WO2023100974A1 - Procédé de culture cellulaire utilisant un milieu de culture exempt de sérum - Google Patents

Procédé de culture cellulaire utilisant un milieu de culture exempt de sérum Download PDF

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WO2023100974A1
WO2023100974A1 PCT/JP2022/044352 JP2022044352W WO2023100974A1 WO 2023100974 A1 WO2023100974 A1 WO 2023100974A1 JP 2022044352 W JP2022044352 W JP 2022044352W WO 2023100974 A1 WO2023100974 A1 WO 2023100974A1
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cells
crosslinked
vinyl alcohol
serum
group
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Japanese (ja)
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聡 山崎
明士 藤田
悟朗 小林
賢 綾野
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株式会社クラレ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • 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

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  • a basal medium mixed with known components such as amino acids, carbohydrates, lipids, vitamins, nucleic acids or their precursors, salts, and trace metals (e.g., Dulbecco's Modified Eagle Medium (DMEM), Ham's F12 medium, etc.) (commercially available as FBS)
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • animal cells cannot be cultured (survival/proliferation) unless animal serum is added to the basal medium.
  • animal sera such as fetal bovine serum (FBS) are (1) expensive and limited in supply, and (2) have large variations in quality between lots. (3) It is difficult to isolate a target physiologically active substance from a medium containing animal serum containing miscellaneous proteins.
  • FBS fetal bovine serum
  • infectious disease risk associated with prion-related diseases such as mad cow disease, bovine spongiform encephalopathy, infectious spongiform brain, and even Creutzfeldt-Jakob disease.
  • PVA polyvinyl alcohol
  • a low-protein medium containing insulin and transferrin containing insulin and transferrin
  • cells can be cultured without using proteins such as animal serum, purified albumin, or recombinant albumin. shown that it can be done.
  • a PVA-added low-protein medium has been disclosed in which induced pluripotent stem cells (hereinafter sometimes abbreviated as iPS cells) can be adhered and cultured on a cell culture dish coated with laminin (Patent Document 3).
  • iPS cells induced pluripotent stem cells
  • iPS cells induced pluripotent stem cells
  • a technique has recently been disclosed in which human hematopoietic stem cells can be cultured for several months while maintaining their undifferentiated state using a low-protein medium supplemented with PVA (Patent Document 4, Non-Patent Document 2).
  • JP-A-7-23780 JP 2007-228815 A Japanese Patent Application Laid-Open No. 2020-89338 WO2021-049617
  • iPS cells can be cultured by adhering to a cell culture dish by using a low-protein medium to which PVA is added, as described in Patent Document 3.
  • the low-protein medium contains proteins. It contains insulin and transferrin, and has problems such as unstable quality as described above, difficulty in refining the desired physiologically active substance, and high cost.
  • Patent Document 4 and Non-Patent Document 2 also have similar problems. In order to solve these problems, a completely protein-free medium, in which all proteins have been removed from the low-protein medium, is used. Especially adhesion-dependent cells cannot even adhere to the cell culture dish, making it difficult to culture. Met.
  • the present invention has been made in view of the above conventional problems, and aims to provide a method that enables adhesion-dependent cells to be cultured in a serum-free medium.
  • the present invention relates to the following [1] to [18].
  • [1] A method for culturing cells, which comprises the step of adhering cells to a crosslinked body of a water-soluble polymer conjugated with a cell adhesion factor using a serum-free medium and culturing the cells.
  • [2] A method for producing a target substance, comprising the step of culturing cells by the method of [1].
  • [3] The method of [1] or [2], wherein the serum-free medium is a protein-free medium.
  • [4] The method according to any one of [1] to [3], wherein the cell adhesion factor is covalently conjugated to the crosslinked water-soluble polymer.
  • the ethylenically unsaturated group is at least one selected from the group consisting of a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, and a norbornenyl group, [7] Or the method according to [8]. [10] Any one of [7] to [9], wherein the introduction rate of the ethylenically unsaturated group is 0.1 to 10 mol% in all structural units constituting the vinyl alcohol polymer. The method described in .
  • Method. [12]
  • the crosslinked body of the water-soluble polymer is an amorphous particle, a spherical particle, a fine molded body, an article of arbitrary shape formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article. 1] The method according to any one of [11].
  • [13] A method for culturing cells using the serum-free medium according to any one of [1] to [12], wherein a physiologically active substance acts on cells cultured using the serum-free medium. .
  • [14] A method for culturing cells using the serum-free medium according to any one of [1] to [13], wherein the cells cultured using the serum-free medium are transfected with a gene vector, A method for producing a bioactive substance.
  • [15] A complex of a cell adhesion factor and a crosslinked water-soluble polymer, wherein the cell adhesion factor and the crosslinked water-soluble polymer are conjugated by non-covalent bonding.
  • [16] A complex of gelatin or collagen and a crosslinked water-soluble polymer for use in cell culture.
  • cells can be cultured (survival/proliferation) in a serum-free medium, preferably a protein-free medium. Therefore, it is natural that there is no risk of infectious diseases derived from the medium, but it is extremely inexpensive, and the quality of the cells is stable with no differences between protein lots, and it is easy to purify useful physiologically active substances produced by the cells. can be provided in high purity.
  • the medium does not contain proteins, it is possible to provide a highly reproducible method for maintaining undifferentiated stem cells and differentiating stem cells, which has not been possible until now due to the influence of contaminants such as proteins contained in the medium. .
  • FIG. 2 shows a photomicrograph of GFP-expressing 293T cells in Example 5.
  • a serum-free medium is a medium containing no animal-derived serum. Furthermore, it is preferred that the serum-free medium of the present invention is a protein-free medium. Conventionally, it has been difficult to culture and grow cells in serum-free media to which no animal-derived serum has been added, but the method of the present invention allows cells to be cultured and grown without using these media. is fundamentally different from the prior art.
  • the term "serum” can be interpreted in the meaning commonly used in the field of biochemistry to which the present invention belongs, but typically it refers to blood, blood cells and several blood coagulation factors (fibrinogen (factor I)). ), prothrombin (factor II), factor V, factor VIII, etc.).
  • the serum-free medium of the present invention does not contain proteins such as ethanolamine, 2-mercaptoethanol, sodium selenite, etc., which are known as serum substitute components other than proteins (insulin, growth factors, transferrin, albumin, etc.). Factors may be added.
  • a basal medium that can be prepared by mixing nutritional components such as amino acids, carbohydrates, lipids, vitamins, nucleic acids or their precursors, salts, and trace metals can be used. used with the addition of antibiotics.
  • the methods of the invention employ liquid media.
  • ⁇ Basic medium examples include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM/F12 medium, and McCoy's 5A medium (McCoy's 5A medium). ), Eagle MEM medium (Eagles's Minimum Essential Medium; EMEM), ⁇ MEM medium (alpha Modified Eagles's Minimum Essential Medium); ⁇ MEM), MEM medium (Minimum Essential Medium), RPM11 640 medium, Iscove's modified Dulbecco's medium (Iscove' s Modified Dubecco's Medium (IMDM)), etc.; a medium or the like in which two or more of these are mixed can be used. Of course, other media suitable for the cells to be cultured can also be used.
  • antibiotics added to the serum-free medium of the present invention include actinomycin D, amphotericin B, ampicillin, antimycin A, bafilomycin A1, bleomycin, carbenicillin, chloramphenicol, concanamycin B, erythromycin, Gentamicin, hygromycin, kanamycin, mitomycin C, neomycin, oligomycin, penicillin, puromycin, rapamycin, streptomycin, tetracycline, tobramycin, valinomycin, etc., and appropriate antibiotics can be used depending on the purpose.
  • the water-soluble polymer crosslinked body in the present invention refers to a so-called water-containing gel that forms a three-dimensional network structure by cross-linking the water-soluble polymer and swells by absorbing an aqueous solvent inside.
  • Examples of cross-linking forms include physical cross-linking such as hydrogen bond, ionic bond, coordinate bond, and hydrophobic bond; chemical cross-linking formed by covalent bonding by chemical reaction; and the like. Both synthetic polymers and natural polymers can be used as water-soluble polymers.
  • Examples of synthetic polymers include vinyl alcohol polymers, (meth)acrylic acid polymers, (meth)acrylamide polymers, N-isopropyl (meth)acrylamide polymers, vinylpyrrolidone polymers, hydroxyethyl (Meth)acrylamide-based polymers, hydroxyethyl (meth)acrylate-based polymers, ethylene oxide-based polymers, propylene oxide-based polymers, polyethyleneimine, polyallylamine, derivatives thereof, and the like.
  • Water-soluble polysaccharides can be used as natural polymers.
  • water-soluble polysaccharides include water-soluble cellulose derivatives such as alginic acid, propylene glycol alginate, agarose, hydroxyethyl cellulose, carboxymethyl cellulose; guar gum, carrageenan, agar, chitosan, gellan gum, dextran, starch, hyaluronic acid, pullulan, heparin. etc.
  • the exemplified water-soluble polymers can be used in the present invention.
  • vinyl alcohol-based polymers, (meth)acrylamide-based polymers, hydroxyethyl (meth)acrylate-based polymers, ethylene oxide-based polymers, water-soluble cellulose derivatives, and dextran are more preferable.
  • vinyl alcohol polymers, water-soluble cellulose derivatives, and dextran are more preferred, and vinyl alcohol polymers are particularly preferred.
  • the vinyl alcohol polymer used in the present invention can be produced by saponifying a polyvinyl ester obtained by polymerizing a vinyl ester monomer and converting the ester group in the polyvinyl ester into a hydroxyl group.
  • the vinyl ester monomer include vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, and vinyl caprate. , vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate.
  • the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.
  • the polyvinyl ester may contain structural units derived from monomers other than vinyl ester-based monomers, if necessary, as long as the effects of the present invention are not impaired.
  • monomers include ⁇ -olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or salts thereof; methyl acrylate, ethyl acrylate, n-propyl acrylate, i-acrylate, Acrylate alkyl esters such as propyl, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate and octadecyl acrylate; methacrylic acid or its salts; methyl methacrylate , ethyl methacrylate, n-propyl methacrylate, i-propyl methacrylate, n-buty
  • Methacrylic acid alkyl esters acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetoneacrylamide, acrylamidopropanesulfonic acid or its salts, acrylamidopropyldimethylamine or its salts or quaternary salts, N - acrylamide derivatives such as methylolacrylamide or derivatives thereof; methacrylamide derivatives such as methylol methacrylamide or derivatives thereof; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i Vinyl ethers such as -butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether and stearyl vinyl ether; Nitriles such as acrylonitrile and
  • the content of the structural unit derived from the other monomer is 20 mol% or less with respect to the total structural units constituting the polyvinyl ester. preferably 10 mol % or less, and even more preferably 5 mol % or less.
  • the method of saponifying the polyvinyl ester it can be carried out in the same manner as before.
  • an alcoholysis method using an alkali catalyst or an acid catalyst, a hydrolysis method, or the like can be applied.
  • a saponification reaction using methanol as a solvent and a caustic soda (NaOH) catalyst is simple and preferable.
  • the range of the average degree of polymerization of the vinyl alcohol polymer is not limited, but is, for example, 300 to 10,000, more preferably 450 to 5,000, still more preferably 500 to 3,000, Most preferably, it is between 500 and 2,500. Two or more kinds of vinyl alcohol polymers having different average degrees of polymerization may be mixed and used. From the viewpoint of suppressing embrittlement of the crosslinked product of the present invention, the average degree of polymerization is preferably 300 or more. In addition, from the viewpoint of keeping the viscosity of the aqueous vinyl alcohol polymer solution within a range that is easy to handle, the average degree of polymerization is preferably 10,000 or less, more preferably 5,000 or less, and still more preferably 3,000 or less. .
  • the average degree of polymerization of the vinyl alcohol polymer in this specification refers to the average degree of polymerization measured according to JIS K 6726:1994. Specifically, since the degree of polymerization of the vinyl alcohol polymer and the raw material PVA described later can be considered to be the same, the intrinsic viscosity can be determined from the intrinsic viscosity measured in water at 30° C. after purifying the raw material PVA.
  • the degree of saponification of the vinyl alcohol polymer is preferably 50 mol% or more, more preferably 60 mol% or more, still more preferably 65 mol% or more.
  • the upper limit of saponification degree is 100 mol % or 99 mol %.
  • the degree of saponification of a vinyl alcohol-based polymer refers to the total number of moles of structural units (for example, vinyl acetate units) that can be converted to vinyl alcohol units by saponification in raw material PVA and vinyl alcohol units. It means the ratio (mol%) of the number of moles of alcohol units, and can be measured according to JIS K 6726:1994.
  • the 4% by mass viscosity at 20° C. of the vinyl alcohol polymer is preferably 0.5 to 110 mPa ⁇ s, more preferably 1 to 80 mPa ⁇ s, and even more preferably 2 to 60 mPa ⁇ s.
  • the viscosity in this specification refers to an aqueous solution containing 4% by mass of a vinyl alcohol polymer, according to the rotational viscometer method of JIS K 6726: 1994, using a B-type viscometer (rotation speed 12 rpm) at a temperature of 20 ° C. refers to the viscosity measured by
  • the crosslinked vinyl alcohol polymer may be a physically crosslinked vinyl alcohol polymer or a covalently crosslinked vinyl alcohol polymer.
  • the total amount of structural units derived from vinyl alcohol and structural units derived from vinyl ester with respect to all structural units constituting the vinyl alcohol polymer used in the present invention is preferably 80 mol% or more, more preferably 90 mol% or more, and further Preferably, it is 95 mol % or more.
  • a method of physically cross-linking the vinyl alcohol polymer for example, a method of freezing and melting an aqueous solution of the vinyl alcohol polymer, dissolving in a mixed solvent of dimethyl sulfoxide and water, and cooling this hot solution to room temperature.
  • saturated or unsaturated fatty acids such as hexanoic acid and octanoic acid are introduced into hydroxyl groups derived from vinyl alcohol by esterification, or hydrophobic aldehydes such as butyraldehyde and benzaldehyde are introduced into 1,3- When introduced into the diol structure by acetalization, physical cross-linking by hydrophobic bonding is also possible.
  • a method for cross-linking the vinyl alcohol polymer by covalent bonding a method using a polyfunctional cross-linking agent that reacts with the side chain hydroxyl groups of the vinyl alcohol polymer or copolymerization, post-modification, etc. There is a method of introducing a functional group into and reacting these. However, since the stability of the cross-linked product when swollen with water increases, it is preferable that the cross-linked product is cross-linked by a covalent bond.
  • a method of using a polyfunctional cross-linking agent that reacts with the side chain hydroxyl groups of the vinyl alcohol polymer a method of reacting polyfunctional aldehyde compounds such as glyoxal, malondialdehyde, and glutaraldehyde under acidic conditions (polyacetal cross-linking).
  • polyfunctional aldehyde compounds such as glyoxal, malondialdehyde, and glutaraldehyde under acidic conditions
  • polyether cross-linking a method of reacting polyfunctional epoxy compounds such as epichlorohydrin and ethylene glycol diglycidyl ether under alkaline conditions
  • polyether cross-linking a method of reacting polyfunctional carboxylic acid compounds such as maleic acid and succinic acid
  • the rate of introduction of the cross-linking compound is 0.0 to the repeating unit of the vinyl alcohol polymer into which the cross-linking compound is introduced. 05 to 5 mol % is preferred, and 0.1 to 2.5 mol % is more preferred.
  • ⁇ Modified PVA> As a method of introducing functional groups into a vinyl alcohol polymer by copolymerization and reacting them, vinyl ester monomers and other A polymerizable monomer is copolymerized with a monomer having a reactive substituent other than a hydroxyl group, and then saponified to obtain copolymerized modified polyvinyl alcohol (hereinafter abbreviated as "copolymerized modified PVA" There is a method of adding a polyfunctional cross-linking agent that reacts with functional groups such as carboxy groups present in the copolymerized modified PVA and amino groups present in the copolymerized modified PVA.
  • Copolymer-modified PVA having a carboxy group is sometimes referred to as "carboxylic acid-modified PVA”
  • copolymer-modified PVA having an amino group is sometimes referred to as "amino-modified PVA”.
  • Examples of monomers constituting carboxylic acid-modified PVA include ⁇ , ⁇ -unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid and itaconic acid; methyl (meth)acrylate and (meth)acrylic acid; Examples include (meth)acrylic acid alkyl esters such as ethyl; ⁇ , ⁇ -unsaturated carboxylic anhydrides such as maleic anhydride and itaconic anhydride, and derivatives thereof.
  • a polyfunctional cross-linking agent that reacts with the introduced carboxyl group to produce a carboxylic acid-modified PVA by copolymerizing a vinyl ester-based monomer and a monomer that constitutes the carboxylic acid-modified PVA, followed by saponification.
  • polyfunctional epoxy compounds such as epichlorohydrin and ethylene glycol diglycidyl ether
  • polyfunctional amino compounds such as ethylenediamine, polyethyleneimine and polyallylamine and carbodiimide condensation such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • a crosslinked product can be obtained by a combination of agents or the like.
  • the carboxyl group introduced into the carboxylic acid-modified PVA can be conjugated by covalently bonding a cell adhesion factor having an amino group with an amide bond (--CONH--).
  • amino-modified PVA is obtained by copolymerizing a vinyl ester monomer and N-vinylformamide or the like, followed by saponification, and a polyfunctional cross-linking agent that reacts with the introduced amino group, such as the polyfunctional epoxy compound
  • a crosslinked product can be obtained by, for example, combining a polyfunctional carboxylic acid compound such as succinic acid or maleic acid with the carbodiimide condensing agent.
  • ⁇ Ethylenically unsaturated group> As a method of introducing a functional group into a vinyl alcohol polymer by post-modification and reacting them, a method of introducing an ethylenically unsaturated group into a side chain of the vinyl alcohol polymer can be exemplified.
  • the introduced ethylenically unsaturated group can be easily obtained as a crosslinked product by inducing a polymerization reaction by adding an additive such as a radical initiator.
  • the ethylenically unsaturated group is preferably formed via the side chain, terminal functional group, etc. of the vinyl alcohol polymer. (which may be abbreviated as an "ethylenically unsaturated group-containing compound”) is more preferably reacted.
  • ethylenically unsaturated groups include vinyl groups, (meth)acryloyl groups, (meth)acryloylamino groups, vinylphenyl groups, norbornenyl groups, and the like.
  • the ethylenically unsaturated groups may be derivatives of these groups.
  • "a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group” means a crosslinked "vinyl alcohol polymer having an ethylenically unsaturated group". In such embodiments, at least a portion of the ethylenically unsaturated groups are consumed by the cross-linking reaction of the vinyl alcohol-based polymer.
  • crosslinked vinyl alcohol polymer having an ethylenically unsaturated group includes the result of consumption of the ethylenically unsaturated groups of the vinyl alcohol polymer before cross-linking and before complexing by the cross-linking reaction.
  • the crosslinked product may include those having no ethylenically unsaturated groups and those having residual ethylenically unsaturated groups.
  • Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group that is the side chain of the vinyl alcohol polymer include (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, ( Examples thereof include (meth)acrylic acid such as meth)acrylic acid esters and derivatives thereof, and a (meth)acryloyl group can be introduced by subjecting these compounds to an esterification reaction or a transesterification reaction in the presence of a base.
  • Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group, which is the side chain of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and a glycidyl group in the molecule.
  • (meth)acryloyl groups and/or allyl groups can be introduced into the vinyl alcohol polymer.
  • the ethylenically unsaturated group-containing compound to be reacted with the 1,3-diol group of the vinyl alcohol polymer includes, for example, acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2- Compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule such as carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde are included.
  • an ethylenically unsaturated group can be introduced into the starting material PVA.
  • 5-norbornene-2-carboxaldehyde, 3-vinylbenzaldehyde, 4-vinylbenzaldehyde, etc. are acetalized to introduce norbornenyl groups and/or vinylphenyl groups into raw material PVA. can be done.
  • a (meth)acryloylamino group can be introduced into the vinyl alcohol polymer by reacting with N-(2,2-dimethoxyethyl)(meth)acrylamide or the like.
  • a method for introducing an ethylenically unsaturated group into a vinyl alcohol polymer can be used in addition to the above-described reactions, and two or more reactions may be used in combination.
  • a method for introducing the ethylenically unsaturated group there is another method of reacting a reactive substituent such as a carboxy group present in the carboxylic acid-modified PVA or an amino group present in the amino-modified PVA with an ethylenically unsaturated group-containing compound.
  • a reactive substituent such as a carboxy group present in the carboxylic acid-modified PVA or an amino group present in the amino-modified PVA
  • an ethylenically unsaturated group-containing compound There is a method to make The carboxy group of the carboxylic acid-modified PVA can be reacted with, for example, glycidyl methacrylate under acidic conditions to form an ester bond and introduce a methacryloyl group.
  • An acryloylamino group can be introduced into the amino group of the amino-modified PVA by amidating acrylic anhydride in the presence of a base, for example, a vinyloxycarbonyl group can be introduced by amidating divinyl adipate.
  • Methods for introducing ethylenically unsaturated groups via copolymerization-modified PVA can also be used in addition to the above-described reactions, and two or more reactions may be used in combination.
  • vinyl alcohol polymer having an ethylenically unsaturated group vinyl alcohol in which an ethylenically unsaturated group is introduced via the hydroxyl group of the side chain of the raw material PVA such as a 1,3-diol group, from the viewpoint of ease of production.
  • -based polymers are preferred, vinyl alcohol-based polymers obtained by subjecting (meth)acrylic acid or derivatives thereof to an esterification reaction or a transesterification reaction with hydroxyl groups on the side chains of raw material PVA, and 1,3- of vinyl alcohol-based polymers.
  • a vinyl alcohol polymer or the like obtained by acetalizing a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule with respect to a diol group is more preferable.
  • the introduction rate of the ethylenically unsaturated groups contained in the vinyl alcohol polymer before cross-linking should It is preferably 10 mol % or less, more preferably 5 mol % or less, still more preferably 3 mol % or less in the structural unit. From the viewpoint of promoting the cross-linking reaction, rapidly forming a cross-linked product, and improving the elastic modulus of the obtained cross-linked product, it is preferably 0.1 mol % or more, more preferably 0.2 mol % or more, More preferably, it is 0.3 mol % or more.
  • the “rate of introduction of ethylenically unsaturated groups” in the “crosslinked product of a vinyl alcohol polymer having an ethylenically unsaturated group” means that the vinyl alcohol polymer constituting the crosslinked product is It also indicates the ratio (mol %) of the ethylenically unsaturated groups in all the structural units before the compounding.
  • the vinyl alcohol polymer having an ethylenically unsaturated group further contains a monomer.
  • the monomer include acrylamides such as acrylamide, N-isopropylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid and N,N-dimethylacrylamide; (meth)acrylic acid, crotonic acid, itaconic acid, maleic acid, ⁇ , ⁇ -unsaturated carboxylic acids such as fumaric acid; water-soluble radically polymerizable monomers such as vinylpyridine, hydroxyethyl (meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; N,N'- Examples include cross-linking agents having two or more ethylenically unsaturated groups in the molecule, such as methylenebisacrylamide,
  • the content of the monomer is preferably 50% by mass or less, more preferably 30% by mass or less, relative to the vinyl alcohol polymer having an ethylenically unsaturated group. , 10% by mass or less is more preferable.
  • a vinyl alcohol polymer having an ethylenically unsaturated group can be gelled by cross-linking the ethylenically unsaturated groups introduced into the vinyl alcohol polymer by actinic energy rays and/or heat.
  • a crosslinked product of the vinyl alcohol polymer of the present invention can be obtained by the above.
  • active energy rays include gamma rays, ultraviolet rays, visible rays, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron beams, plasma currents, ionizing rays, and particle beams.
  • the uncrosslinked polymer solution described later may contain a radical polymerization initiator.
  • Radical polymerization initiators include radical photopolymerization initiators and thermal radical polymerization initiators.
  • the photoradical polymerization initiator is not particularly limited as long as it initiates radical polymerization by irradiation with active energy rays such as ultraviolet rays and visible light.
  • active energy rays such as ultraviolet rays and visible light.
  • those exhibiting water solubility are preferable.
  • 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (trade name "Omnirad2959", manufactured by IGM Resins B.V.
  • the thermal radical polymerization initiator is not particularly limited as long as it initiates radical polymerization by heat, and examples thereof include azo initiators and peroxide initiators commonly used in radical polymerization. From the viewpoint of improving the transparency and physical properties of the vinyl alcohol polymer, a peroxide initiator that does not generate gas is preferred. From the viewpoint of removing the thermal radical polymerization initiator by washing after crosslinking as described above, those exhibiting water solubility are preferable. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate and sodium persulfate.
  • a redox polymerization initiator combined with a reducing agent may also be used. If it is a redox polymerization initiator, it can be crosslinked by stimulus of mixing of a peroxide initiator and a reducing agent.
  • a reducing agent to be combined as a redox polymerization initiator known reducing agents can be used. Sodium hydrosulfite and the like are preferred.
  • azo initiator those exhibiting water solubility are also preferable.
  • -044 2,2′-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate (trade name “VA-044B”), 2,2′-azobis[2 -methylpropionamidine] dihydrochloride (trade name “V-50”), 2,2′-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] tetrahydrate (trade name “VA- 057”), 2,2′-azobis[2-(2-imidazolin-2-yl)propane] (trade name “VA-061”), 2,2′-azobis[2-methyl-N-(2- Hydroxyethyl)propionamide] (trade name “VA-086”), 4,4′-azobis(4-cyanopentanoic acid) (trade name “V-501”) (manufactured by Wako Pure Chemical Industries, Ltd.) etc.
  • a polythiol having two or more thiol groups in the molecule is added.
  • polythiol those exhibiting water solubility are preferable.
  • polythiol having a hydroxyl group such as dithiothreitol
  • terminal thiols such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol.
  • polythiols containing ether bonds such as
  • the most suitable method for cross-linking a vinyl alcohol polymer by covalent bonding can be selected according to need. Therefore, the method of introducing functional groups into the vinyl alcohol polymer by post-modification and reacting them, particularly the method of introducing the ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer, is preferred.
  • Forms of molded products of the crosslinked water-soluble polymer of the present invention include general amorphous particles, spherical particles, films, threads, hollow fibers, porous monoliths, and the like.
  • the form of the molded article may be a micromolded article, an article of arbitrary shape molded by a 3D printer, or an article coated with a crosslinked body of a water-soluble polymer (coated article).
  • the micromolded body is a molded body having fine irregularities on the surface and/or inside thereof, and the size of the microfabrication is 10 to 1000 ⁇ m.
  • the arbitrary shape formed by a 3D printer is an arbitrary shape that can be formed by, for example, a stereolithography method, an inkjet method, a nozzle extrusion type 3D printer, or the like.
  • a coated article is, for example, a film, a tray, a petri dish, a well plate, a thread, a hollow fiber, a porous monolith, a micromolded body, a crosslinked body composited on a substrate of an arbitrary shape molded by a 3D printer, etc.
  • the substrate material can be freely selected from glass, polyolefin, polymethyl methacrylate, polystyrene, polyester, polyolefin, polyethylene vinyl alcohol copolymer, polyamide, polyimide, and the like.
  • the cell adhesion factor is not particularly limited as long as it is a molecule having the property of cell adhesion.
  • Examples include gelatin, collagen, laminin, fibronectin, vitronectin, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, Cell adhesion proteins such as fibrin, elastin, netrin, entactin, proteoglycan, retronectin; synthetic peptides containing cell adhesion sequences such as RGD, REDV, YIGSR, IKVAV peptides; heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan Acidic polysaccharides containing glycosaminoglycans such as sulfuric acid; polycations such as ⁇ -polylysine, ⁇ -polylysine, ⁇ -polyargin
  • the cell adhesion protein may be naturally derived, recombinant, or synthetic peptide chemically synthesized by a solid-phase method or the like.
  • the molecular weight of the cell adhesion protein is not particularly limited. Those in the range of 10,000 to 800,000 are more preferable.
  • the molecular weight of the synthetic peptide is not particularly limited, but preferably has a weight average molecular weight of 200 to 10,000, more preferably 250 to 7,000.
  • the molecular weight of the growth factor is not particularly limited. is more preferred.
  • the molecular weight of the acidic polysaccharide is not particularly limited. more preferred.
  • the molecular weight of the polycations is not particularly limited, but those having a weight average molecular weight of 2000 to 1,000,000 are preferred, and those having a weight average molecular weight of 4000 to 500,000 are preferred. more preferred.
  • the cell adhesion factor preferably has a functional group that can bind to the crosslinked water-soluble polymer.
  • Such functional groups include primary or secondary amino groups, carboxy groups, hydroxyl groups and the like, preferably primary amino groups or carboxy groups, more preferably primary amino groups.
  • the cell adhesion factor may be conjugated on the surface, inside, or both of the crosslinked water-soluble polymer.
  • the network structure of the crosslinked water-soluble polymer is dense, so the crosslinked water-soluble polymer usually cannot penetrate to the inside, and the surface are often compounded.
  • This structure can vary depending on the crosslink density of the crosslinked water-soluble polymer and the molecular weight of the cell adhesion factor. It is also possible to combine cell adhesion factors inside and on the surface by mixing a water-soluble polymer with a cell adhesion factor in advance and cross-linking it, and an optimum method can be selected depending on the purpose.
  • the present invention provides a complex of a cell adhesion factor and a crosslinked water-soluble polymer.
  • examples of the water-soluble polymer include vinyl alcohol-based polymers.
  • the present invention provides a composite of a crosslinked vinyl alcohol polymer and a cell adhesion factor.
  • the crosslinked body of the cell adhesion factor and the water-soluble polymer may be conjugated by covalent bonding or by non-covalent bonding (for example, by intermolecular forces such as hydrogen bonding and van der Waals force). may have changed.
  • the crosslinked vinyl alcohol polymer of the present invention may simply include the cell adhesion factor, or the crosslinked vinyl alcohol polymer and the cell adhesion factor may be It may be a covalently bonded composite, but is preferably a covalently bonded vinyl alcohol-based polymer crosslinked product.
  • the cell adhesion factor can be held in the crosslinked vinyl alcohol polymer and function stably.
  • a covalent bond is formed by activating the hydroxyl group of PVA and reacting it with the functional group of the cell adhesion factor; It is possible to select, for example, forming a covalent bond by reacting with the functional group of the cell adhesion factor using acid-modified PVA and/or amino-modified PVA.
  • the functional group of the vinyl alcohol polymer is preferably at least one selected from the group consisting of an amino group, a carboxylic acid group and a thiol group, and more preferably a carboxylic acid group.
  • Specific methods for activating hydroxyl groups of PVA to introduce cell adhesion factors include, for example, 1,1′-carbonyldiimidazole, di(N-succimidyl) carbonate, p-toluenesulfonyl chloride, 2,2,2 - A method using a hydroxyl group-activating reagent such as trifluoroethanesulfonyl chloride and cyanuric chloride.
  • a hydroxyl group-activating reagent such as trifluoroethanesulfonyl chloride and cyanuric chloride.
  • Acid anhydride reagents such as succinic anhydride capable of introducing carboxylic acid by ester bonding to the hydroxyl group of the vinyl alcohol polymer; Acetal reagents such as 2,2-dimethoxyethylamine that can be introduced can also be used. These carboxylic acid and amino groups can form an amide bond using the amino group of the cell adhesion factor, carboxylic acid and the carbodiimide condensing agent.
  • an acid anhydride reagent such as succinic anhydride is reacted with a crosslinked vinyl alcohol polymer, a carboxy group (COOH) derived from succinic acid is introduced at the terminal, and this carboxy group and the amino group of the cell adhesion factor are combined by 1.
  • the cell adhesion factor can be conjugated to the crosslinked vinyl alcohol polymer via an amide bond.
  • an amide bond is formed using an amino group or carboxylic acid of the cell adhesion factor and the carbodiimide condensing agent. can be formed.
  • the vinyl alcohol polymer When the crosslinked vinyl alcohol polymer and the cell adhesion factor are covalently bonded, the vinyl alcohol polymer preferably has a carboxy group.
  • the introduction rate of the carboxyl group contained in the vinyl alcohol polymer before being composited is such that the degree of swelling of the crosslinked product of the vinyl alcohol polymer does not become too large and the gel strength is kept high. It is preferably 50 mol % or less, more preferably 30 mol % or less, still more preferably 15 mol % or less, of all structural units constituting the polymer.
  • the cell adhesion factor is preferably 0.1 mol % or more, more preferably 0.5 mol % or more, and still more preferably It is 1.0 mol % or more.
  • the crosslinked body of the water-soluble polymer to which the cell adhesion factor is conjugated is a crosslinked body of a vinyl alcohol polymer having a carboxy group
  • the "vinyl alcohol polymer having a carboxy group "crosslinked product” means a crosslinked product of "a vinyl alcohol polymer having a carboxy group”.
  • at least part of the carboxy groups may be consumed by the conjugation reaction between the crosslinked product and the cell adhesion factor.
  • crosslinked vinyl alcohol polymer having a carboxy group includes a part of the carboxy groups of the vinyl alcohol polymer before cross-linking and before complexing is consumed by the complexing reaction, but cross-linked It can also include those in which a part of the carboxy group remains in the body and complex. Therefore, in the present invention, the "ratio of carboxy group introduction" in the "crosslinked product of a vinyl alcohol polymer having a carboxy group” means that the vinyl alcohol polymer constituting the crosslinked product has It shows the ratio (mol%) of carboxy groups in all structural units at the time of .
  • the composite may be formed after the step of crosslinking the uncrosslinked polymer solution as described below, or may be composited at the stage of the uncrosslinked polymer solution. Conjugation of the cell adhesion factor is preferably carried out after the cross-linking step in order to avoid the reduction or loss of activity of the cell adhesion factor due to cross-linking.
  • the method for producing the crosslinked vinyl alcohol polymer of the present invention is not particularly limited, but first, a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol polymer (uncrosslinked polymer solution preparation step), Thereafter, a step of molding the uncrosslinked polymer solution (molding step), and then a step of crosslinking and gelling the vinyl alcohol polymer contained in the uncrosslinked polymer solution (crosslinking step). preferably.
  • a specific method will be described below.
  • the uncrosslinked polymer solution preparation step in the present invention is a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol polymer, and can be obtained by dissolving the vinyl alcohol polymer in a solvent.
  • the solvent is preferably water, and may further contain a water-soluble organic solvent.
  • water-soluble organic solvents include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol and isopropanol; ethylene glycol, diethylene glycol, triethylene glycol and glycerin. and polyhydric alcohols such as
  • the water-soluble organic solvents may be used singly or in combination of two or more, or a mixture of water-soluble organic solvents and water may be used.
  • the content is preferably 30% by mass or less, more preferably 20% by mass or less, and even more preferably 10% by mass or less.
  • the content of the solvent in the uncrosslinked polymer solution is preferably 50% by mass or more, more preferably 55% by mass or more, still more preferably 60% by mass or more, and preferably 99.999% by mass or less. 99% by mass or less is more preferable, and 99.9% by mass or less is even more preferable.
  • the content of the vinyl alcohol polymer in the uncrosslinked polymer solution is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. From the viewpoint of suppressing the viscosity increase of the uncrosslinked polymer solution and obtaining good moldability, it is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less.
  • the content of the vinyl alcohol polymer is preferably within the above range from the viewpoint of increasing the strength and film thickness of the obtained gel and suppressing the viscosity of the uncrosslinked polymer solution to an extent that facilitates molding.
  • the method for molding the uncrosslinked polymer solution is not particularly limited. It can be molded into shapes such as arbitrarily shaped articles, films, threads, hollow fibers, porous monoliths, coated articles, etc., formed by 3D printers.
  • the crosslinked vinyl alcohol polymer of the present invention is preferably produced by subjecting the vinyl alcohol polymer to a crosslinking step after the molding step.
  • Crosslinking in this step can be performed by the method described above.
  • the cross-linking step may be carried out while the uncross-linked polymer solution contains the solvent, or may be carried out after removing the solvent from the un-cross-linked polymer solution.
  • a cross-linked gel containing a solvent as an uncross-linked polymer solution shrinks violently in the drying step described later, and it may be difficult to form, for example, a film, thread, hollow fiber, coating, or the like.
  • the uncrosslinked polymer solution after molding, and then crosslink it.
  • the degree of dryness of the uncrosslinked polymer solution can be selected to suit the molded product and/or the crosslinking method, but the solvent content is preferably 50% or less, more preferably 25% or less, and still more preferably 10% or less. . From the viewpoint of suppressing shrinkage in the drying step, which will be described later, it is preferable to set the solvent content to the above upper limit or less.
  • the uncrosslinked polymer solution may contain not only the vinyl alcohol polymer but also components necessary for crosslinking such as the crosslinking agent and initiator before molding. As described above, when the uncrosslinked polymer solution is dried once after molding, the components necessary for crosslinking may be added after molding. As described above, the most suitable crosslinking conditions are selected according to the vinyl alcohol polymer used and/or the crosslinking method. It is preferable to perform the molding before the crosslinked vinyl alcohol polymer is formed rather than after the crosslinked vinyl alcohol polymer is formed after the completion of the crosslinking. In some of the above-described crosslinking methods, the reaction is initiated at a temperature below room temperature only by mixing the uncrosslinked polymer solution and the components necessary for crosslinking, so mixing immediately before molding is desirable.
  • the temperature of the cross-linking reaction is preferably 100° C. or lower, more preferably 60° C. or lower, still more preferably 37° C. or lower, from the viewpoint of maintaining the activity of the cell adhesion factor.
  • the compounding step in the present invention is a step of compounding a crosslinked vinyl alcohol polymer with a cell adhesion factor to obtain a crosslinked composite vinyl alcohol polymer. Therefore, as already explained, when the composite is formed at the stage of the uncrosslinked polymer solution, the composite step is basically unnecessary.
  • a carboxylic acid-modified vinyl alcohol-based copolymer and/or an amino-modified vinyl alcohol-based polymer is used as the vinyl alcohol-based polymer, a cell adhesion factor is added to the remaining functional groups by the above-described conjugation method. can be introduced.
  • a hydroxyl-activating reagent As a method for introducing a functional group, a hydroxyl-activating reagent, an acid anhydride reagent, an acetal reagent, or the like may be used as described above, and a cell adhesion factor is introduced into the functional group by the above-described conjugation method. can.
  • the crosslinked body of the water-soluble polymer to which the cell adhesion factor is conjugated in the method of the present invention is used for cell culture applications, for example, autoclave sterilization, ethylene oxide gas sterilization, hydrogen peroxide low temperature plasma sterilization, It is preferably sterilized by dry heat sterilization, chemical sterilization using glutaraldehyde or the like, radiation sterilization using gamma rays or electron beams, or the like.
  • ethylene oxide gas sterilization, hydrogen peroxide low-temperature plasma sterilization, and radiation sterilization are preferably adopted from the viewpoint of easy maintenance of the activity of the complexed cell adhesion factor, and no residue is left. From this point of view, radiation sterilization is more preferable.
  • the concentration of the cell adhesion factor to be complexed is not particularly limited.
  • the density is preferably 0.005-30 ⁇ g/cm 2 , more preferably 0.01-10 ⁇ g/cm 2 .
  • the ratio of the cell adhesion factor to the water-soluble polymer is not particularly limited when the cell adhesion factor is complexed inside the water-soluble polymer crosslinked body or inside and on the surface, but for example, 100 parts by weight of the cell adhesion factor
  • the polymer is preferably 50 to 10,000 parts by weight, more preferably 100 to 5,000 parts by weight.
  • cells may be attached to the surface of a crosslinked body of water-soluble polymer conjugated with a cell adhesion factor, or cells may be included.
  • the term "cell” in the present specification is not particularly limited, but is preferably derived from mammals used for producing useful substances such as pluripotent stem cells, tissue stem cells, somatic cells, medicines, etc., for treatment, etc. cell lines and insect cells.
  • Cells include adherent cells and floating cells.
  • Adherent cells refer to cells that proliferate by adhering to a scaffold such as a crosslinked body of a water-soluble polymer conjugated with the cell adhesion factor of the present invention in cell culture.
  • Suspended cells refer to cells that basically do not require attachment to a scaffold for cell growth.
  • Planktonic cells include cells that are capable of weakly adhering to a carrier.
  • pluripotent stem cells are stem cells that have the ability to differentiate into cells of any tissue (pluripotency). These include germ stem cells (EG cells), germ stem cells (GS cells), and the like.
  • EG cells germ stem cells
  • GS cells germ stem cells
  • tissue stem cells refer to stem cells that are limited in differentiating tissues but have the ability to differentiate into various cell types (pluripotency). , skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, liver stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, pancreatic stem cells (pancreatic ductal epithelial stem cells, etc.), leukocytic stem cells, lymphocytic stem cells, corneal stem cells, etc. are mentioned.
  • the somatic cells refer to cells that constitute multicellular organisms. , fibroblasts, mesenchymal-derived cells, cardiomyocytes, myogenic cells, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fiber cells, EB virus mutant cells, hepatocytes, renal cells, bone marrow cells, macrophages, Liver parenchymal cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, pancreatic ⁇ cells, and the like, but are not limited to these.
  • mammalian-derived established cell lines include CRFK cells, 3T3 cells, A549 cells, AH130 cells, B95-8 cells, BHK cells, BOSC23 cells, BS-C-1 cells, C3H10T1/2 cells, and C-6 cells.
  • CHO cells COS cells, CV-1 cells, F9 cells, FL cells, FL5-1 cells, FM3A cells, G-361 cells, GP + E-86 cells, GP + envAm12 cells, H4-II-E cells, HEK293 cells, HeLa cells, HEp-2 cells, HL-60 cells, HTC cells, HUVEC cells, IMR-32 cells, IMR-90 cells, K562 cells, KB cells, L cells, L5178Y cells, L-929 cells, MA104 cells, MDBK cells , MDCK cells, MIA PaCG-2 cells, N18 cells, Namalwa cells, NG108-15 cells, NRK cells, OC10 cells, OTT6050 cells, P388 cells, PA12 cells, PA317 cells, PC-12 cells, PER.
  • C6 cells C6 cells, PG13 cells, QGH cells, Raji cells, RPMI-1788 cells, SGE1 cells, Sp2/O-Ag14 cells, ST2 cells, THP-1 cells, U-937 cells, V79 cells, VERO cells, WI-38 cells , ⁇ 2 cells, and ⁇ CRE cells.
  • insect cells examples include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, sakusan cells, cinjusang cells, Spodoptera cells (Sf9 cells, Sf21 cells, etc.), mulberry butterfly cells, leaf beetle cells, Drosophila cells, centipedes
  • the cells include flesh fly cells, Aedes albopictus cells, swallowtail cells, American cockroach cells, and stinging nettle cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.).
  • the above cells may aggregate with each other or may be differentiated.
  • Aggregated cells may have organ function. Cells may be those immediately after being collected from a living body, or may be cultured cells. Cells collected from a living body may form an organ.
  • cells are cultured by adhering to a crosslinked water-soluble polymer complexed with a cell adhesion factor.
  • a cell adhesion factor-complexed water-soluble polymer crosslinked body is attached to a cell, a cell is included in a cell adhesion factor-complexed water-soluble polymer crosslinked body, or the like. to culture the cells.
  • the serum-free medium is used during the culture.
  • a typical embodiment includes a method in which a crosslinked body of a water-soluble polymer conjugated with a cell adhesion factor is made into particles, and the particles and cells are allowed to adhere in a medium using the particles as carriers.
  • a water-soluble polymer in which a cell adhesion factor is conjugated to a core material e.g., polystyrene particles, polymethyl(meth)acrylate particles, polylactic acid particles, dextran particles, agarose particles, silica particles, etc.
  • the crosslinked body may be attached to form particles, and the particles may be used as carriers to attach the particles and the cells in a culture medium.
  • a method of forming a crosslinked body of a water-soluble polymer conjugated with a cell adhesion factor into a film, and using the film as a carrier attaches the film and cells in a medium.
  • Core materials in the embodiment for example, polyethylene film, polyvinyl chloride film, polypropylene film, polyester film, polycarbonate film, polystyrene film, ethylene-vinyl alcohol copolymer film, polyvinyl alcohol film, polymethyl methacrylate film, nylon film, cellophane, etc.
  • the film and cells may be attached in a medium.
  • the inner surface and/or the bottom surface of the culture vessel may be coated with a crosslinked water-soluble polymer conjugated with a cell adhesion factor.
  • cells may be cultured by placing cells in a serum-free medium suspension in a culture vessel coated with a crosslinker.
  • the inner surface and/or the bottom surface of the culture vessel may be coated with a crosslinked body of a water-soluble polymer conjugated with a cell adhesion factor, and cells may be included in the crosslinked body.
  • a serum-free medium is placed in the culture vessel and cultured in the medium.
  • culture conditions such as culture temperature, conditions generally employed in conventional cell culture can be widely used, and examples thereof include an environment of 37° C. and 5% CO 2 . If necessary, the cells grown in the serum-free medium are collected and/or subcultured by common methods.
  • the cell adhesion factor to which the cells are attached can be detached from the crosslinked body of the complexed water-soluble polymer, and the cells can be collected in the state of floating cells.
  • the cells thus obtained may be seeded on a water-soluble polymer complexed with a cell adhesion factor together with fresh serum-free medium, and subcultured.
  • cells can be cultured and grown only in a basal medium without adding serum or serum substitutes in the culture step.
  • the method of the present invention includes a step of culturing cells using a serum-free medium
  • the method may include a step of adding a predetermined protein of interest other than cell culture before or after the culture step.
  • a predetermined protein of interest other than cell culture before or after the culture step.
  • cell growth factors/differentiation factors, cytokines, hormones, polyanions, polyvinyl alcohol, transferrin, etc. are added for the purpose of inducing signal transduction analysis, differentiation, etc. in cells cultured/proliferated in these serum-free media. be able to.
  • serum since it may be difficult for cells to adhere to the water-soluble polymer gel complexed with cell adhesion factors in the early stage of culture, serum , serum substitutes, cell growth factors/differentiation factors, cytokines, hormones, polyanions, polyvinyl alcohol, transferrin, etc., to adhere cells to crosslinked water-soluble polymers conjugated with cell adhesion factors. may be performed.
  • an embodiment in which the cultured cells produce a protein as a result of culturing the cells using a serum-free medium can also be included in the method of the present invention.
  • Cell growth factors/differentiation factors that may be added to the serum-free medium of the present invention include, for example, vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor- ⁇ ( TGF- ⁇ ), osteonectin, angiopoietin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), Ciliary neurotrophic factor (CNTF), glial cell lineage-derived neurotrophic factor (GDNF), nerve growth factor (NGF), leukemia inhibitory factor (LIF), stem cell growth factor (SCF), bone morphogenetic protein (BMP), interferon - ⁇ , interferon- ⁇ , interferon- ⁇ , interferon- ⁇ , tumor necrosis factor- ⁇ (TNF- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), Notch ligands (Delthelial cell growth factor
  • Cytokines that may be added to the serum-free medium of the present invention include interleukin-1 ⁇ , interleukin-1 ⁇ , interleukin-2, interleukin-4, interleukin-5, interleukin-6, and interleukin-7.
  • G-CSF granulocyte-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • M-CSF macrophage colony-stimulating factor
  • EPO erythropoietin
  • TPO thrombopoie
  • Hormones that may be added to the serum-free medium of the present invention include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and Angiotensin, antidiuretic hormone, atrial natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadothrombin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placenta Sexual lactogen, growth hormone, inhibin, insulin, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone
  • polyanions examples include heparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate, and the like. These polyanions are stabilized by, for example, binding to the cell growth factor/differentiation factor, and promote cell proliferation more efficiently.
  • Polyvinyl alcohol (PVA) that may be added to the serum-free medium of the present invention can be freely selected from the above vinyl alcohol-based polymers and modified PVA.
  • BSA bovine serum albumin
  • human recombinant albumin, etc. are often added to the medium for the purpose of cell protection, growth factor stabilization, and the like.
  • PVA a serum-free culture environment can be constructed in which proteins such as BSA are excluded.
  • the content of PVA in the serum-free medium is preferably 0.01-0.5% by mass, more preferably 0.05-0.2% by mass.
  • a cell culture can be obtained by culturing cells by the method of the present invention.
  • Substances such as proteins can also be produced by utilizing the metabolism of cultured cells.
  • the present invention provides a method for producing a target substance, which comprises the step of culturing cells by allowing them to adhere to a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor using a serum-free medium. offer.
  • the method of the present invention can be widely applied to substance production and the like in the field of biotechnology to which the present invention belongs. Therefore, the target substance is not particularly limited, but includes cell cultures themselves, substances produced by cells, and the like.
  • Cell cultures include, for example, cultured cells, cell masses, tissues and the like.
  • Substances produced by cells include, for example, physiologically active substances such as antibodies, viral vaccines, viral vectors, hormones, cytokines, and enzymes.
  • the method of the present invention may include a step of recovering the target substance obtained by the culture step. As the recovery step, a wide range of methods commonly used in the field to which the present invention belongs can be used.
  • RNA DNA or RNA
  • Plasmid vectors and/or messenger RNAs encoding proteins to be expressed such as antibodies, adenoviruses, adeno-associated viruses (AAV), lentiviruses, etc., are subjected to retroviral methods, liposome methods, cationic liposome methods, adenoviral methods, etc. can be introduced into the cells by
  • the method of the present invention can be used as a production system for protein production and/or expression in serum-free medium.
  • Proteins can be obtained by similarly culturing cells into which DNA and/or messenger RNA has been introduced by the above method in a serum-free medium.
  • the protein thus obtained can be isolated from intracellularly or extracellularly (medium, etc.) and purified as a substantially pure and homogeneous protein. Separation and purification of proteins may be carried out using separation and purification methods commonly used in protein purification, and are not limited in any way.
  • chromatography column filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
  • proteins can be separated and purified.
  • conventional serum and/or serum substitutes are used, a large amount of contaminating proteins are present, requiring a great deal of effort in the purification process. Purification of the expressed protein becomes easier.
  • cells can be cultured (survival/proliferation) in a serum-free medium, more specifically, a protein-free medium. Therefore, it is possible to easily purify useful proteins produced by the cells and to provide them with high purity.
  • the medium does not contain proteins, it is possible to reproducibly elucidate the molecular mechanism in the undifferentiated maintenance and differentiation of stem cells, which was not possible until now due to the influence of contaminants such as serum-derived proteins contained in the medium.
  • a high evaluation method can be provided.
  • the method of the present invention can contribute not only to regenerative medicine but also to mass production of cells including cultured meat.
  • ⁇ Complex reagent> ⁇ 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. ⁇ N-hydroxysuccinimide: manufactured by Tokyo Chemical Industry Co., Ltd.
  • ⁇ Cell adhesion factor> ⁇ Gelatin (porcine origin, type A): manufactured by Sigma-Aldrich Japan Co., Ltd.
  • ⁇ Collagen trade name “Cell Matrix”, type I-C): manufactured by Nitta Gelatin Co., Ltd.
  • ⁇ Solvent> Ion-exchanged water: Ion-exchanged water with an electrical conductivity of 0.08 ⁇ 10 -4 S / m or less
  • PBS Prepared by dissolving a PBS tablet (manufactured by Takara Bio Inc.) in a specified amount of ion-exchanged water .
  • ⁇ MES buffer 0.1 mol/L aqueous solution of 2-morpholinoethanesulfonic acid monohydrate (manufactured by Dojindo Laboratories) was prepared and neutralized with 0.1 mol/L NaOH aqueous solution. , pH was 5.6.
  • ⁇ Cell> - 293T cells cell lines established by the inventors - Mouse ES cells: cell lines established by the inventors - Mouse fetal fibroblasts (MEF): cell lines established by the inventors
  • FBS and serum replacement> ⁇ Fetal bovine serum (FBS): manufactured by Thermo Fisher Scientific Co., Ltd. ⁇ "StemSure" Serum Replacement: manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd. ⁇ Albumin, derived from bovine serum, fraction V: FUJIFILM Wako Pure Chemical Industries, Ltd. ) N-2 Supplement: Thermo Fisher Scientific Co., Ltd. B-27 Supplement: Thermo Fisher Scientific Co., Ltd.
  • Penicillin-streptomycin (5,000 U/mL): manufactured by Thermo Fisher Scientific Co., Ltd.
  • GFP Green fluorescent protein
  • Saponified product of polyvinyl acetate, average degree of polymerization 1700, degree of saponification about 98.0 to 99.0 mol%, viscosity (4%, 20 ° C.) 25 0 to 31.0 mPa ⁇ s, manufactured by Kuraray Co., Ltd.) was placed in a 1 L separable flask equipped with a Dimroth condenser, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was started with a mechanical stirrer. After the temperature was raised to 80°C by a water bath, stirring was continued at 80°C for 4 hours.
  • DMSO dimethyl sulfoxide
  • the rate of introduction of ethylenically unsaturated groups (methacryloyl groups) in the methacryloyl PVA117 was 2.0 mol% relative to the repeating units of the starting PVA (hereinafter abbreviated as "MA-PVA117 (2.0)” ).
  • the washing water was removed and the plate was dried at room temperature for 24 hours or more to obtain a collagen-complexed MA-PVA117(2.0)-SA(3.4) coated well plate (hereinafter referred to as “collagen-MA-PVA117(2 .0)-SA(3.4)-coated plate”).
  • the wells were immersed in 1 mL of PBS overnight and the amount of collagen immobilized per well was measured by the bicinchoninic acid (BCA) method (BCA Protein Assay Kit (manufactured by Takara Bio Inc.)). The density was 20.6 ⁇ g/well.
  • the well plate was sealed in an aluminum deposition bag and sterilized by irradiation with gamma rays (25 kGy) at Koga Isotope Co., Ltd.
  • the amount of collagen immobilized per well was also measured in the same manner as in Synthesis Example A, and the conjugation density of cell adhesion factor was 29.3 ⁇ g/well.
  • the well plate was sealed in an aluminum deposition bag and sterilized by irradiation with gamma rays (25 kGy) at Koga Isotope Co., Ltd.
  • the amount of immobilized gelatin per well was also measured in the same manner as in Synthesis Example A, and was 53.8 ⁇ g/well.
  • the well plate was sealed in an aluminum deposition bag and sterilized by irradiation with gamma rays (25 kGy) at Koga Isotope Co., Ltd.
  • Preparation of 293T cells were cultured at 37° C. in 5% CO 2 in DMEM supplemented with 10% FBS and penicillin-streptomycin (concentration of 100 U/mL penicillin, 100 ⁇ g/mL streptomycin). The proliferated 293T cells were treated with trypsin/EDTA solution and collected, and DMEM without fetal bovine serum and serum replacement (hereinafter abbreviated as “DMEM basal medium”. 100 U/mL penicillin and 100 ⁇ g/mL streptomycin were added. (supplemented)), and the 293T cells were dispersed in the DMEM basal medium to a final concentration of 1 ⁇ 10 5 cells/5 mL.
  • mice ES cells were maintained according to the following method and adapted to feeder-free culture. 15% StemSure Serum Replacement for StemSure D-MEM (high glucose) (phenol red, containing sodium pyruvate) on feeder cells prepared by treating mouse embryonic fibroblasts (MEF) with mitomycin C. , 2 mM L-glutamine, 1% non-essential amino acid, penicillin-streptomycin (concentration of 50 U/mL penicillin and 50 ⁇ g/mL streptomycin), 0.1 mM 2-mercaptoethanol, 103 U/mL StemSure LIF.
  • StemSure Serum Replacement for StemSure D-MEM high glucose
  • phenol red containing sodium pyruvate
  • feeder cells prepared by treating mouse embryonic fibroblasts (MEF) with mitomycin C. , 2 mM L-glutamine, 1% non-essential amino acid, penicillin-streptomycin (concentration of 50 U/mL penicillin and 50
  • Mouse ES cells maintained on these feeders were seeded on a gelatin-coated culture dish, and added 0.5% to a 1:1 mixture of DMEM/F-12, GlutaMAX supplement and Neurobasal Medium.
  • Proliferated mouse ES cells were treated with a trypsin/EDTA solution, collected, washed with DMEM basal medium three times, and finally dispersed in DMEM basal medium to 1 ⁇ 10 5 cells/5 mL.
  • Example 1 293T cells dispersed in DMEM basal medium were placed on the collagen-MA-PVA117(2.0)-SA(3.4)-coated plate prepared in Synthesis Example A so that 1 ⁇ 10 5 cells/well, and 5 Cultured at 37° C., % CO 2 . The cells were cultured for 7 days without changing the medium, 293T cells were collected by trypsin/EDTA treatment, and the number of cells per well was counted. Table 1 shows the results.
  • Example 2 and 3 The collagen-MA-PVA117(2.0)-SA(1.0)-coated plate and the gelatin-MA-PVA117(2.0)-SA(3.4)-coated plate prepared in Synthesis Example B and C respectively were used. Evaluation of serum-free culture was performed in the same manner as in Example 1, except that the serum was used. Table 1 shows the results.
  • Example 4 Mouse ES cells dispersed in DMEM basal medium were added to the collagen-MA-PVA117(2.0)-SA(3.4)-coated plate prepared in Synthesis Example A so that 1 ⁇ 10 5 cells/well, Cultured at 37° C., 5% CO 2 . The cells were cultured for 7 days while changing the DMEM basal medium once every 3 days, mouse ES cells were collected by trypsin/EDTA treatment, and the number of cells per well was counted.
  • Example 1 Serum-free culture was evaluated in the same manner as in Example 1 using a commercially available cell culture surface-treated well plate (made of polystyrene). Table 1 shows the results.
  • PEI polyethyleneimine

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Abstract

La présente invention a pour objet de fournir un procédé qui permet de mettre en culture des cellules dépendant d'un ancrage avec un milieu de culture exempt de sérum. La présente invention concerne un procédé de culture cellulaire comprenant une étape de culture de cellules avec un milieu de culture exempt de sérum tout en faisant adhérer les cellules à un produit réticulé d'un polymère soluble dans l'eau qui est composé avec un facteur d'adhérence cellulaire.
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Citations (3)

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Publication number Priority date Publication date Assignee Title
JP2006314285A (ja) * 2005-05-13 2006-11-24 Kuraray Co Ltd 細胞培養用担体及び該細胞培養用担体を用いた細胞培養方法
US20080153134A1 (en) * 2006-12-22 2008-06-26 Willy Wiyatno Methods and apparatus for generating hydrophilic patterning of high density microplates using an amphiphilic polymer
WO2020116623A1 (fr) * 2018-12-07 2020-06-11 関東化学株式会社 Milieu de culture maintenant l'état de non différentiation destiné à des cellules souches pluripotentes

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
JP2006314285A (ja) * 2005-05-13 2006-11-24 Kuraray Co Ltd 細胞培養用担体及び該細胞培養用担体を用いた細胞培養方法
US20080153134A1 (en) * 2006-12-22 2008-06-26 Willy Wiyatno Methods and apparatus for generating hydrophilic patterning of high density microplates using an amphiphilic polymer
WO2020116623A1 (fr) * 2018-12-07 2020-06-11 関東化学株式会社 Milieu de culture maintenant l'état de non différentiation destiné à des cellules souches pluripotentes

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MATSUMURA, K. HYON, S.-H. NAKAJIMA, N. IWATA, H. WATAZU, A. TSUTSUMI, S.: "Surface modification of poly(ethylene-co-vinyl alcohol): hydroxyapatite immobilization and control of periodontal ligament cells differentiation", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, vol. 25, no. 19, 1 August 2004 (2004-08-01), AMSTERDAM, NL , pages 4817 - 4824, XP004505493, ISSN: 0142-9612, DOI: 10.1016/j.biomaterials.2003.11.055 *
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