtained myocardial three-dimensional tissue was 40 times / min.
  • Example 4 Coated cells were prepared using human iPS-derived cardiomyocytes (5 ⁇ 10 5 cells), and myocardial tissue chips (5 layers) were prepared in the same manner as in Example 2.
  • Calcium imaging was performed using the myocardial tissue chips of Examples 4-6. Calcium imaging was performed according to the following procedure. That is, 4 ⁇ M Fluo3AM was added to the myocardial tissue chip after 5 days of culture and incubated for 45 minutes. Thereafter, imaging was performed using a fluorescence microscope under conditions of an excitation wavelength of 508 nm and a fluorescence wavelength of 527 nm.
  • the change of the fluorescence intensity accompanying the pulsation was strong in the order of Example 5 and Example 6, and the pulsation was large in the order of Example 5 and Example 4.
  • Example 4 pulsated, no change in fluorescence intensity due to pulsation was observed. In Example 6, pulsation was hardly observed.
  • Example 8 [Production of myocardial tissue chip having vascular network] A 24-well cell in which human iPS-derived coated cardiomyocytes (1.0 ⁇ 10 6 cells) and coated normal human microvascular endothelial cells (NHCMEC) are mixed so that the ratio of NHCMEC is 10% of the total number of cells. A myocardial tissue chip was prepared by seeding on a culture insert and culturing (Example 8-1).
  • FIG. 7A shows a phase contrast micrograph of the myocardial three-dimensional tissue (after 6 days of culture) in the obtained myocardial tissue chip.
  • Example 8 except that human iPS-derived coated cardiomyocytes (5.0 ⁇ 10 5 cells) and coated fibroblasts (5.0 ⁇ 10 5 cells) were used instead of only human iPS-derived coated cardiomyocytes.
  • FIG. 7B shows a phase contrast micrograph of the myocardial three-dimensional tissue (after 6 days of culture) in the obtained myocardial tissue chip.
  • the myocardial tissue chip of Example 8-2 prepared by mixing coated fibroblasts was compared with the myocardial tissue chip of Example 8-1 that did not contain coated fibroblasts. A finer vascular network was formed.
  • NHCF normal human cardiac fibroblast

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Abstract

L'invention porte sur un procédé ou analogue de fabrication d'une puce tissulaire de muscle cardiaque, dans lequel une structure 3D du muscle cardiaque est formée, et qui peut être utilisée pour le criblage de médicaments. La présente invention concerne un procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage de médicaments, le procédé de fabrication d'une puce tissulaire de muscle cardiaque comprenant le dépôt, sur un substrat, de cellules d'essai dans lesquelles la surface des cellules est revêtue d'un film contenant un composant de matrice extracellulaire, et la formation d'une construction de tissu 3D de muscle cardiaque sur le substrat par dépôt répétitif des cellules d'essai, les cellules comprenant des cellules du muscle cardiaque dérivant de cellules souches multipotantes artificielles.
PCT/JP2014/072029 2013-08-23 2014-08-22 Procédé de fabrication d'une puce tissulaire de muscle cardiaque utilisée pour le criblage d'un composé candidat médicamenteux WO2015025957A1 (fr)

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JPWO2017073794A1 (ja) * 2015-10-30 2018-08-16 国立大学法人京都大学 多能性幹細胞から３次元の心筋組織を製造する方法
WO2018164098A1 (fr) * 2017-03-06 2018-09-13 国立大学法人大阪大学 Population de cellules à utiliser en tant que modèle de fibrage de cœur, procédé pour sa production, procédé de criblage l'utilisant, procédé d'évaluation de modèle de fibrage de cœur l'utilisant et système d'évaluation de modèle de fibrage de cœur
JPWO2018164098A1 (ja) * 2017-03-16 2020-01-16 国立大学法人大阪大学 心臓線維化モデルとして用いられる細胞集団、その製造方法、それを利用したスクリーニング方法及び心臓線維化モデルの評価方法、並びに心臓線維化モデルの評価システム
JP2019034126A (ja) * 2017-08-10 2019-03-07 国立大学法人大阪大学 心筋組織−コラーゲンの自己可動膜
JP7133172B2 (ja) 2017-08-10 2022-09-08 国立大学法人大阪大学 心筋組織－コラーゲンの自己可動膜
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CN111051501A (zh) * 2017-09-04 2020-04-21 凸版印刷株式会社 异常跳动心肌模型及其制造方法、异常跳动心肌模型的造模剂以及心脏病治疗药的药效评价方法
WO2019088224A1 (fr) * 2017-11-02 2019-05-09 国立大学法人大阪大学 Puce cellulaire et puce tissulaire tridimensionnelle et procédé pour leur production
JP2020072691A (ja) * 2017-11-02 2020-05-14 国立大学法人大阪大学 細胞チップおよび三次元組織チップ、およびその製造方法
JPWO2019088224A1 (ja) * 2017-11-02 2019-11-14 国立大学法人大阪大学 細胞チップおよび三次元組織チップ、およびその製造方法
JP7333543B2 (ja) 2017-11-02 2023-08-25 国立大学法人大阪大学 細胞チップおよび三次元組織チップ、およびその製造方法
WO2020129774A1 (fr) * 2018-12-20 2020-06-25 日本ゼオン株式会社 Procédé d'évaluation de la cardiotoxicité
JPWO2020129774A1 (ja) * 2018-12-20 2021-11-04 日本ゼオン株式会社 心毒性評価方法
WO2020179929A1 (fr) * 2019-03-06 2020-09-10 国立大学法人大阪大学 Procédé de production de tissu cellulaire, ensemble de production de tissu cellulaire, et récipient de culture contenant un tissu cellulaire produit par ledit procédé de production

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