WO2023282273A1 - 細胞培養方法 - Google Patents

細胞培養方法 Download PDF

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Publication number
WO2023282273A1
WO2023282273A1 PCT/JP2022/026773 JP2022026773W WO2023282273A1 WO 2023282273 A1 WO2023282273 A1 WO 2023282273A1 JP 2022026773 W JP2022026773 W JP 2022026773W WO 2023282273 A1 WO2023282273 A1 WO 2023282273A1
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cell
formula
producing
group
substrate
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French (fr)
Japanese (ja)
Inventor
周吾 遠山
恵一 福田
康平 鈴木
祐揮 上田
美耶 廣飯
佳臣 広井
さやか 金編
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Nissan Chemical Corp
Keio University
Heartseed Inc
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Nissan Chemical Corp
Keio University
Heartseed Inc
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/10—Esters
    • C08F20/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing human induced pluripotent stem cell (hiPS cell)-derived cardiomyocyte aggregates.
  • Cardiac disease is the leading cause of death in humans worldwide, and heart failure due to contraction failure of the left ventricle is cited as the cause.
  • Transplanting a sufficient number of ventricular muscles could be a radical cure for heart failure with asystole failure (HFrEF).
  • HFrEF asystole failure
  • a method for efficiently producing a sufficient number of ventricular myocytes has not been known so far.
  • treatment with cell aggregates spheroids
  • establishment of an industrial technique capable of stably producing large amounts of spheroids is desired.
  • Various materials have been proposed so far as base film-forming agents for efficient cell culture.
  • Patent Document 1 discloses a method for producing a polymer used as a base film for cell culture and a cell culture vessel
  • Patent Document 2 discloses a method for producing a cell structure.
  • serum derived from living organisms for the base membrane of these cell cultures for uniform adhesion of cells to the base membrane.
  • There have been problems such as quality variations, including the ability, and safety risks such as allergies and virus contamination when using serum derived from animals other than humans.
  • the present invention provides a method for mass production of human induced pluripotent stem cell (hiPS cell)-derived cardiomyocyte aggregates, which can produce homogeneous and high-quality cardiomyocyte aggregates without using biological serum.
  • hiPS cell human induced pluripotent stem cell
  • the present inventors have found that by seeding and culturing hiPS cell-derived cardiomyocytes using a cell aggregate-producing substrate comprising a base film containing a specific polymer and a cell-adhesive substance, The present invention has been completed based on the finding that homogeneous and high-quality cardiomyocyte aggregates can be produced without using serum.
  • the present invention includes the following.
  • the following formula (I) is applied onto a substrate having the ability to suppress adhesion of cells:
  • U a1 and U a2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
  • R a1 is a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
  • R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms.
  • hiPS cells human induced pluripotent stem cells
  • the polymer further has the formula (II): [In the formula, R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. method for producing cardiomyocyte aggregates.
  • the method for producing hiPS cell-derived cardiomyocyte aggregates of the present invention uniform adhesion of cardiomyocytes can be achieved even under animal-derived serum-free culture conditions, and cell aggregates can be produced subsequently. As a result, mass production of homogeneous and high-quality cardiomyocyte aggregates for use in the field of regenerative medicine can be achieved.
  • FIG. 1 is a photograph of the entire surface of a well after seeding cardiomyocytes in each well and culturing for 24 hours in the seeding/culturing step of Example 1.
  • FIG. 1 is a photograph of the entire surface of a well after seeding cardiomyocytes in each well and culturing for 48 hours in the seeding/culturing step of Example 1.
  • FIG. 1 is a photograph of the entire surface of a well after seeding cardiomyocytes in each well and culturing for 72 hours in the seeding/culturing step of Example 1.
  • FIG. FIG. 10 is a photograph of the entire surface of the well after seeding cardiomyocytes in each well of substrate 2 and culturing for 72 hours in the seeding/culturing step of Example 2.
  • FIG. 10 is a photograph of the entire surface of the well after seeding cardiomyocytes in each well of the substrate 3 and culturing for 72 hours in the seeding/culturing step of Example 2.
  • FIG. 10 is a graph showing the results of measuring the diameter of cardiomyocytes after seeding cardiomyocytes on substrate 2 and culturing them for 96 hours in the seeding/culturing step of Example 2.
  • FIG. 10 is a graph showing the results of measuring the diameter of cardiomyocytes after seeding cardiomyocytes on the substrate 3 and culturing them for 96 hours in the seeding/culturing step of Example 2.
  • FIG. 10 is a photograph of the entire surface of the well after seeding cardiomyocytes in each well of the substrate 3 and culturing for 72 hours in the seeding/culturing step of Example 2.
  • FIG. 10 is a graph showing the results of measuring the diameter of cardiomyocytes after seeding cardiomyocytes on substrate 2 and culturing them for 96 hours in the seeding/culturing
  • Example 3 cardiomyocytes (stocks: CM1, CM2 and CM3) were seeded in each well of substrate 2, and the entire surface of the wells was photographed after 48 hours and 72 hours of culture.
  • cardiomyocytes (stocks: CM1, CM2 and CM3) were seeded in each well of an Elplasia plate, and the entire surface of the wells was photographed after culturing for 48 hours and 7 days.
  • the degree of maturity of the cell clusters (cardiomyocyte spheres) cultured on the substrates 2 and 3 of the present invention and the cell clusters (cardiomyocyte spheres) cultured on the Elplasia plate was analyzed by quantitative PCR (comparative Ct method).
  • FIG. 2 is a graph showing the expression levels of the maturation markers (TNNI3/TNNI1) that were determined.
  • 4 is a photograph of the whole surface of the well after seeding 4 ⁇ 10 6 cells of cardiomyocytes in each well of the substrate 4 and culturing for 72 hours in the seeding/culturing step of Example 4.
  • FIG. 3 is a photograph of the entire surface of the well after seeding 3 ⁇ 10 6 cells of cardiomyocytes in each well of the substrate 4 and culturing for 72 hours in the seeding/culturing step of Example 4.
  • FIG. 4 is a photograph of the entire surface of the well after seeding 4 ⁇ 10 6 cells of cardiomyocytes in each well of substrate 5 and culturing for 72 hours in the seeding/culturing step of Example 4.
  • FIG. 3 is a photograph of the entire surface of the well after seeding 3 ⁇ 10 6 cells of cardiomyocytes in each well of substrate 5 and culturing for 72 hours in the seeding/culturing step of Example 4.
  • FIG. 4 is a graph showing the results of measuring the diameter of 4 ⁇ 10 6 cells of cardiomyocytes seeded on substrate 4 in the seeding/culturing step of Example 4, harvested after culturing for 96 hours, and measuring the diameter.
  • 10 is a graph showing the results of measuring the diameter of 3 ⁇ 10 6 cardiomyocytes seeded on substrate 4, collected after culturing for 96 hours, and measured in the seeding/culturing step of Example 4.
  • FIG. 4 is a graph showing the results of measuring the diameter of 4 ⁇ 10 6 cells of myocardial cells seeded on substrate 5 in the seeding/culturing step of Example 4, harvested after culturing for 96 hours, and measuring the diameter.
  • 3 is a graph showing the results of measuring the diameter of 3 ⁇ 10 6 cells of myocardial cells seeded on substrate 5 in the seeding/culturing step of Example 4, harvested after culturing for 96 hours, and measuring the diameter.
  • the method for producing hiPS cell-derived cardiomyocyte aggregates of the present invention comprises the following formula (I): [In the formula, U a1 and U a2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R a1 is a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms and R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms]. Forming a basement membrane followed by seeding cardiomyocytes.
  • the base film for cardiomyocyte culture according to the production method of the present invention is formed using a base film-forming agent.
  • the base film former typically has the following formula (I): [In the formula, U a1 and U a2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R a1 is a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms and R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms].
  • the polymer contained in the base film-forming agent for cell culture of the present application is a polymer containing a repeating unit derived from the monomer represented by formula (I) above.
  • the polymer is represented by the following formula (II) together with the cationic monomer represented by the above formula (I): [In the formula, R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].
  • the polymer containing repeating units derived from the monomer represented by the formula (I) has the following formula (Ia): [wherein U a1 , U a2 , R a1 and R a2 have the same meanings as in formula (I)].
  • the polymer obtained by polymerizing the anionic monomer represented by the formula (II) together with the cationic monomer represented by the formula (I) is the repeating unit represented by the formula (Ia) together with the following formula (IIa): It can also be expressed as a polymer containing a repeating unit represented by [wherein Rb has the same meaning as in formula (II)].
  • the production method of the present invention includes a polymer containing a repeating unit represented by the above formula (Ia), or a repeating unit represented by the above formula (IIa) together with a repeating unit represented by the above formula (Ia).
  • hiPS cells human induced pluripotent stem cells
  • the "linear or branched alkyl group having 1 to 5 carbon atoms” includes, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2 , 2-dimethylpropyl group or 1-ethylpropyl group.
  • R a1 and R b are each independently selected from a hydrogen atom and a methyl group.
  • U a1 and U a2 are preferably each independently selected from hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group and n-butyl group, but are preferably methyl group or ethyl group. Preferred are methyl groups.
  • the "linear or branched alkylene group having 1 to 5 carbon atoms" includes, for example, methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methyl propylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1,1-dimethyl-trimethylene group, 1,2- dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group and the like.
  • R a2 is preferably selected from an ethylene group and a propylene group.
  • cationic monomers represented by formula (I) above include 2-N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminomethyl methacrylate and the like, and 2-N,N-dimethylaminoethyl Methacrylates are preferred.
  • anionic monomer represented by formula (II) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.
  • the molar ratio of units derived from the monomer represented by formula (I)/units derived from the monomer represented by formula (II) in the polymer is 100/0 to 50/50. It is preferably 98/2 to 50/50. More preferably 98/2 to 60/40, particularly preferably 98/2 to 70/30. When the molar ratio of the formula (II) is 50 or less, it is possible to suppress a decrease in cell adhesive force due to the anionicity of the polymer.
  • the polymer is not particularly limited as long as it contains units derived from the monomer represented by formula (I) and optionally units derived from the monomer represented by formula (II), which impairs the object of the present invention. It may contain repeating units other than the units derived from the monomers represented by formula (I)/formula (II), within the range of not included.
  • the polymer contains, as repeating units, 50 mol% or more, preferably 75 mol% or more, more preferably 80 mol% or more, more preferably 80 mol% or more, and more preferably It may be 90 mol % or more.
  • it may be a polymer obtained by polymerizing a monomer represented by Formula (I)/Formula (II) and a monomer having two or more carbon-carbon unsaturated bonds.
  • a monomer having two or more carbon-carbon unsaturated bonds is specifically a monomer having two or more carbon-carbon double bonds, such as polyfunctional acrylate compounds, polyfunctional acrylamide compounds, polyfunctional A polyester or an isoprene compound may be used.
  • Preferred specific examples include monomers represented by the following formulas (III) to (V).
  • R c and R d each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
  • R e is a linear or branched alkylene group having 1 to 5 carbon atoms. group
  • n represents a number from 1 to 50.
  • the monomer represented by formula (III) is preferred.
  • the molar ratio of the monomers represented by formulas (III)-(V) to the total polymer is preferably 0-50%, more preferably 2-25%.
  • the molar ratio of the formulas (III) to (V) is 50% or less, gelation of the solid content during production due to high molecular weight due to excessive cross-linking can be suppressed, and production can be facilitated.
  • R c and R d are each independently selected from a hydrogen atom and a methyl group.
  • R e is preferably selected from a methylene group, an ethylene group and a propylene group, most preferably an ethylene group.
  • n is a number from 1 to 50, n is preferably a number from 1 to 30, and n is preferably a number from 1 to 10.
  • Difference between the mol% value of the monomer represented by the formula (II) with respect to the entire polymer and the mol% value of the monomer represented by the formula (II) with respect to the total amount of monomer charged during the preparation step is 0 to 10 mol %.
  • the polymer of the present application has a small difference between the charged ratio of the monomer and the measured value of the polymer produced by the production method described later, and is 0 to 10 mol %, more preferably 0 to 8 mol %.
  • the number average molecular weight (Mn) of the polymer is 20,000 to 1,000,000, more preferably 50,000 to 800,000.
  • the ratio (Mw/Mn) between the weight average molecular weight (Mw) and the number average molecular weight (Mn) of the polymer is 1.01 to 10.00, preferably 1.2 to 8.0, It is preferably 1.4 to 6.0, preferably 1.5 to 5.0, preferably 1.6 to 4.5.
  • the number average molecular weight (Mn) and the number average molecular weight (Mn) can be determined, for example, by Gel Filtration Chromatography described in Examples.
  • cell aggregate refers to a structure formed as a result of aggregation of cells, and its shape is not limited to a spherical shape, a ring shape, or the like.
  • the size adjustment of cell aggregates by defining the adhesion area can be produced.
  • the base film-forming agent for cell culture of the present invention contains a cell adhesive substance.
  • a cell adhesive substance By containing a cell adhesive substance, cell adhesion, spreading, proliferation and differentiation can be promoted.
  • known substances such as biological substances such as extracellular matrix (ECM) proteins, glycoproteins, and peptides, and synthetic compounds (low molecular weight, high molecular weight) can be used. It is preferably a non-substance compound, such as a synthetic compound (low molecular weight, high molecular weight).
  • a low molecular weight compound is, for example, a compound having a weight average molecular weight of 2,000 or less, and a high molecular weight compound is, for example, a weight average molecular weight of 2,000 or more, and the upper limit is, for example, 1,000,000.
  • extracellular matrix (ECM) proteins examples include collagen (e.g. Merck type I collagen (product numbers C9791, C7661, C1809, C2249, C2124), type II collagen (product number C9301), type IV collagen (product numbers C0543, C5533). ), elastin (e.g. Merck product numbers: E1625, E6527), fibronectin (e.g. Merck product numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g.
  • the cell adhesive substance is preferably a glycoprotein. Specifically, it is preferably selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osthiopontin and bone sialoprotein. Also, a protein having an RGD sequence as an amino acid sequence is preferred.
  • peptides examples include ECM peptide (MAPTrix (registered trademark) from Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries: 180-01531).
  • ECM peptide MAPTrix (registered trademark) from Kollodis Bio Sciences
  • RGD peptide manufactured by Fujifilm Wako Pure Chemical Industries: 180-01531.
  • Examples of synthetic compounds include polylysine (eg Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (eg Merck product number P4975).
  • Examples of synthetic compounds low molecular weight include adhesamine (eg AD-00000-0201 manufactured by Nagase & Co., Ltd.) and synthetic cyclic RGD peptide (eg LS-3920.0010 manufactured by IRIS BIOTECH).
  • the ratio (mass basis) of the polymer and the cell adhesive substance in the base film-forming agent for cell culture of the present invention is not limited as long as the base-forming agent capable of cell culture can be formed, but is 100:0.1. ⁇ 100:100, preferably 100:10 to 100:30.
  • the cell adhesion substance is 0.1 or more, the cell adhesiveness is sufficiently exhibited, and when the cell adhesion substance is 100 or less, cell aggregation after cell adhesion (formation of cell aggregates) is facilitated. can.
  • the base film-forming agent for cell culture of the present invention contains a solvent.
  • the solvent is not limited as long as it can dissolve the polymer, but it is preferably a water-containing solution.
  • the aqueous solution includes water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent in which water or a salt-containing aqueous solution and alcohol are combined.
  • Alcohols include alcohols having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol.
  • the base film-forming agent may optionally contain other substances within the range that does not impair the performance of the obtained base film.
  • Other substances include pH adjusters, cross-linking agents, preservatives, surfactants, primers to improve adhesion to containers or substrates, antifungal agents and sugars.
  • the production method of the present invention comprises a substrate for producing cardiomyocyte aggregates, which is provided with spots of the basement membrane for cardiomyocyte culture formed with the above-mentioned basement membrane-forming agent on a substrate having the ability to suppress adhesion of cells.
  • the substrate for producing cardiomyocyte aggregates of the present invention is produced using a substrate having the ability to suppress adhesion of cells. Prior to the formation of the spots (underlying film), the substrate may be treated to inhibit adhesion of cells.
  • the substrate having the ability to suppress cell adhesion may be a commercially available cell culture dish treated with low cell adhesion, a cell culture vessel having the ability to inhibit cell adhesion, or the like.
  • the cell culture vessel described above can be used, it is not limited to this.
  • a substrate manufactured through a step of applying a coating film-forming composition having a known ability to suppress adhesion of cells may be used.
  • the coating film-forming composition for example, the coating film-forming composition described in International Publication No. 2014/196650 can be used.
  • U a11 , U a12 , U b11 , U b12 and U b13 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
  • An ⁇ is a halide ion
  • an inorganic acid Represents an anion selected from the group consisting of ion, hydroxide ion and isothiocyanate ion] and a solvent, and a step of applying the composition for forming a coating film onto the surface of the container or the substrate and drying the composition.
  • the coating film may be included on at least a part of the substrate surface, but it is applied over the entire surface where cell aggregates are produced (i.e., the surface where the spots of the present application exist) or over the entire substrate surface. is preferred.
  • the entire disclosures of WO2014/196650 and WO2016/093293 are hereby incorporated by reference.
  • the relative absorbance WST O.D. 450 nm (%) ((Absorbance of Example (WST O.D. 450 nm)) / (Absorbance of Comparative Example (WST O.D. 450 nm)) is 50% or less, preferably 30% Hereinafter, more preferably, it means 20% or less.
  • copolymers of ethylenically unsaturated monomers or polysaccharides or derivatives thereof may be used.
  • ethylenically unsaturated monomers include one or two selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and hydrophilic functional derivatives thereof. More than one species of ethylenically unsaturated monomers may be mentioned.
  • polysaccharides or derivatives thereof include cellulosic polymers such as hydroxyalkylcellulose (eg, hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan.
  • hydrophilic functional derivative refers to an ethylenically unsaturated monomer having a hydrophilic functional group or structure.
  • hydrophilic functional groups or structures include betaine structures; amide structures; alkylene glycol residues; amino groups;
  • a betaine structure means a monovalent or divalent group of compounds having an amphoteric center with a quaternary ammonium type cationic structure and an acidic anionic structure, such as the phosphorylcholine group: can be mentioned.
  • ethylenically unsaturated monomers having such a structure include 2-methacryloyloxyethylphosphorylcholine (MPC).
  • the amide structure has the formula: [Here, R 16 , R 17 and R 18 are each independently a hydrogen atom or an organic group (e.g., a methyl group, a hydroxymethyl group, a hydroxyethyl group, etc.)] means a group represented by Examples of ethylenically unsaturated monomers having such structures include (meth)acrylamide, N-(hydroxymethyl)(meth)acrylamide, N-isopropyl(meth)acrylamide and the like. Furthermore, monomers or polymers having such a structure are disclosed in, for example, JP-A-2010-169604.
  • An alkylene glycol residue is an alkylene glycol (HO-Alk-OH; where Alk is an alkylene group having 1 to 10 carbon atoms).
  • the hydroxyl groups at one or both terminals of the alkylene glycol remain after the condensation reaction with other compounds. It means an alkyleneoxy group (--Alk--O--) and also includes a poly(alkyleneoxy) group in which the alkyleneoxy unit is repeated.
  • Examples of ethylenically unsaturated monomers having such structures include 2-hydroxyethyl (meth)acrylate, methoxypolyethylene glycol (meth)acrylate, and the like. Further, monomers or polymers having such structures are disclosed in, for example, JP-A-2008-533489.
  • An amino group has the formula: —NH 2 , —NHR 19 or —NR 20 R 21 [wherein R 19 , R 20 and R 21 are each independently an organic group (for example, linear or branched alkyl group, etc.)].
  • Amino groups in the present invention include quaternary or salified amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacryloylcholine chloride, and the like.
  • a sulfinyl group has the formula: [Here, R 22 is an organic group (eg, an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms and having one or more hydroxy groups, etc.)] means a group represented by Examples of polymers having such a structure include copolymers disclosed in Japanese Unexamined Patent Application Publication No. 2014-48278.
  • a water-insoluble copolymer that is difficult to dissolve in phosphate-buffered saline can be used as a coating film that has the ability to suppress cell adhesion.
  • Water-soluble as used herein means that 1.0 g or more can be dissolved in 100 g of water at 25°C.
  • Water-insoluble means that it does not correspond to “water-soluble”, that is, the solubility in 100 g of water at 25°C is less than 1.0 g.
  • water-insoluble copolymer means a copolymer having a solubility of less than 1.0 g in 100 g of water at 25°C, particularly a solubility in 100 g of phosphate buffered saline at 25°C It means a copolymer that is less than 1.0 g.
  • water-insoluble copolymers examples include copolymers containing repeating units (A) represented by the following formula (A) and repeating units (B) represented by the following formula (B).
  • R 1 to R 3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
  • X 1 and X 2 each independently represent a single bond, an ester It represents a bond, an ether bond, an amide bond, or a linear or branched alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.
  • the water-insoluble copolymer may contain two or more types of repeating units (A), and may contain two or more types of repeating units (B), but one type of repeating unit (A ) and one type of repeating unit (B).
  • R 1 to R 3 are each independently preferably a hydrogen atom, a methyl group, or an ethyl group.
  • ether bond means -O-
  • linear or branched alkylene group having 1 to 5 carbon atoms optionally interrupted by an oxygen atom means a linear or branched alkylene group having 1 to 5 carbon atoms. It means an alkylene group or a group in which one or more carbon-carbon bonds of a linear or branched alkylene group having 1 to 5 carbon atoms are linked through an ether bond.
  • X 1 and X 2 are each independently preferably a methylene group, an ethylene group, or a propylene group.
  • R 1 and R 2 are hydrogen atoms
  • R 3 is a methyl group
  • X 1 and X 2 are single bonds.
  • the molar ratio (A:B) of the repeating unit (A) and the repeating unit (B) in the water-insoluble copolymer is from 89:11 to 50:50.
  • the molar ratio of the repeating unit (A) and the repeating unit (B) (A: B) can be expressed as (100 ⁇ m):m.
  • the range of m is 11-50. and the lower limit of m may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • An upper limit for m may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 38, 37, 36, or 35.
  • the range of m is, for example, 12-49, 12-48, 15-48, 20-49, 20-45, 22-49, or 22-45.
  • the total mol% of the repeating unit (A) and the repeating unit (B) in all repeating units in the water-insoluble copolymer is not particularly limited, but is preferably 90 mol% or more, more preferably 95 mol% or more. Preferably, 99.5 mol % or more is more preferable, and 100% is particularly preferable.
  • the copolymer By setting the molar ratio of the repeating unit (A) and the repeating unit (B) in the water-insoluble copolymer to a specific range, the copolymer can be dissolved in phosphate buffered saline without cross-linking. A hard-to-remove coating film is obtained. Therefore, the water-insoluble copolymer need not have photosensitive groups for cross-linking the copolymer. That is, it is preferable that the water-insoluble copolymer does not have a photosensitive group. Photosensitive groups include, for example, an azide group.
  • the water-insoluble copolymer does not need to have a photosensitive group for cross-linking the copolymer, it is necessary to perform light irradiation for cross-linking the copolymer when forming the coating film. There is no Therefore, the use of the water-insoluble copolymer simplifies the process of forming a coating film having the ability to inhibit cell adhesion.
  • the viscosity-average degree of polymerization of the water-insoluble copolymer (hereinafter sometimes simply referred to as "degree of polymerization") is not particularly limited, but from the viewpoint of suitably obtaining the cell adhesion inhibitory ability, 200 to 3,000. Preferably, 200 to 2,500 is more preferable, and 200 to 2,000 is particularly preferable.
  • the viscosity-average degree of polymerization is measured in a completely saponified state of the water-insoluble copolymer.
  • the "viscosity average degree of polymerization" of polyvinyl alcohol obtained by complete saponification is determined by the following formula from the intrinsic viscosity [ ⁇ ] (g / dL) when measured at 30 ° C. with an Ostwald viscometer using ion-exchanged water as a solvent. It is a calculated value.
  • P indicates the viscosity average degree of polymerization.
  • the viscosity average degree of polymerization can be determined according to JIS K6726.
  • the method for producing the water-insoluble copolymer is not particularly limited. A method of partially hydrolyzing by reaction to obtain a copolymer can be mentioned.
  • R 1 , R 3 and X 1 are defined as above.
  • a compound represented by the following formula (C) and a compound represented by the following formula (D) are copolymerized to obtain a copolymer. method to obtain.
  • R 1 to R 3 , X 1 and X 2 have the same meanings as above.
  • the water-insoluble copolymer may be a random copolymer or a block copolymer.
  • a commercially available product may be used as the water-insoluble copolymer.
  • Specific examples of commercially available copolymers include polyvinyl acetate (manufactured by Nippon Acetate & Poval Co., Ltd., trade name JMR-150L (registered trademark)).
  • the content of the copolymer in the film-forming component in the coating film-forming composition used in the production of the substrate capable of inhibiting adhesion of cells according to the present invention is not particularly limited, but is preferably 80% by mass or more. , more preferably 90% by mass or more, and particularly preferably 95% by mass or more.
  • the film-forming component refers to a component excluding the solvent component from all components of the composition.
  • the content of the copolymer in the coating film-forming composition used in the production of the substrate having the ability to suppress cell adhesion according to the present invention is not particularly limited, but from the viewpoint of facilitating the formation of a coating film having a desired thickness. Therefore, 0.1 to 10% by mass is preferable, 0.3 to 8% by mass is more preferable, and 0.5 to 5% by mass is particularly preferable.
  • the content of the copolymer in the coating film-forming composition may be 0.02 to 2% by mass, or may be 0.05 to 1% by mass.
  • the ratio of the total area of the spots, preferably a plurality of spots (underlying membrane), the diameter of each spot, and the distance between the centers of the spots, provided on the substrate for producing cardiomyocyte aggregates of the present invention, is determined by the type of cells and substrate used, the type of cells, and the Depending on the desired size of the agglomerate, etc., it can be appropriately selected from a predetermined range. preferably 99% or less, the diameter of each spot is 50 to 5000 ⁇ m, preferably 300 to 3000 ⁇ m, more preferably 300 to 1000 ⁇ m, most preferably 300 to 600 ⁇ m, the distance between the center of the spot , 30 to 1000 ⁇ m, preferably 50 to 800 ⁇ m, more preferably 400 to 600 ⁇ m.
  • the present invention by arranging independent micro-sized regions (spots) to which cardiomyocytes can adhere at a high density, preferably regularly, on a substrate having the ability to suppress adhesion of cells, cells of uniform size can be obtained.
  • a plurality of spheroids can be formed on one substrate (container) at once.
  • the spot can be formed by applying the base film forming agent.
  • a method for applying the base film-forming agent for example, an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like can be used, but printing techniques such as an inkjet method or screen printing are preferable.
  • a substrate optionally protected at non-formed spots is immersed in the base film-forming agent, or the undercoat film-forming agent is added to the substrate (container) optionally protected at non-spotted locations.
  • a substrate one aspect of which is a cell culture vessel
  • a base film-forming agent is optionally added to the vessel in which the non-spotted areas are protected, and then left to stand for a predetermined period of time. It is done by the method of standing for a while.
  • the addition can be performed, for example, by adding the base film-forming agent in an amount of 0.5 to 1 times the total volume of the container using a syringe or the like.
  • the standing is carried out by appropriately selecting the time and temperature depending on the material of the vessel or substrate and the type of base film-forming agent for cell culture. It is carried out at 10-80°C.
  • the spots on the surface of the substrate obtained by such a method can be washed as they are without a drying step, or washed with water or a sample medium (e.g., water, buffer solution, medium, etc.) to be subjected to cell culture. Later, it can be used as a substrate for manufacturing cardiomyocyte aggregates.
  • a sample medium e.g., water, buffer solution, medium, etc.
  • a sample medium subjected to cell culture e.g., water, buffer, medium, etc., particularly preferably a medium (e.g., DMEM medium (Dulbecco's modified Eagle medium)) After washing, it can be used as a substrate for producing cardiomyocyte aggregates.
  • a sample medium subjected to cell culture e.g., water, buffer, medium, etc., particularly preferably a medium (e.g., DMEM medium (Dulbecco's modified Eagle medium)
  • DMEM medium Dulbecco's modified Eagle medium
  • the cell aggregate-producing substrate may be subjected to a drying process.
  • the drying step is carried out in the atmosphere or under vacuum, preferably at a temperature in the range of -200°C to 200°C.
  • the base film-forming agent is completely fixed to the substrate.
  • the spots can also be formed by drying at room temperature (10° C. to 35° C., preferably 20° C. to 30° C., eg 25° C.), but in order to form the spots more rapidly, for example, at 40° C. to 80° C. It may be dried at °C.
  • drying temperature is less than ⁇ 200° C., an uncommon refrigerant must be used, which lacks versatility, and drying takes a long time due to solvent sublimation, resulting in poor efficiency. If the drying temperature is above 200°C, thermal decomposition of the polymer will occur. A more preferable drying temperature is 10°C to 180°C, and a more preferable drying temperature is 20°C to 150°C.
  • the cell aggregate-producing substrate of the present application is produced through the simple steps described above.
  • a step of washing with at least one solvent selected from aqueous solutions containing water and electrolytes may be carried out. Cleaning is preferably performed with running water, ultrasonic cleaning, or the like.
  • the aqueous solution containing the water and the electrolyte may be heated, for example, in the range of 40.degree. C. to 95.degree.
  • Aqueous solutions containing electrolytes are preferably PBS, physiological saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered physiological saline, HEPES-buffered physiological saline, and Veronal-buffered physiological saline, and PBS is preferred. Especially preferred.
  • the coating film After being fixed, the coating film is not eluted even if it is washed with water, PBS, alcohol, etc., and remains firmly fixed to the substrate.
  • the film thickness of the spot (underlying film) of the present application is such that the maximum film thickness and the minimum film thickness are in the range of 1 to 1000 nm, preferably in the range of 5 to 500 nm.
  • the substrate for producing cell aggregates of the present invention can be produced by coating the base film-forming agent on the surface of the substrate and drying it.
  • the term “surface” refers to a surface that contacts contents such as cells or cell culture medium.
  • the shape of the substrate surface may be flat or uneven, but is preferably flat.
  • substrate materials include glass, metals, metal-containing compounds or metalloid-containing compounds, activated carbon, and resins.
  • Typical metals (aluminum group elements: Al, Ga, In; iron group elements: Fe, Co, Ni; chromium group elements: Cr, Mo, W, U; manganese group elements: Mn, Re; noble metals: Cu , Ag, Au, etc.
  • the metal-containing compound or semi-metal-containing compound is, for example, a metal oxide as a basic component, and is a sintered body sintered by heat treatment at high temperature, such as ceramics, semiconductors such as silicon, metals Inorganic compounds such as oxides or metalloid oxides (silicon oxide, alumina, etc.), metal carbides, metalloid carbides, metal nitrides, metalloid nitrides (silicon nitride, etc.), metal borides, metalloid borides, etc. inorganic solid materials such as molded bodies, aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).
  • the resin may be a natural resin or a derivative thereof, or a synthetic resin.
  • the natural resin or a derivative thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), cellulose with immobilized dextran sulfate, and synthetic resins.
  • Resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA).
  • polyurethane PU
  • EVAL ethylene vinyl alcohol
  • PE polyethylene
  • PET polyester
  • PP polypropylene
  • PVDF polyvinylidene fluoride
  • PES polyethersulfone
  • PC polycarbonate
  • COP cycloolefin polymer
  • PVC polyvinyl chloride
  • PTFE polytetrafluoroethylene
  • UHPE ultra-high molecular weight polyethylene
  • PDMS polydimethylsiloxane
  • ABS acrylonitrile-butadiene-styrene resin
  • Teflon registered trademark
  • the material of the substrate may be one kind or a combination of two or more kinds. It is preferable that the substrate has flexibility so that it can be wound up (roll system) as shown in FIG.
  • Materials for the substrate used in the roll method include synthetic resins and natural polymers.
  • the substrate of the present application may be a substrate used in a so-called cell culture vessel.
  • Petri dishes commonly used for cell culture tissue culture dishes, petri dishes or dishes such as multi-dishes, flasks such as cell culture flasks, spinner flasks, multi-layered flasks, plastic bags, Teflon (registered trademark) bags, culture bags bags, plates such as microplates, microwell plates, multiplates, multiwell plates, chamber slides, tubes, trays, bottles such as roller bottles, and the like.
  • the production method of the present invention includes a step of seeding cardiomyocytes and a step of culturing cardiomyocytes.
  • the step of seeding and culturing cardiomyocytes can be performed by a known method suitable for cardiomyocytes.
  • the steps of seeding and culturing cardiomyocytes can be performed in the presence or absence of biological serum. .
  • the present invention relates to a method for producing human induced pluripotent stem cell (hiPS cell)-derived cardiomyocyte aggregates.
  • the hiPS cell-derived cardiomyocytes used in the present invention means cardiomyocytes that have been induced to differentiate from hiPS cells.
  • hiPS cells available from reagent suppliers or from cell banks are differentiated into cardiomyocytes.
  • Derived, purified and purified products can be used, and such methods are described in various literatures (e.g., Tohyama, et al. Cell Stem Cell 2013, Tohyama, et al. Cell Metabolism 2016, Tohyama, et al. Stem Cell Reports 2017), and hiPS cell-derived cardiomyocytes can be obtained accordingly.
  • GFC Gel Filtration Chromatography
  • a polymer was synthesized by dropwise polymerization in 166.62 g of 2-propanol.
  • the reaction product was reprecipitated with hexane, which is a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure.
  • the weight average molecular weight of this polymer by GFC was 228,000 (hereinafter referred to as "Synthetic Example Polymer 1").
  • a culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface (registered trademark) ) Plate 24F, model number: MS-90240) was coated with 13 spots of a base film-forming agent at about 0.75 nL/spot (spot diameter: 500 ⁇ m, center-to-spot spacing: 600 ⁇ m). It was dried in a constant temperature dryer at 70° C. for one day to prepare a cell aggregate-producing substrate 1 . Sterilization was performed by irradiating 25 kGy of gamma rays.
  • ⁇ Production example 1> (Preparation of Substrate with Inhibitory Cell Adhesion Ability by Inkjet) Using an inkjet device (manufactured by Seiko Epson Corporation, inkjet device for R & D) and an inkjet head (manufactured by Seiko Epson Corporation, Precision Core Head S800-A1), a polystyrene substrate having a size of 79 mm ⁇ 121 mm was prepared. An appropriate amount of the composition for forming a coating film prepared in Example 1 was applied in a perfect circle with a diameter of 37 mm. It was dried in an oven at 70° C. for 24 hours to produce a substrate capable of suppressing cell adhesion.
  • Example 2 (Base film forming step) To 6.50 g of the diluent composition obtained in Preparation Example 2 above, 34.20 g of sterile water and 2.61 g of Recombinant Human Vitronectin (manufactured by Peprotech) diluted to 0.5 mg/mL with sterile water were added and thoroughly mixed. The mixture was stirred to prepare a base film forming agent 2. Using an inkjet device (manufactured by Seiko Epson Corporation, inkjet device for R & D) and an inkjet head (manufactured by Seiko Epson Corporation, Precision Core head S800-A1), the ability to suppress adhesion of the cells prepared in Preparation Example 1 was evaluated.
  • an inkjet device manufactured by Seiko Epson Corporation, inkjet device for R & D
  • an inkjet head manufactured by Seiko Epson Corporation, Precision Core head S800-A1
  • An appropriate amount of the prepared base film forming agent 2 was applied to the culture surface of the substrate so that the spot diameter was 400 ⁇ m and the spot center-to-center interval was 500 ⁇ m. It was dried in a constant temperature dryer at 70°C for 1 day. Affixed to a bottomless 6-well plate (manufactured by CES Tech) to prepare a substrate 2 for producing cell aggregates. Sterilization was performed by irradiating 25 kGy of gamma rays.
  • an appropriate amount of the prepared base film forming agent 2 was applied so that the spot diameter was 400 ⁇ m and the spot center-to-center spacing was 500 ⁇ m. After drying for 5 minutes at room temperature, it was applied again to carry out overcoating. It was dried in a constant temperature dryer at 70°C for 1 day. Affixed to a bottomless 6-well plate (manufactured by CES Tech) to prepare a substrate 3 for producing cell aggregates. Sterilization was performed by irradiating 25 kGy of gamma rays.
  • the cell suspension was slowly added so that 4 ⁇ 10 6 cells were seeded in each well of the substrate 2 for producing cell aggregates, and 3 ⁇ 10 6 cells were seeded in each well of the substrate 3 for producing cell aggregates. It was gently shaken by hand to evenly distribute the mixture. After that, it was allowed to stand in a 37° C./CO 2 incubator while maintaining a carbon dioxide concentration of 5%. After 72 hours, the entire surface of the well was photographed. The results for substrates 2 and 3 are shown in FIGS. 4A and 4B, respectively. After that, 1 mL of AS301 medium was slowly added, and the culture was continued. After 96 hours from seeding, a photograph of the entire surface of the well was taken, and the cells were collected by pipetting. was used to measure cell diameter.
  • FIGS. 5A and B The results for substrates 2 and 3 are shown in FIGS. 5A and B, respectively.
  • 72 hours after seeding some of the cardiomyocyte clusters were detached from the bottom of the plate and were floating, but uniform cardiomyocyte clusters were observed to be formed in all wells. Cardiomyocyte spheres with a diameter of about 150 ⁇ m were formed in all the cells collected 96 hours after seeding.
  • Example 3> (Maturity of cells contained in cardiomyocyte spheres)
  • the cardiomyocyte stock frozen in the same manner as above was thawed in a water bath set at 37°C, suspended in clinical serum-free medium AS301 (manufactured by Ajinomoto Co., Inc.), and used for the production of cell aggregates obtained in the above step.
  • the cell suspension was slowly added to each well of Substrates 2 and 3 so that 4 ⁇ 10 6 cells were seeded, and the cells were gently shaken by hand to evenly spread the cells. After that, the cells were collected by pipetting after standing in a 37° C./CO 2 incubator for 48 hours and 72 hours while maintaining a carbon dioxide concentration of 5%.
  • FIG. 6A shows the appearance of the spheres after 48 hours and 72 hours from the cell aggregate-producing substrate 2
  • FIG. 6B shows the appearance of the spheres from the Elplasia plate after 48 hours and 7 days.
  • the ratio of TNNI3/TNNI1 was almost the same between the sample cultured for 48 hours and the sample cultured on the Elplasia plate for 7 days. It was shown that the degree of maturity was similar to that of myocardial spheres prepared over 7 days on Elplasia plates.
  • Example 4 (Base film forming step) To 1.00 g of the composition for dilution obtained in Preparation Example 2 above, 5.59 g of sterilized water and 0.10 g of iMatrix-221 (manufactured by Matrixome Co., Ltd.) were added and thoroughly stirred. prepared.
  • an inkjet device manufactured by Microjet Co., Ltd., model number: LaboJet-600
  • an inkjet head model number: IJHBS-1000
  • An appropriate amount of the base film forming agent 2 prepared above was applied so that the spot diameter was 400 ⁇ m and the center-to-center interval of the spots was 500 ⁇ m. It was dried in a constant temperature dryer at 70°C for 1 day.
  • a base film-forming agent 3 was applied in the same manner to prepare a substrate 5 for producing cell aggregates. Sterilization was performed by irradiating 25 kGy of gamma rays.
  • FIGS. 8A to 8D The results for each cell seeding amount for Substrates 4 and 5 are shown in FIGS. 8A to 8D, respectively.
  • FIG. 7 72 hours after seeding, some cells were separated from the bottom of the plate and were floating, but uniform cardiomyocyte clusters were observed to be formed in all wells. Cardiomyocyte spheres with a diameter of about 120 to 160 ⁇ m were formed in all the cells collected 96 hours after seeding (FIG. 8).
  • the method for producing hiPS cell-derived cardiomyocyte aggregates of the present invention uniform adhesion of cardiomyocytes can be achieved even under animal-derived serum-free culture conditions, and cell aggregates can be produced subsequently. As a result, mass production of homogeneous and high-quality cardiomyocyte aggregates for use in the field of regenerative medicine can be achieved.

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WO2017131241A1 (ja) * 2016-01-29 2017-08-03 国立研究開発法人国立循環器病研究センター 細胞塊、細胞構造体及び立体組織体

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JPH05292957A (ja) * 1992-04-21 1993-11-09 Kurabo Ind Ltd 接着性細胞培養用被覆組成物
JP2014162865A (ja) * 2013-02-26 2014-09-08 National Cerebral & Cardiovascular Center 温度応答性ポリマーの製造方法、温度応答性ポリマー、細胞培養器の製造方法、及び細胞培養器
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JP2017055744A (ja) * 2015-09-18 2017-03-23 国立研究開発法人国立循環器病研究センター カプセル化細胞構造体の製造方法、及びカプセル化細胞構造体
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