Cell culture method using serum-free culture medium

Abstract

The problem to be solved by the present invention is to provide a method which makes it possible to culture anchorage-dependent cells with a serum-free culture medium. The present invention provides a cell culture method including a step for culturing cells with a serum-free culture medium while adhering the cells to a crosslinked product of a water-soluble polymer that is compounded with a cell adhesion factor.

Description

Cell culture method using serum-free medium

[0001] [Cross-reference to related applications] This application claims priority to Japanese Patent Application No. 2021-195494, filed on December 1, 2021, the entire disclosure of which is incorporated herein by reference. The present invention relates to a cell culture method using a serum-free medium.

[0002] In recent years, the production of useful physiologically active substances (antibodies, viral vaccines, viral vectors, hormones, cytokines, enzymes, etc.) using mass-cultured animal cells has become common on an industrial scale. Cell culture is carried out in a medium in which approximately 10 to 20% animal serum, such as fetal bovine serum (FBS), which contains large amounts of proteins including growth factors, is added to a basal medium (e.g., commercially available Dulbecco's Modified Eagle's Medium (DMEM) or Ham's F12 medium) containing known components such as amino acids, carbohydrates, lipids, vitamins, nucleic acids or their precursors, salts, and trace metals. Generally, animal cells cannot be cultured (survive and grow) unless animal serum is added to the basal medium.

[0003] However, animal sera such as fetal bovine serum (FBS) have the following problems: (1) they are expensive and have limited supply, (2) there is a large variation in quality between lots, which requires a great deal of effort for lot checks before use and for securing lots, and (3) it is difficult to isolate the desired physiologically active substance from a medium containing animal sera containing various proteins. In addition to the above, animal sera pose a risk of infection with prion-related diseases such as mad cow disease, bovine spongiform encephalopathy, transmissible spongiform encephalopathy, and Creutzfeldt-Jakob disease.

[0004] In order to solve the problems associated with animal serum, studies have been conducted in recent years on culture media that do not use animal serum (serum-free media). However, it is usually difficult to culture animal cells using only basal media, and serum-free media are simply basal media supplemented with serum substitutes containing proteins such as insulin, growth factors, transferrin, and even albumin (e.g., Patent Document 1, Patent Document 2, Non-Patent Document 1). Some media use recombinant proteins, achieving xeno-free (no xenogeneic animal origin) and animal-free (no animal-derived components), partially resolving the problems associated with animal serum. However, the cost of proteins remains high, and proteins added to these basal media must be removed when purifying the desired physiologically active substance. Furthermore, even purified proteins still have issues, such as promoting unintended differentiation of stem cells during culture.

[0005] In light of this environment, the development of a completely protein-free medium is desired. Recently, it has been shown that adding polyvinyl alcohol (hereinafter sometimes abbreviated as PVA) to a low-protein medium containing insulin and transferrin enables cell culture without the use of proteins such as animal serum, purified albumin, or recombinant albumin. For example, a low-protein medium supplemented with PVA has been disclosed that allows induced pluripotent stem cells (hereinafter sometimes abbreviated as iPS cells) to be attached and cultured on a laminin-coated cell culture dish (Patent Document 3). Furthermore, a technique has recently been disclosed that enables the culture of human hematopoietic stem cells for several months while maintaining their undifferentiated state using a low-protein medium supplemented with PVA (Patent Document 4, Non-Patent Document 2).

[0006] Japanese Patent Application Laid-Open No. 7-23780 Japanese Patent Application Laid-Open No. 2007-228815 Japanese Patent Application Laid-Open No. 2020-89338 WO2021-049617

[0007] Biotechnology, 2014, Vol. 92, pp. 487-490 Nature, 2019, Vol. 571, pp. 117-121

[0008] It has been shown that iPS cells can indeed be attached to cell culture dishes and cultured by using the low-protein medium supplemented with PVA described in Patent Document 3, but the low-protein medium contains the proteins insulin and transferrin, which have problems such as unstable quality as mentioned above, difficulty in purifying the target physiologically active substance, and high cost. Patent Document 4 and Non-Patent Document 2 also have similar problems. When a completely protein-free medium in which all proteins have been removed from the low-protein medium is used to solve these problems, adhesion-dependent cells in particular are unable to even attach to the cell culture dish, making it difficult to culture them.

[0009] The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a method that enables the culture of adhesion-dependent cells in a serum-free medium.

[0010] As a result of extensive research, the present inventors have found that a method of adhering cells to a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor and culturing them makes it possible to culture (survive and grow) cells in a serum-free medium, preferably a protein-free medium, and have thus completed the present invention.

[0011] That is, the present invention relates to the following items [1] to

[18] . [1] A method for culturing cells, comprising the step of adhering cells to a crosslinked body of a water-soluble polymer complexed 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 described in [1]. [3] The method described in [1] or [2], wherein the serum-free medium is a protein-free medium. [4] The method described in any one of [1] to [3], wherein the cell adhesion factor is complexed to the crosslinked body of the water-soluble polymer by a covalent bond. [5] The method described in any one of [1] to [4], wherein the crosslinked body of the water-soluble polymer is a crosslinked body of a vinyl alcohol-based polymer. [6] The method described in [5], wherein the crosslinked body of the vinyl alcohol-based polymer is crosslinked by a covalent bond. [7] The method described in [5] or [6], wherein the crosslinked body of the vinyl alcohol-based polymer is a crosslinked body of a vinyl alcohol-based polymer having an ethylenically unsaturated group and a carboxy group. [8] The method described in [7], wherein the carboxy group of the crosslinked body and the amino group of the cell adhesion factor are covalently bonded by an amide bond. [9] The method according to [7] or [8], wherein 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.

[10] The method according to any one of [7] to [9], wherein the introduction rate of the ethylenically unsaturated group is 0.1 to 10 mol% based on all structural units constituting the vinyl alcohol-based polymer.

[11] The method according to any one of [7] to

[10] , wherein the introduction rate of the carboxy group is 0.1 to 50 mol% based on all structural units constituting the vinyl alcohol-based polymer.

[12] The method according to any one of [1] to

[11] , wherein the crosslinked product of the water-soluble polymer is an amorphous particle, a spherical particle, a micromolded product, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article.

[13] A method for culturing cells using the serum-free medium according to any one of [1] to

[12] , wherein a physiologically active substance is allowed to act on the 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 in the serum-free medium are transfected with a gene vector to produce a physiologically active 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 a non-covalent bond.

[16] A complex of gelatin or collagen and a crosslinked water-soluble polymer for use in cell culture.

[17] The complex according to

[16] , wherein, when the complex contains collagen, the collagen has a weight-average molecular weight of 100,000 to 400,000.

[18] A complex of a cell adhesion factor and a crosslinked water-soluble polymer for use in the method according to any one of [1] to

[14] .

[0012] According to the present invention, cells can be cultured (survive and proliferate) in a serum-free medium, preferably a protein-free medium. Therefore, not only is there no risk of infection from the medium, but the medium is also extremely inexpensive and stable in quality with no lot-to-lot variation in proteins. Furthermore, useful physiologically active substances produced by the cells can be easily purified to high purity. Furthermore, because the medium does not contain proteins, a highly reproducible method can be provided for maintaining the undifferentiated state and differentiating stem cells, which has previously been impossible due to the influence of protein impurities contained in the medium.

[0013] 1 shows a micrograph of 293T cells expressing GFP in Example 5.

[0014] [Serum-Free Medium] In the present invention, a serum-free medium refers to a medium that does not contain animal-derived serum. Furthermore, the serum-free medium of the present invention is preferably a protein-free medium. Conventionally, it has been difficult to culture and grow cells in serum-free media that do not contain animal-derived serum. However, the method of the present invention is fundamentally different from conventional techniques in that it allows cells to be cultured and grown without the use of serum. In the present invention, the term "serum" may be interpreted in the sense commonly used in the field of biochemistry to which the present invention pertains, but typically refers to blood from which blood cells and several blood coagulation factors (e.g., fibrinogen (factor I), prothrombin (factor II), factor V, and factor VIII) have been removed. The serum-free medium of the present invention may contain non-protein factors, such as ethanolamine, 2-mercaptoethanol, and sodium selenite, which are known as serum substitute components other than proteins (e.g., insulin, growth factors, transferrin, and even albumin). Typically, the serum-free medium of the present invention is a basal medium prepared by mixing nutritional components such as amino acids, carbohydrates, lipids, vitamins, nucleic acids or their precursors, salts, trace metals, etc., and is usually prepared by adding antibiotics to the basal medium. In a typical embodiment, a liquid medium is used in the method of the present invention.

[0015] <Basal medium> Examples of basal media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12 medium, McCoy's 5A medium, Eagle's Minimum Essential Medium (EMEM), αMEM (alpha Modified Eagle's Minimum Essential Medium; αMEM), and MEM (Minimum Essential Medium; αMEM). Medium), RPM11640 medium, Iscove's Modified Dulbecco's Medium (IMDM), etc.; a medium comprising two or more of these in combination, etc. Of course, other media suitable for the cells to be cultured can also be used.

[0016] <Antibiotics> Examples of antibiotics that can be 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, and valinomycin, and any appropriate antibiotic can be used depending on the purpose.

[0017] [Crosslinked Water-Soluble Polymer] The crosslinked water-soluble polymer in the present invention refers to a so-called hydrogel in which a three-dimensional network structure is formed by crosslinking the water-soluble polymer, and the structure absorbs an aqueous solvent and swells. Examples of the crosslinking form include physical crosslinking such as hydrogen bonding, ionic bonding, coordination bonding, and hydrophobic bonding; and chemical crosslinking in which covalent bonding is achieved by chemical reaction, and the most suitable form can be selected depending on the purpose. Both synthetic and natural polymers can be used as the water-soluble polymer.

[0018] 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 polymers, hydroxyethyl(meth)acrylate polymers, ethylene oxide polymers, propylene oxide polymers, polyethyleneimine, polyallylamine, and derivatives thereof.

[0019] The natural polymer may be a water-soluble polysaccharide, such as alginic acid, propylene glycol alginate, agarose, hydroxyethyl cellulose, carboxymethyl cellulose, or other water-soluble cellulose derivatives; guar gum, carrageenan, agar, chitosan, gellan gum, dextran, starch, hyaluronic acid, pullulan, or heparin.

[0020] The water-soluble polymers exemplified above can be used in the present invention. However, from the viewpoints of the stability of the crosslinked body during cell culture, the controllability of the crosslinked body's physical properties, the ease of complexing with the cell adhesion factor, and the cell proliferation in serum-free medium, among the water-soluble polymers, 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 preferred, vinyl alcohol-based polymers, water-soluble cellulose derivatives, and dextran are even more preferred, and vinyl alcohol-based polymers are particularly preferred.

[0021] <Vinyl Alcohol Polymer and Production Method Thereof> 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 groups in the polyvinyl ester to hydroxyl groups. Examples of the vinyl ester monomer include aliphatic vinyl esters such as vinyl formate, vinyl acetate, vinyl propionate, vinyl n-butyrate, vinyl isobutyrate, vinyl pivalate, vinyl versatate, vinyl caproate, vinyl caprylate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate, vinyl stearate, and vinyl oleate; and aromatic vinyl esters such as vinyl benzoate. These may be used alone or in combination of two or more. Among the vinyl ester monomers, aliphatic vinyl esters are preferred, and vinyl acetate is more preferred from the viewpoint of production costs. That is, the polyvinyl ester is preferably polyvinyl acetate obtained by polymerizing vinyl acetate.

[0022] Furthermore, the polyvinyl ester may contain structural units derived from monomers other than the vinyl ester-based monomers, if necessary, within the range that does not impair the effects of the present invention.Examples of the other monomers include α-olefins such as ethylene, propylene, n-butene, and isobutylene; acrylic acid or a salt thereof; alkyl acrylates such as methyl acrylate, ethyl acrylate, n-propyl acrylate, i-propyl acrylate, n-butyl acrylate, i-butyl acrylate, t-butyl acrylate, 2-ethylhexyl acrylate, dodecyl acrylate, and octadecyl acrylate; methacrylic acid or a salt thereof; methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, and methacrylic acid; methacrylic acid alkyl esters such as i-propyl methacrylate, n-butyl methacrylate, i-butyl methacrylate, t-butyl methacrylate, 2-ethylhexyl methacrylate, dodecyl methacrylate, and octadecyl methacrylate; acrylamide, N-methylacrylamide, N-ethylacrylamide, N,N-dimethylacrylamide, diacetone acrylamide, acrylamidopropanesulfonic acid or a salt thereof, acrylamidopropyldimethylamine or a salt or quaternary salt thereof, N-methylolacrylamide or a salt thereof methacrylamide derivatives such as methacrylamide, N-methylmethacrylamide, N-ethylmethacrylamide, methacrylamidepropanesulfonic acid or a salt thereof, methacrylamidepropyldimethylamine or a salt or quaternary salt thereof, N-methylolmethacrylamide or a derivative thereof; N-vinylamide derivatives such as N-vinylformamide and N-vinylacetamide; vinyl ethers such as methyl vinyl ether, ethyl vinyl ether, n-propyl vinyl ether, i-propyl vinyl ether, n-butyl vinyl ether, i-butyl vinyl ether, t-butyl vinyl ether, dodecyl vinyl ether, and stearyl vinyl ether; nitriles such as acrylonitrile and methacrylonitrile; vinyl halides such as vinyl chloride and vinyl fluoride; vinylidene halides such as vinylidene chloride and vinylidene fluoride; allyl compounds such as allyl acetate and allyl chloride; maleic acid or a salt, ester, or acid anhydride thereof; vinylsilyl compounds such as vinyltrimethoxysilane; and isopropenyl acetate. These may be used alone or in combination of two or more.

[0023] When the polyvinyl ester contains structural units derived from other monomers, the content of the structural units derived from other monomers is preferably 20 mol % or less, more preferably 10 mol % or less, and even more preferably 5 mol % or less, based on the total structural units constituting the polyvinyl ester.

[0024] The method for saponifying the polyvinyl ester is not particularly limited, and can be a conventional method. For example, an alcoholysis method using an alkali catalyst or an acid catalyst, a hydrolysis method, or the like can be applied. Among them, a saponification reaction using methanol as a solvent and caustic soda (NaOH) as a catalyst is simple and preferable.

[0025] 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, even more preferably 500 to 3,000, and most preferably 500 to 2,500. Two or more types of vinyl alcohol polymers with different average degrees of polymerization may be mixed and used. From the viewpoint of suppressing embrittlement of the crosslinked body of the present invention, the average degree of polymerization is preferably 300 or more. Furthermore, 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 even more preferably 3,000 or less.

[0026] The average degree of polymerization of the vinyl alcohol polymer in this specification refers to the average degree of polymerization measured in accordance with JIS K 6726: 1994. Specifically, since the degree of polymerization of the vinyl alcohol polymer and the PVA, which will be described later as a raw material, can be considered to be the same, the average degree of polymerization can be determined from the limiting viscosity measured in water at 30°C after purifying the PVA, which will be used as a raw material.

[0027] The degree of saponification of the vinyl alcohol polymer is preferably 50 mol% or more, more preferably 60 mol% or more, and even more preferably 65 mol% or more, from the viewpoint of improving the water solubility of the vinyl alcohol polymer. The upper limit of the degree of saponification is 100 mol% or 99 mol%. In this specification, the degree of saponification of the vinyl alcohol polymer means the ratio (mol%) of the number of moles of vinyl alcohol units to the total number of moles of structural units (e.g., vinyl acetate units) that can be converted to vinyl alcohol units by saponification in the raw material PVA and vinyl alcohol units, and can be measured in accordance with JIS K 6726:1994.

[0028] The 4 mass% viscosity of the vinyl alcohol polymer at 20°C is preferably 0.5 to 110 mPa·s, more preferably 1 to 80 mPa·s, and even more preferably 2 to 60 mPa·s. When the viscosity is within the above range, the ease of production of the crosslinked body is improved, and the strength of the composite crosslinked body can be improved. Note that the viscosity in this specification refers to the viscosity measured at 20°C for a 4 mass% aqueous solution of the vinyl alcohol polymer using a B-type viscometer (rotation speed: 12 rpm) in accordance with the rotational viscometer method of JIS K 6726:1994.

[0029] <Crosslinked vinyl alcohol polymer> The crosslinked vinyl alcohol polymer may be one in which the vinyl alcohol polymer is physically crosslinked or may be crosslinked by a covalent bond. The total amount of vinyl alcohol-derived structural units and vinyl ester-derived structural units relative 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 even more preferably 95 mol% or more.

[0030] Methods for physically crosslinking vinyl alcohol polymers include, for example, freezing and thawing an aqueous solution of a vinyl alcohol polymer, dissolving the polymer in a mixed solvent of dimethyl sulfoxide and water, and cooling the hot solution to room temperature. In addition, physical crosslinking via hydrophobic bonds is also possible by introducing saturated or unsaturated fatty acids such as hexanoic acid and octanoic acid to hydroxyl groups derived from vinyl alcohol by esterification, or by introducing hydrophobic aldehydes such as butyraldehyde and benzaldehyde to 1,3-diol structures derived from vinyl alcohol monomers by acetalization. Methods for covalently crosslinking vinyl alcohol polymers include using a multifunctional crosslinking agent that reacts with the side-chain hydroxyl groups of the vinyl alcohol polymer, or by introducing functional groups into the vinyl alcohol polymer by copolymerization, post-modification, or the like, and then reacting the functional groups. However, crosslinking via covalent bonds is preferred because it increases the stability of the crosslinked product when swollen in water.

[0031] Methods using a polyfunctional crosslinking agent that reacts with the side chain hydroxyl groups of a vinyl alcohol polymer include a method of reacting a polyfunctional aldehyde compound such as glyoxal, malondialdehyde, or glutaraldehyde under acidic conditions (polyacetal crosslinking), a method of reacting a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether under alkaline conditions (polyether crosslinking), and a method of reacting a polyfunctional carboxylic acid compound such as maleic acid or succinic acid (polyester crosslinking). When a polyfunctional crosslinking agent that reacts with the side chain hydroxyl groups of a vinyl alcohol polymer is used, the introduction rate of the crosslinking compound is preferably 0.05 to 5 mol%, more preferably 0.1 to 2.5 mol%, relative to the repeating units of the vinyl alcohol polymer into which the crosslinking compound is introduced.

[0032] <Modified PVA> A method for introducing functional groups into a vinyl alcohol polymer by copolymerization and reacting them includes copolymerizing a vinyl ester monomer with a polymerizable monomer other than the vinyl ester monomer, which is a monomer having a reactive substituent other than a hydroxyl group, during the production process of the vinyl alcohol polymer, followed by saponification to obtain a copolymerized modified polyvinyl alcohol (hereinafter sometimes abbreviated as "copolymerized modified PVA"). Then, a multifunctional crosslinking agent that reacts with functional groups, such as carboxy groups and amino groups, present in the copolymerized modified PVA is added. A copolymerized modified PVA having a carboxy group may be referred to as a "carboxylic acid-modified PVA," and a copolymerized modified PVA having an amino group may be referred to as an "amino-modified PVA."

[0033] Examples of monomers constituting carboxylic acid-modified PVA include α,β-unsaturated carboxylic acids such as (meth)acrylic acid, maleic acid, fumaric acid, and itaconic acid; (meth)acrylic acid alkyl esters such as methyl (meth)acrylate and ethyl (meth)acrylate; and α,β-unsaturated carboxylic acid anhydrides such as maleic anhydride and itaconic anhydride, and derivatives thereof. A carboxylic acid-modified PVA is produced by copolymerizing a vinyl ester monomer with a monomer constituting the carboxylic acid-modified PVA and then saponifying the copolymer. A crosslinked product can be obtained by combining a polyfunctional crosslinking agent that reacts with the introduced carboxyl groups, for example, a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether, or a polyfunctional amino compound such as ethylenediamine, polyethyleneimine, or polyallylamine, with a carbodiimide condensing agent such as 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The carboxyl group introduced into the carboxylic acid-modified PVA can be conjugated with a cell adhesion factor having an amino group by covalently bonding it via an amide bond (-CONH-).

[0034] Furthermore, amino-modified PVA can be obtained by copolymerizing a vinyl ester monomer with N-vinylformamide or the like, followed by saponification, and then combining a polyfunctional crosslinking agent that reacts with the introduced amino group, such as the polyfunctional epoxy compound or a polyfunctional carboxylic acid compound such as succinic acid or maleic acid, with the carbodiimide condensing agent.

[0035] <Ethylenically Unsaturated Groups> Examples of methods for introducing functional groups into vinyl alcohol polymers by post-modification and reacting them include a method for introducing ethylenically unsaturated groups into the side chains of the vinyl alcohol polymer. The introduced ethylenically unsaturated groups can be easily crosslinked by adding an additive such as a radical initiator to induce a polymerization reaction. The ethylenically unsaturated groups are preferably introduced via the side chains or terminal functional groups of the vinyl alcohol polymer, and more preferably by reacting a compound containing an ethylenically unsaturated group (hereinafter sometimes abbreviated as "ethylenically unsaturated group-containing compound") with a hydroxyl group in the side chain of the vinyl alcohol polymer. In the present invention, examples of the ethylenically unsaturated group include a vinyl group, a (meth)acryloyl group, a (meth)acryloylamino group, a vinylphenyl group, and a norbornenyl group. Furthermore, in the present invention, the ethylenically unsaturated group may be a derivative of these groups. In the present invention, the term "crosslinked vinyl alcohol polymer having an ethylenically unsaturated group" refers to a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group. In such an embodiment, at least a part of the ethylenically unsaturated groups is consumed by the crosslinking reaction of the vinyl alcohol polymer. Therefore, the term "crosslinked product of a vinyl alcohol polymer having an ethylenically unsaturated group" may include both a crosslinked product that no longer has ethylenically unsaturated groups as a result of the ethylenically unsaturated groups of the vinyl alcohol polymer before crosslinking and before conjugation being consumed by the crosslinking reaction, and a crosslinked product in which the ethylenically unsaturated groups remain.

[0036] Examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include (meth)acrylic acid or derivatives thereof such as (meth)acrylic acid, (meth)acrylic anhydride, (meth)acrylic acid halide, and (meth)acrylic acid ester. A (meth)acryloyl group can be introduced by subjecting these compounds to an esterification reaction or an ester exchange reaction in the presence of a base.

[0037] Furthermore, examples of the ethylenically unsaturated group-containing compound to be reacted with the hydroxyl group in the side chain of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and a glycidyl group in the molecule, such as glycidyl (meth)acrylate, allyl glycidyl ether, etc. By subjecting these compounds to an etherification reaction in the presence of a base, it is possible to introduce a (meth)acryloyl group and / or an allyl group into the vinyl alcohol polymer.

[0038] Furthermore, examples of ethylenically unsaturated group-containing compounds to be reacted with the 1,3-diol group of the vinyl alcohol polymer include compounds containing an ethylenically unsaturated group and an aldehyde group in the molecule, such as acrylaldehyde (acrolein), methacrylaldehyde (methacrolein), 5-norbornene-2-carboxaldehyde, 7-octenal, 3-vinylbenzaldehyde, and 4-vinylbenzaldehyde. By subjecting these compounds to an acetalization reaction in the presence of an acid catalyst, ethylenically unsaturated groups can be introduced into the starting PVA. More specifically, by subjecting, for example, 5-norbornene-2-carboxaldehyde, 3-vinylbenzaldehyde, 4-vinylbenzaldehyde, or the like to an acetalization reaction, norbornenyl groups and / or vinylphenyl groups can be introduced into the starting PVA. Furthermore, it is possible to introduce a (meth)acryloylamino group into a vinyl alcohol polymer by reacting it with N-(2,2-dimethoxyethyl)(meth)acrylamide, etc. Methods for introducing an ethylenically unsaturated group into a vinyl alcohol polymer other than the above-exemplified reactions can also be used, and two or more types of reactions may be used in combination.

[0039] Other methods for introducing the ethylenically unsaturated group include reacting a reactive substituent, such as a carboxy group present in a carboxylic acid-modified PVA or an amino group present in an amino-modified PVA, with an ethylenically unsaturated group-containing compound. The carboxy group of a 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. The amino group of an amino-modified PVA can be introduced with an acryloylamino group by amidation reaction with, for example, acrylic anhydride in the presence of a base, or with a vinyloxycarbonyl group by amidation reaction with, for example, divinyl adipate. Methods for introducing an ethylenically unsaturated group via a copolymerized modified PVA can be other than the reactions exemplified above, and two or more reactions may be used in combination.

[0040] From the viewpoint of ease of production, the vinyl alcohol polymer having an ethylenically unsaturated group is preferably a vinyl alcohol polymer in which an ethylenically unsaturated group has been introduced via a hydroxyl group in a side chain of the raw material PVA, such as a 1,3-diol group. More preferred are vinyl alcohol polymers obtained by esterifying or transesterifying the hydroxyl group in the side chain of the raw material PVA with (meth)acrylic acid or a derivative thereof, and vinyl alcohol polymers obtained by acetalizing the 1,3-diol group of a vinyl alcohol polymer with a compound containing an ethylenically unsaturated group and an aldehyde group in the molecule.

[0041] <Introduction rate of ethylenically unsaturated groups> The introduction rate of the ethylenically unsaturated groups contained in the vinyl alcohol polymer before crosslinking is preferably 10 mol% or less, more preferably 5 mol% or less, and even more preferably 3 mol% or less, based on the total structural units constituting the vinyl alcohol polymer, from the viewpoint of suppressing embrittlement of the crosslinked vinyl alcohol polymer. From the viewpoint of accelerating the crosslinking reaction, rapidly forming a crosslinked body, and improving the elastic modulus of the resulting crosslinked body, the introduction rate is preferably 0.1 mol% or more, more preferably 0.2 mol% or more, and even more preferably 0.3 mol% or more. As described above, at least a portion of the ethylenically unsaturated groups is consumed by the crosslinking reaction of the vinyl alcohol polymer having ethylenically unsaturated groups. In the present invention, the "introduction rate of ethylenically unsaturated groups" in the "crosslinked vinyl alcohol polymer having ethylenically unsaturated groups" refers to the proportion (mol%) of ethylenically unsaturated groups in the total structural units of the vinyl alcohol polymer constituting the crosslinked body before crosslinking and before conjugation.

[0042] From the viewpoint of improving the mechanical strength of the crosslinked vinyl alcohol polymer and introducing functional groups different from the hydroxyl groups of PVA, the vinyl alcohol polymer having an ethylenically unsaturated group may further contain a monomer. Examples of the monomer include acrylamides such as acrylamide, N-isopropylacrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and N,N-dimethylacrylamide; α,β-unsaturated carboxylic acids such as (meth)acrylic acid, crotonic acid, itaconic acid, maleic acid, and fumaric acid; water-soluble radically polymerizable monomers such as vinylpyridine, hydroxyethyl(meth)acrylate, styrenesulfonic acid, and polyethylene glycol mono(meth)acrylate; and crosslinkers having two or more ethylenically unsaturated groups in the molecule such as N,N'-methylenebisacrylamide, ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, and polyethylene glycol di(meth)acrylate. From the viewpoint of improving the mechanical strength of the crosslinked body, the content of the monomer is preferably 50% by mass or less, more preferably 30% by mass or less, and even more preferably 10% by mass or less, based on the vinyl alcohol polymer having an ethylenically unsaturated group.

[0043] <Crosslinking by Polymerization of Ethylenically Unsaturated Groups> A vinyl alcohol polymer having an ethylenically unsaturated group can be gelled by crosslinking the ethylenically unsaturated group introduced into the vinyl alcohol polymer using active energy rays and / or heat, thereby obtaining a crosslinked vinyl alcohol polymer of the present invention. Examples of active energy rays include gamma rays, ultraviolet rays, visible light, infrared rays (heat rays), radio waves, alpha rays, beta rays, electron beams, plasma flow, ionizing rays, and particle beams. When the vinyl alcohol polymer is crosslinked using active energy rays such as ultraviolet rays, visible light, infrared rays (heat rays), and / or heat, the uncrosslinked polymer solution described below preferably contains a radical polymerization initiator. Examples of radical polymerization initiators include photoradical polymerization initiators and thermal radical polymerization initiators.

[0044] The photoradical polymerization initiator is not particularly limited as long as it initiates radical polymerization by irradiation with active energy rays such as ultraviolet light or visible light. However, from the viewpoint of washing and removing the photoradical polymerization initiator after crosslinking, a water-soluble initiator is preferred. Specific examples include 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one (trade name "Omnirad 2959", manufactured by IGM RESINS B.V.), α-ketoglutaric acid, lithium phenyl(2,4,6-trimethylbenzoyl)phosphinate (trade name "L0290", manufactured by Tokyo Chemical Industry Co., Ltd.), and Eosin Y.

[0045] The thermal radical polymerization initiator is not particularly limited as long as it initiates radical polymerization by heat, and examples thereof include azo initiators, peroxide initiators, and the like that are commonly used in radical polymerization. From the viewpoint of improving the transparency and physical properties of the vinyl alcohol polymer, peroxide initiators that do not generate gas are preferred. From the viewpoint of washing and removing the thermal radical polymerization initiator after crosslinking as described above, those that are water-soluble are preferred. Specific examples include inorganic peroxides such as ammonium persulfate, potassium persulfate, and sodium persulfate.

[0046] Alternatively, a redox polymerization initiator may be used in combination with a reducing agent. The redox polymerization initiator can be crosslinked by the stimulus of mixing a peroxide initiator with a reducing agent. Known reducing agents can be used as the reducing agent to be combined with the redox polymerization initiator. Among these, highly water-soluble N,N,N',N'-tetramethylethylenediamine, sodium sulfite, sodium hydrogensulfite, sodium hydrosulfite, and the like are preferred.

[0047] Similarly, the azo initiator is preferably one that is water-soluble, and specific examples thereof include 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (trade name "VA-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- 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") (all manufactured by Wako Pure Chemical Industries, Ltd.), and the like.

[0048] <Polythiol> In the crosslinking step described below, when a vinyl alcohol polymer having a vinyl group as the ethylenically unsaturated group is used, from the viewpoint of promoting crosslinking, for example, a polythiol having two or more thiol groups in the molecule may be added to perform crosslinking using a thiol-ene reaction. The polythiol is preferably water-soluble, and examples thereof include polythiols having a hydroxyl group such as dithiothreitol; and polythiols containing an ether bond, such as terminal thiolated products such as 3,6-dioxa-1,8-octanedithiol, polyethylene glycol dithiol, and multi-arm polyethylene glycol.

[0049] As described above, an optimum method for crosslinking a vinyl alcohol polymer by a covalent bond can be selected as needed. However, from the viewpoints of the toxicity of the unreacted crosslinking agent and condensing agent contained in the crosslinked product and ease of molding, a method in which functional groups are introduced into the vinyl alcohol polymer by post-modification and then reacted, particularly a method in which an ethylenically unsaturated group is introduced into the side chain of the vinyl alcohol polymer, is preferred.

[0050] [Shape of the crosslinked water-soluble polymer] Examples of the molded product of the crosslinked water-soluble polymer of the present invention include general amorphous particles, spherical particles, films, threads, hollow fibers, porous monoliths, etc. Furthermore, the molded product may be in the form of a micromolded product, an arbitrarily shaped article molded using a 3D printer, or an article coated with the crosslinked water-soluble polymer (coated article). The micromolded product is a molded product having fine irregularities on the surface and / or inside, and the size of the microfabrication is 10 to 1000 μm. Furthermore, an arbitrary shape molded using a 3D printer is, for example, an arbitrary shape that can be molded using a stereolithography, inkjet, or nozzle extrusion 3D printer. The term "coated article" refers to a substrate having a shape such as a film, tray, petri dish, well plate, thread, hollow fiber, porous monolith, micromolded body, or arbitrarily shaped article formed by a 3D printer, on which a composite crosslinked body is coated. The substrate material can be freely selected from glass, polyolefin, polymethyl methacrylate, polystyrene, polyester, polyolefin, polyethylene vinyl alcohol copolymer, polyamide, polyimide, and other materials.

[0051] [Conjugation of Cell Adhesion Factors] The cell adhesion factor is not particularly limited as long as it is a molecule that has the property of adhering cells, and examples thereof include cell adhesion proteins such as gelatin, collagen, laminin, fibronectin, vitronectin, nidogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, fibrin, elastin, netrin, entactin, proteoglycan, and retronectin; synthetic peptides containing a cell adhesion sequence such as RGD, REDV, YIGSR, and IKVAV peptide; acidic polysaccharides including glycosaminoglycans such as heparin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; polycations such as α-polylysine, ε-polylysine, α-polyarginine, and chitosan; antibodies such as anti-integrin antibody, anti-VCAM1 antibody, anti-E-selectin antibody, anti-CD3 antibody, and anti-CD28 antibody; cell growth factors and differentiation factors described below; and cytokines described below. The cell adhesion protein may be naturally occurring or recombinant, or may be a synthetic peptide chemically synthesized by solid-phase methods or the like. When a cell adhesion protein is used as the cell adhesion factor, the molecular weight of the cell adhesion protein is not particularly limited, but preferably has a weight-average molecular weight of 5,000 to 1,000,000, and more preferably has a weight-average molecular weight of 6,000 to 800,000. When a synthetic peptide is used as the cell adhesion factor, the molecular weight of the synthetic peptide is not particularly limited, but preferably has a weight-average molecular weight of 200 to 10,000, and more preferably has a weight-average molecular weight of 250 to 7,000. When a cell growth factor / differentiation factor is used as the cell adhesion factor, the molecular weight of the growth factor is not particularly limited, but preferably has a weight-average molecular weight of 3,000 to 200,000, and more preferably has a weight-average molecular weight of 5,000 to 100,000. When an acidic polysaccharide is used as the cell adhesion factor, the molecular weight of the acidic polysaccharide is not particularly limited, but the weight-average molecular weight is preferably 3,000 to 2,000,000, and more preferably 4,000 to 1,200,000.When a polycation is used as the cell adhesion factor, the molecular weight of the polycation is not particularly limited, but the weight-average molecular weight is preferably 2,000 to 1,000,000, and more preferably 4,000 to 500,000.

[0052] The cell adhesion factor preferably has a functional group capable of binding to the crosslinked water-soluble polymer, such as a primary or secondary amino group, a carboxy group, or a hydroxyl group, preferably a primary amino group or a carboxy group, and more preferably a primary amino group.

[0053] The cell adhesion factor may be complexed to the surface, the interior, or both of the crosslinked water-soluble polymer. For example, when a crosslinked water-soluble polymer is produced and then a cell adhesion factor is added to the crosslinked water-soluble polymer for complexation, the mesh structure of the crosslinked water-soluble polymer is so dense that it usually cannot penetrate to the interior, and the cell adhesion factor is often complexed only on the surface. This structure can vary depending on the crosslinking density of the crosslinked water-soluble polymer and the molecular weight of the cell adhesion factor. Alternatively, the cell adhesion factor can be complexed to the interior and the surface by mixing the cell adhesion factor with the water-soluble polymer in advance and crosslinking, and the optimal method can be selected depending on the purpose.

[0054] [Conjugate] In one embodiment, the present invention provides a conjugate of a cell adhesion factor and a crosslinked water-soluble polymer. In a preferred embodiment, the water-soluble polymer is a vinyl alcohol polymer, etc. Accordingly, in a preferred embodiment, the present invention provides a conjugate of a cell adhesion factor and a crosslinked vinyl alcohol polymer. In the present invention, the cell adhesion factor and the crosslinked water-soluble polymer may be conjugated by a covalent bond or by a non-covalent bond (e.g., by intermolecular forces such as hydrogen bonds or van der Waals forces). In a more specific embodiment, the crosslinked vinyl alcohol polymer of the present invention may simply contain the cell adhesion factor, or may be a complex in which the crosslinked vinyl alcohol polymer and the cell adhesion factor are covalently bonded. However, a crosslinked vinyl alcohol polymer covalently bonded is preferred. By forming a covalent bond between the crosslinked vinyl alcohol polymer and the cell adhesion factor, the cell adhesion factor can be retained in the crosslinked vinyl alcohol polymer, allowing it to function stably. Methods for covalently bonding a crosslinked vinyl alcohol polymer to a cell adhesion factor include, for example, activating the hydroxyl groups of PVA and reacting them with the functional groups of the cell adhesion factor to form a covalent bond, or using carboxylic acid-modified PVA and / or amino-modified PVA to react with the functional groups of the cell adhesion factor to form a covalent bond. From the viewpoint of reaction efficiency, it is preferable to use at least one functional group selected from the group consisting of an amino group, a carboxylic acid, and a thiol group as the functional group of the vinyl alcohol polymer, and carboxylic acid is more preferable.

[0055] Specific methods for activating the hydroxyl groups of PVA to introduce cell adhesion factors include methods using hydroxyl group activating reagents such as 1,1'-carbonyldiimidazole, di(N-succimidyl)carbonate, p-toluenesulfonyl chloride, 2,2,2-trifluoroethanesulfonyl chloride, and cyanuric acid chloride. Reacting these hydroxyl group activating reagents with the hydroxyl groups of PVA enables the formation of covalent bonds with functional groups such as primary or secondary amino groups, carboxyl groups, and hydroxyl groups of the cell adhesion factor. Other methods include acid anhydride reagents such as succinic anhydride, which can introduce carboxylic acids via ester linkages to the hydroxyl groups of the vinyl alcohol polymer; and acetal reagents such as 2,2-dimethoxyethylamine, which can introduce amino groups via acetal linkages to the 1,3-diol groups of the vinyl alcohol polymer. These carboxylic acids and amino groups can form amide bonds with the amino groups and carboxylic acids of the cell adhesion factor using the carbodiimide condensing agents, etc. For example, when a crosslinked vinyl alcohol polymer is reacted with an acid anhydride reagent such as succinic anhydride, a carboxy group (COOH) derived from succinic acid is introduced at the end, and by condensing this carboxy group with the amino group of the cell adhesion factor using a condensing agent such as 1,1'-carbonyldiimidazole, dicyclohexylcarbodiimide, or water-soluble carbodiimide, the cell adhesion factor can be conjugated to the crosslinked vinyl alcohol polymer via an amide bond.

[0056] A specific method for introducing a cell adhesion factor into carboxylic acid-modified PVA and / or amino-modified PVA is to form an amide bond using the amino group of the cell adhesion factor, carboxylic acid, and the carbodiimide condensing agent, etc., as described above. Although the above examples are possible, other methods, including other methods, can be used as the optimum method depending on the application.

[0057] When a crosslinked vinyl alcohol polymer and a cell adhesion factor are covalently bonded, the vinyl alcohol polymer preferably has a carboxy group. The introduction rate of the carboxy group contained in the vinyl alcohol polymer before complexation is preferably 50 mol% or less, more preferably 30 mol% or less, and even more preferably 15 mol% or less, based on the total structural units constituting the vinyl alcohol polymer, from the viewpoint of preventing the swelling degree of the crosslinked vinyl alcohol polymer from becoming too large and maintaining high gel strength. Furthermore, from the viewpoint of maintaining a high complexation density of the cell adhesion factor and maintaining good cell adhesiveness, as described below, the introduction rate is preferably 0.1 mol% or more, more preferably 0.5 mol% or more, and even more preferably 1.0 mol% or more.

[0058] In the present invention, in an embodiment in which the crosslinked water-soluble polymer complexed with a cell adhesion factor is a crosslinked vinyl alcohol polymer having a carboxy group, the term "crosslinked vinyl alcohol polymer having a carboxy group" refers to a crosslinked vinyl alcohol polymer having a carboxy group. In such an embodiment, at least a portion of the carboxy groups may be consumed by the complexation reaction between the crosslinked polymer and the cell adhesion factor. Therefore, the term "crosslinked vinyl alcohol polymer having a carboxy group" may also include a vinyl alcohol polymer before crosslinking and complexation in which a portion of the carboxy groups has been consumed by the complexation reaction, but in which some of the carboxy groups remain in the crosslinked polymer and complex. Therefore, in the present invention, the "introduction rate of carboxy groups" in the "crosslinked vinyl alcohol polymer having a carboxy group" refers to the proportion (mol %) of carboxy groups in all structural units of the vinyl alcohol polymer constituting the crosslinked polymer before crosslinking and complexation.

[0059] The complex may be formed after the crosslinking step of the uncrosslinked polymer solution, as described below, or may be conjugated at the stage of the uncrosslinked polymer solution. The conjugation of the cell adhesion factor is preferably carried out after the crosslinking step to avoid a decrease or loss of activity of the cell adhesion factor due to crosslinking.

[0060] <Method for producing a crosslinked vinyl alcohol polymer> There is no particular limitation on the method for producing a crosslinked vinyl alcohol polymer of the present invention, but it is preferable to produce the crosslinked vinyl alcohol polymer by first going through a step of preparing an uncrosslinked polymer solution containing the vinyl alcohol polymer (uncrosslinked polymer solution preparation step), then a step of molding the uncrosslinked polymer solution (molding step), and then a step of crosslinking the vinyl alcohol polymer contained in the uncrosslinked polymer solution to form a gel (crosslinking step). A specific method will be described below.

[0061] (Uncrosslinked Polymer Solution Preparation Step) 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. Examples of the water-soluble organic solvent include aprotic polar solvents such as dimethylformamide, dimethylacetamide, dimethyl sulfoxide, and N-methylpyrrolidone; monoalcohols such as methanol, ethanol, propanol, and isopropanol; and polyhydric alcohols such as ethylene glycol, diethylene glycol, triethylene glycol, and glycerin. The water-soluble organic solvent may be used alone or in combination of two or more, or a mixture of the water-soluble organic solvent and water may be used.

[0062] When the uncrosslinked polymer solution contains the water-soluble organic solvent, the content thereof 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, and even more preferably 60% by mass or more, and is preferably 99.999% by mass or less, more preferably 99.99% by mass or less, and even more preferably 99.9% by mass or less.

[0063] 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 increase in viscosity of the uncrosslinked polymer solution and obtaining good moldability, the content is preferably 50% by mass or less, more preferably 45% by mass or less, and even more preferably 40% by mass or less. From the viewpoint of increasing the strength and film thickness of the obtained gel and suppressing the viscosity of the uncrosslinked polymer solution to a level that makes it easy to mold, the content of the vinyl alcohol polymer is preferably within the above range.

[0064] (Molding step) In the production of the crosslinked vinyl alcohol polymer of the present invention, the method for molding the uncrosslinked polymer solution is not particularly limited, and the solution can be molded into the shape of the amorphous particles, spherical particles, fine molded bodies, arbitrarily shaped articles molded with a 3D printer, films, threads, hollow fibers, porous monoliths, coated articles, and the like, using known methods.

[0065] (Crosslinking Step) The crosslinked vinyl alcohol polymer of the present invention is preferably produced by a crosslinking step of crosslinking the vinyl alcohol polymer after the molding step. The crosslinking in this step can be carried out by the method described above. The crosslinking step may be carried out while the uncrosslinked polymer solution still contains a solvent, or may be carried out after removing the solvent from the uncrosslinked polymer solution. In the crosslinking step, a gel crosslinked while the uncrosslinked polymer solution still contains a solvent may shrink significantly in the drying step described below, making it difficult to form shapes such as films, threads, hollow fibers, and coatings. In this case, it is preferable to dry the uncrosslinked polymer solution once after molding and then crosslink it. The degree of drying of the uncrosslinked polymer solution can be selected according to the molded product and / or crosslinking method, but the solvent content is preferably 50% or less, more preferably 25% or less, and even more preferably 10% or less. From the viewpoint of suppressing shrinkage in the drying step described below, it is preferable to set the solvent content to the above upper limit or less.

[0066] Before molding, 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. As described above, if the uncrosslinked polymer solution is dried after molding, the components necessary for crosslinking may be added after molding. As described above, optimal crosslinking conditions are selected depending on the vinyl alcohol polymer and / or crosslinking method used. However, in order to reduce the influence of the crosslinked vinyl alcohol polymer on molding after crosslinking is completed, it is preferable to mold before the crosslinked vinyl alcohol polymer is formed, rather than after crosslinking is completed and the crosslinked vinyl alcohol polymer is formed. Among the above-mentioned crosslinking methods, some involve simply mixing the uncrosslinked polymer solution with the components necessary for crosslinking, which initiates the reaction at temperatures below room temperature. Therefore, it is desirable to mix them immediately before molding. Furthermore, in the case of crosslinking that requires heat, the temperature of the crosslinking reaction is preferably 100°C or lower, more preferably 60°C or lower, and even more preferably 37°C or lower, from the viewpoint of maintaining the activity of the cell adhesion factor.

[0067] (Conjugation Step) The conjugation step in the present invention is a step of conjugating a cell adhesion factor to a crosslinked vinyl alcohol polymer to obtain a crosslinked conjugated vinyl alcohol polymer. Therefore, as already explained, if conjugation is performed at the uncrosslinked polymer solution stage, the conjugation step is basically unnecessary. When a carboxylic acid-modified vinyl alcohol copolymer and / or an amino-modified vinyl alcohol polymer is used as the vinyl alcohol polymer, the cell adhesion factor can be introduced to the remaining functional groups by the above-mentioned conjugation method. Alternatively, it is also possible to first introduce functional groups for conjugation to hydroxyl groups, etc. contained in the crosslinked vinyl alcohol polymer after the crosslinking step, and then perform the conjugation step. As a method for introducing functional groups, hydroxyl group activating reagents, acid anhydride reagents, acetal reagents, etc. may be used as described above, and the cell adhesion factor can also be introduced to the functional groups by the above-mentioned conjugation method.

[0068] The crosslinked water-soluble polymer complexed with a cell adhesion factor in the method of the present invention is used for cell culture applications, and therefore is preferably sterilized by, for example, autoclave sterilization, ethylene oxide gas sterilization, low-temperature hydrogen peroxide plasma sterilization, dry heat sterilization, chemical sterilization using glutaraldehyde or the like, radiation sterilization using gamma rays or electron beams, etc. Among these, ethylene oxide gas sterilization, low-temperature hydrogen peroxide plasma sterilization, and radiation sterilization are preferred from the viewpoint of easily maintaining the activity of the complexed cell adhesion factor, and radiation sterilization is more preferred because it leaves no residue.

[0069] The density of the cell adhesion factor to be complexed is not particularly limited. For example, when the cell adhesion factor is complexed on the surface of a crosslinked water-soluble polymer, the density of the cell adhesion factor to be complexed per unit surface area is 0.005 to 30 μg / cm. ２ is preferred, and 0.01 to 10 μg / cm ２ When the cell adhesion factor is complexed inside or inside and on the surface of a crosslinked water-soluble polymer, the ratio of the water-soluble polymer to the cell adhesion factor is not particularly limited, but for example, 50 to 10,000 parts by weight of the water-soluble polymer per 100 parts by weight of the cell adhesion factor is preferred, and 100 to 5,000 parts by weight is more preferred.

[0070] [Cells] In the method of the present invention, cells may be attached to the surface of a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor, or the cells may be encapsulated. The term "cells" in this specification is not particularly limited, but preferably includes pluripotent stem cells, tissue stem cells, somatic cells, mammalian cell lines used for the production of useful substances such as pharmaceuticals, treatments, etc., and insect cells.

[0071] The cells include adherent cells and suspension cells. Adherent cells are cells that grow by adhering to a scaffold, such as a crosslinked water-soluble polymer complexed with the cell adhesion factor of the present invention, during cell culture. Suspension cells are cells that do not essentially require attachment to a scaffold for cell growth. Suspension cells include cells that can weakly adhere to a carrier.

[0072] The pluripotent stem cells are stem cells that have the ability to differentiate into cells of any tissue (pluripotency), and include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ stem cells (EG cells), and germ stem cells (GS cells).

[0073] The above-mentioned tissue stem cells refer to stem cells that are limited in the tissues they differentiate into but have the ability to differentiate into various cell types (pluripotency). Examples of tissue stem cells include bone marrow undifferentiated mesenchymal stem cells, skeletal muscle stem cells, hematopoietic stem cells, neural stem cells, hepatic stem cells, adipose tissue stem cells, epidermal stem cells, intestinal stem cells, spermatogonial stem cells, pancreatic stem cells (pancreatic duct epithelial stem cells, etc.), leukocyte stem cells, lymphocyte stem cells, and corneal stem cells.

[0074] The somatic cells refer to cells that constitute a multicellular organism, and examples thereof include, but are not limited to, osteoblasts, chondrocytes, hematopoietic cells, epithelial cells (such as mammary epithelial cells), endothelial cells (such as vascular endothelial cells), epidermal cells, fibroblasts, mesenchymal-derived cells, cardiac muscle cells, myoblasts, smooth muscle cells, skeletal muscle cells derived from living organisms, human tumor cells, fibrocytes, EB virus mutant cells, hepatocytes, kidney cells, bone marrow cells, macrophages, hepatic parenchymal cells, small intestinal cells, mammary gland cells, salivary gland cells, thyroid cells, skin cells, plasma cells, T cells, B cells, killer cells, lymphoblasts, and pancreatic β cells.

[0075] The above mammalian-derived 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, 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. Examples include 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, ψ2 cells, and ψCRE cells.

[0076] Examples of the insect cells include silkworm cells (BmN cells, BoMo cells, etc.), mulberry cells, Anemone cells, Anemone cells, Mamestra frugiperda cells (Sf9 cells, Sf21 cells, etc.), mulberry fly cells, leafroller cells, Drosophila cells, Boettcheris caudatus cells, Aedes albopictus cells, swallowtail butterfly cells, American cockroach cells, and nettle looper cells (Tn-5 cells, HIGH FIVE cells, MG1 cells, etc.).

[0077] The cells may be aggregated together or differentiated. The aggregated cells may have the function of an organ. The cells may be immediately collected from a living body or may be cultured. The cells collected from a living body may have formed an organ.

[0078] [Culturing Method] In the method of the present invention, cells are cultured by adhering to a crosslinked water-soluble polymer complexed with a cell adhesion factor. In a typical embodiment of the present invention, cells are cultured by adhering to a crosslinked water-soluble polymer complexed with a cell adhesion factor, or by encapsulating cells in a crosslinked water-soluble polymer complexed with a cell adhesion factor. In the method of the present invention, the serum-free medium is used for the culture. In a typical embodiment, a method is used in which a crosslinked water-soluble polymer complexed with a cell adhesion factor is formed into particles, and the particles are used as carriers to adhere the particles and cells in a medium. In this embodiment, a crosslinked water-soluble polymer complexed with a cell adhesion factor may be attached to a core material (e.g., polystyrene particles, polymethyl (meth)acrylate particles, polylactic acid particles, dextran particles, agarose particles, silica particles, etc.) to form particles, and the particles and cells are used as carriers to adhere the particles and cells in a medium. In another preferred embodiment, a method is used in which a crosslinked water-soluble polymer complexed with a cell adhesion factor is formed into a film, and the film is used as a carrier to adhere the film and cells in a medium. In this embodiment, a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor may be attached to a core material (e.g., 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.) to form a film, and cells may be attached to the film as a carrier in a culture medium. In another embodiment, the inner surface and / or bottom surface of a culture vessel may be coated with a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor. In this embodiment, a serum-free medium suspension of cells may be placed in the culture vessel coated with the crosslinked body, and the cells may be encapsulated in the crosslinked body. In this embodiment, a serum-free medium is placed in the culture vessel, and the cells are cultured in the medium.The culture conditions, such as the culture temperature, can be broadly those generally used in conventional cell culture, for example, 37°C, 5% CO. ２ Examples of suitable environments include those described above. If necessary, cells grown in serum-free medium are recovered and / or passaged using conventional methods. For example, trypsin treatment or the like can detach the cell adhesion factor attached to the cells from the crosslinked water-soluble polymer complex, allowing the cells to be recovered in a suspended state. The cells thus obtained can be seeded on a water-soluble polymer complexed with a cell adhesion factor together with fresh serum-free medium and passaged.

[0079] According to the method of the present invention, cells can be cultured and grown in a basal medium alone, without the addition of serum or serum substitutes, during the culturing step. However, as long as the method of the present invention includes a step of culturing cells using a serum-free medium, it may also include a step of adding a specific target protein other than cell culture before or after the culturing step. For example, cell growth factors / differentiation factors, cytokines, hormones, polyanions, polyvinyl alcohol, transferrin, etc. can be added to cells cultured and grown in such serum-free medium for the purpose of signal transduction analysis, differentiation, etc. In addition, in the method of the present invention, cells may have difficulty adhering to a water-soluble polymer gel complexed with a cell adhesion factor at the initial stage of culture. Therefore, prior to the culturing step, a step of adhering cells to a crosslinked water-soluble polymer complexed with a cell adhesion factor may be carried out in a medium supplemented with serum, serum substitutes, cell growth factors / differentiation factors, cytokines, hormones, polyanions, polyvinyl alcohol, transferrin, etc., in order to promote cell adhesion. Furthermore, the method of the present invention also encompasses embodiments in which the cultured cells produce proteins as a result of culturing cells using a serum-free medium.

[0080] <Growth Factors and Differentiation Factors> Examples of growth factors and differentiation factors that may be added to the serum-free medium of the present invention include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), 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 line-derived neurotrophic factor (GLN). Examples of such factors include growth factor (GDNF), nerve growth factor (NGF), leukemia inhibitory factor (LIF), stem cell factor (SCF), bone morphogenetic protein (BMP), interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), notch ligands (Delta-1, Delta-3, Delta-4, Jagged-1, Jagged-2), and the like.

[0081] <Cytokines> Examples of cytokines that may be added to the serum-free medium of the present invention include interleukin-1α, interleukin-1β, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-15, interleukin-16, interleukin-17, interleukin-18, interferon α, interferon β, interferon γ, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), MCP-1, erythropoietin (EPO), thrombopoietin (TPO), Flk-2 / Flt-3 ligand (FL), and the like.

[0082] <Hormones> Examples of hormones that may be added to the serum-free medium of the present invention include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Müllerian 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 placental lactogen, growth hormone, inhibin, insulin, leptin, luteinizing hormone, and the like. Examples of hormones that may be involved in the production of steroid hormones include steroid hormones, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

[0083] <Polyanions> Examples of polyanions that may be added to the serum-free medium of the present invention include heparin, dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin sulfate, etc. These polyanions stabilize the cell growth factors and differentiation factors by binding to them, thereby more efficiently promoting cell proliferation.

[0084] <Polyvinyl Alcohol> The polyvinyl alcohol (PVA) that may be added to the serum-free medium of the present invention can be freely selected from the vinyl alcohol polymers or modified PVA described above. When culturing cells, bovine serum albumin (BSA), human recombinant albumin, etc. are often added to the medium for purposes such as cell protection and growth factor stabilization. The use of PVA allows for the creation of a serum-free culture environment that excludes proteins such as BSA. The content of PVA in the serum-free medium is preferably 0.01 to 0.5% by mass, more preferably 0.05 to 0.2% by mass.

[0085] A cell culture can be obtained by culturing cells using the method of the present invention. Furthermore, substances such as proteins can be produced by utilizing the metabolism of the cultured cells. Thus, in one embodiment, the present invention provides a method for producing a target substance, comprising 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. The method of the present invention can be widely applied to substance production, etc., performed in the field of biotechnology, to which the present invention pertains. Therefore, the target substance can include, but is not limited to, the cell culture itself, substances produced by the cells, etc. Examples of cell cultures include cultured cells, cell masses, tissues, etc. Furthermore, examples of substances produced by the cells include physiologically active substances such as antibodies, viral vaccines, viral vectors, hormones, cytokines, and enzymes. The method of the present invention may also include a step of recovering the target substance obtained by the above-mentioned culturing step. For the recovery step, methods commonly used in the field to which the present invention pertains can be widely used.

[0086] [Protein Production by Transfection] A method for producing a protein using cells will be described below as an example of a method for producing a target substance of the present invention. Specifically, nucleic acids (DNA or RNA) can be artificially introduced (transfected) into the cells cultured and grown in a serum-free medium by the method of the present invention. The introduction of nucleic acids into the cells may be by transient transfection or stable transfection. A plasmid vector and / or messenger RNA encoding a protein to be expressed, such as an antibody, adenovirus, adeno-associated virus (AAV), or lentivirus, can be introduced into the cells by, for example, the retrovirus method, the liposome method, the cationic liposome method, or the adenovirus method.

[0087] The method of the present invention can be used as a production system for producing and / or expressing a protein in a serum-free medium. Cells into which DNA and / or messenger RNA have been introduced by the above method can be similarly cultured in a serum-free medium to obtain the protein. The protein thus obtained can be isolated from the inside or outside of the cells (such as the medium) and purified as a substantially pure and homogeneous protein. Protein separation and purification can be performed using separation and purification methods commonly used in protein purification, and are not limited in any way.

[0088] For example, proteins can be separated and purified by appropriately selecting and combining methods such as chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. When conventional serum and / or serum substitutes are used, the purification process requires a great deal of effort due to the presence of large amounts of contaminating proteins, but the method of the present invention uses a serum-free medium, particularly a protein-free medium, which simplifies the purification of expressed proteins.

[0089] The method of the present invention allows cells to be cultured (survive and proliferate) in serum-free medium, more specifically, protein-free medium, which naturally eliminates the risk of infection from the medium. It also provides inexpensive cells with stable quality without variations in protein between lots, and useful proteins produced by the cells can be easily purified to high purity. Furthermore, because the medium does not contain proteins, it can provide a highly reproducible evaluation method for elucidating the molecular mechanisms underlying the maintenance of undifferentiated state and differentiation of stem cells, which has previously been impossible due to the influence of serum-derived protein impurities contained in the medium. The method of the present invention can contribute not only to regenerative medicine but also to the mass production industry of cells, including cultivated meat.

[0090] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0091] [Raw Materials Used] The main components used in the Synthesis Examples, Examples, and Comparative Examples are listed below. <Raw Material PVA> PVA117: Polyvinyl alcohol (trade name "PVA117", degree of polymerization 1700, degree of saponification approximately 98.0 to 99.0 mol%, viscosity (4%, 20°C) 25.0 to 31.0 mPa s, manufactured by Kuraray Co., Ltd.) The degree of polymerization of the raw material PVA was measured in accordance with JIS K 6726:1994.

[0092] <Ethylenically unsaturated group-containing compound> Vinyl methacrylate: manufactured by Tokyo Chemical Industry Co., Ltd.

[0093] <Synthetic reagents> Triethylamine: Fujifilm Wako Pure Chemical Industries, Ltd.

[0094] <Radical polymerization initiator> Omnirad 2959: 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (photoradical polymerization initiator, trade name "Omnirad 2959", manufactured by IGM Resins B.V.)

[0095] <Hydroxyl group activation reagent> Succinic anhydride: manufactured by Tokyo Chemical Industry Co., Ltd.

[0096] <Complexing reagents> 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. N-hydroxysuccinimide: manufactured by Tokyo Chemical Industry Co., Ltd.

[0097] <Cell adhesion factors> Gelatin (porcine-derived, type A): manufactured by Sigma-Aldrich Japan Co., Ltd. Collagen (product name "Cell Matrix", type IC): manufactured by Nitta Gelatin Co., Ltd.

[0098] <Cell culture surface treated well plate (made of polystyrene)> ・Violamo cell culture plate VTC-P6 (6 wells): AS ONE Corporation <Collagen coated well plate (made of polystyrene)> ・Pegsin solubilized type I collagen coated microplate (6 wells) derived from porcine tendon: Asahi Technoglass Corporation

[0099] <Solvent> Ion-exchanged water: Electrical conductivity 0.08 x 10 －４ Ion-exchanged water of S / m or less PBS: Prepared by dissolving PBS tablets (manufactured by Takara Bio Inc.) in a specified amount of ion-exchanged water. MES buffer: Prepared a 0.1 mol / L aqueous solution of 2-morpholinoethanesulfonic acid monohydrate (manufactured by Dojindo Laboratories Ltd.), and neutralized with a 0.1 mol / L aqueous NaOH solution to adjust the pH to 5.6.

[0100] <Cells> ・293T cells: cell line established by the inventors ・Mouse ES cells: cell line established by the inventors ・Mouse embryonic fibroblasts (MEF): cell line established by the inventors

[0101] <Culture media> Dulbecco's modified Eagle's medium (DMEM, high glucose, HEPES): manufactured by Thermo Fisher Scientific Co., Ltd. "StemSure" D-MEM (high glucose) (containing phenol red and sodium pyruvate): manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. DMEM / F-12, GlutaMAX supplement: manufactured by Thermo Fisher Scientific Co., Ltd. Neurobasal Medium: manufactured by Thermo Fisher Scientific Co., Ltd.

[0102] <FBS and serum substitutes> Fetal bovine serum (FBS): manufactured by Thermo Fisher Scientific Co., Ltd. "StemSure" Serum Replacement: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. Albumin, bovine serum-derived, fraction V: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. N-2 Supplement: manufactured by Thermo Fisher Scientific Co., Ltd. B-27 Supplement: manufactured by Thermo Fisher Scientific Co., Ltd.

[0103] <Culture additives> L-Glutamine: manufactured by Thermo Fisher Scientific Co., Ltd. Non-essential amino acid solution: manufactured by Thermo Fisher Scientific Co., Ltd. 2-mercaptoethanol: manufactured by Thermo Fisher Scientific Co., Ltd. "StemSure" LIF, mouse, recombinant, solution: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. PD0325901: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd. CHIR99021: manufactured by Sigma-Aldrich Co., Ltd.

[0104] <Antibiotics> Penicillin-streptomycin (5,000 U / mL): manufactured by Thermo Fisher Scientific Co., Ltd.

[0105] <Cell recovery reagents> Trypsin / EDTA solution (concentration: 1x): manufactured by Thermo Fisher Scientific Co., Ltd.

[0106] <Transfection Reagent> Polyethyleneimine MAX: manufactured by Polysciences, Inc. Green fluorescent protein (GFP) expression vector: prepared by incorporating GFP into pcDNA 3.3-TOPO TA mammalian expression vector manufactured by Thermo Fisher Scientific, Inc.

[0107] [Methods for measuring compounds synthesized in synthesis examples] <Introduction rate of ethylenically unsaturated group and carboxy group> The introduction rate of ethylenically unsaturated group and carboxy group of the vinyl alcohol polymer having an ethylenically unsaturated group obtained in the synthesis examples below was １The introduction rate was measured by H-NMR. The introduction rate was determined from the ratio of the integrated values ​​of the signals of the ethylenically unsaturated group and methylene succinate group to the integrated value of the signal of the vinyl alcohol polymer. １ H-NMR measurement conditions Equipment: Nuclear magnetic resonance apparatus “JNM-ECX400” manufactured by JEOL Ltd. Temperature: 25°C

[0108] Synthesis Examples Synthesis of Vinyl Alcohol-Based Polymer Having Ethylenically Unsaturated Groups Synthesis Example 1 40 g (monomer repeating unit: 908 mmol) of PVA117 (raw material PVA; saponified polyvinyl acetate, average degree of polymerization: 1700, degree of saponification: approximately 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 separable flask equipped with a 1 L Dimroth condenser, 350 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the raw material PVA had dissolved, 2.1 g (18.7 mmol) of vinyl methacrylate was added while heating and stirring at 80°C, and stirring was continued for an additional 3 hours at 80°C. After allowing to cool, the reaction solution was poured into 2 L of methanol while stirring. Stirring was stopped and the mixture was left standing for 1 hour. The resulting solid was collected and then further immersed in 1 L of methanol for 1 hour to wash. This washing procedure was repeated a total of three times. The collected solid was vacuum dried overnight at room temperature to obtain methacryloylated PVA117. The introduction rate of ethylenically unsaturated groups (methacryloyl groups) in the methacryloylated PVA117 was 2.0 mol % relative to the repeating units of the raw material PVA (hereinafter referred to as "MA-PVA117(2.0)").

[0109] Synthesis of a vinyl alcohol polymer having a carboxy group Synthesis Example 2 10 g (monomer repeating unit: 220 mmol) of MA-PVA117(2.0) prepared in Synthesis Example 1 was placed in a separable flask equipped with a 0.5 L Dimroth condenser, 90 mL of dimethyl sulfoxide (DMSO) was added, and stirring was initiated with a mechanical stirrer. The temperature was raised to 80°C in a water bath, and stirring was continued at 80°C for 4 hours. After visually confirming that the starting PVA had dissolved, the water bath was set to 60°C. After confirming that the internal temperature had reached 60°C, 1.2 g (12.1 mmol) of triethylamine and 1.1 g (11 mmol) of succinic anhydride were added, and stirring was continued for an additional 5 hours at 60°C. After allowing to cool, the reaction solution was poured into 0.5 L of methanol with stirring. Stirring was stopped and the mixture was left to stand for 1 hour. The resulting solid was collected and then immersed in 0.5 L of methanol for 1 hour to wash. This washing process was repeated three times. The collected solid was dried overnight under vacuum at room temperature to obtain methacryloylated PVA117 containing succinic acid. The succinic acid (SA) introduction rate was 3.4 mol% relative to the repeating units of MA-PVA117(2.0) (hereinafter referred to as "MA-PVA117(2.0)-SA(3.4)").

[0110] Synthesis Example 3 A methacryloylated PVA117 into which succinic acid had been introduced was obtained in the same manner as in Synthesis Example 2, except that 0.32 g (3.2 mmol) of triethylamine and 0.29 g (2.9 mmol) of succinic anhydride were added. The introduction rate of succinic acid (SA) was 1.0 mol % relative to the repeating unit of MA-PVA117(2.0) (hereinafter abbreviated as "MA-PVA117(2.0)-SA(1.0)").

[0111] <Production of Molded Articles (Coatings)> [Synthesis Example A] 99.85 mL of ion-exchanged water was added to 0.15 g of MA-PVA117(2.0)-SA(3.4) and the mixture was dissolved at 80°C for 4 hours with stirring to prepare a 0.15 wt% aqueous solution. After cooling to room temperature, this 0.15 wt% MA-PVA117(2.0)-SA(3.4) aqueous solution was added at 1 mL / well to a 6-well well plate that had been treated with a cell culture surface, and the wells were coated by drying at room temperature for 72 hours or more. Thereafter, 1 mL / well of a 0.1 wt% Omnirad 2959 methanol solution was added, and the plate was allowed to stand at room temperature for 30 minutes. The mixture was then irradiated with a GS Yuasa metal halide lamp (103 mW / cm ２ ) and the irradiation energy amount was 520 mJ / cm ２ The plate was irradiated with ultraviolet (UV) rays for 1000 s. The Omnirad 2959 methanol solution was removed, and 2 mL of ion-exchanged water was added and washed for 30 minutes. This procedure was repeated twice. The washing water was removed, and the plate was dried at room temperature for 24 hours or more to obtain a crosslinked MA-PVA117(2.0)-SA(3.4) coated well plate.

[0112] Synthesis Example B A crosslinked MA-PVA117(2.0)-SA(1.0)-coated well plate was obtained in the same manner as in Synthesis Example A, except that MA-PVA117(2.0)-SA(1.0) obtained in Synthesis Example 3 was used.

[0113] <Complexation> <Collagen Complexation> [Synthesis Example A] 2 mL / well of MES buffer (pH = 5.6) was added to the crosslinked MA-PVA117(2.0)-SA(3.4)-coated well plate prepared in Synthesis Example A. The plate was left to stand at room temperature for 30 minutes, after which the MES buffer was removed. 0.51 g (4.4 mmol) of N-hydroxysuccinimide and 0.85 g (4.4 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added to 50 mL of MES buffer, mixed quickly, and added to a polystyrene well plate coated with MA-PVA117(2.0)-SA(3.4) at 1 mL / well. After incubation at room temperature for 1 hour, the wells were washed twice with 1 mL of MES buffer for 5 minutes. 1 mL of a 1 mg / mL collagen PBS solution was added to the wells. After reacting for 2 hours at room temperature, the collagen-PBS solution was removed, and the plate was washed twice with 2 mL of ion-exchanged water for 5 minutes. The washing water was removed, and the plate was dried at room temperature for at least 24 hours to obtain a collagen-complexed MA-PVA117(2.0)-SA(3.4)-coated well plate (hereafter 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 using the bicinchoninic acid (BCA) method (BCA Protein Assay Kit, manufactured by Takara Bio Inc.). The cell adhesion factor complexation density was found to be 20.6 μg / well. The well plate was sealed in an aluminum-coated bag and sterilized by gamma irradiation (25 kGy) at Koga Isotope Corporation.

[0114] Synthesis Example B: A collagen-complexed MA-PVA117(2.0)-SA(1.0)-coated well plate was obtained in the same manner as in Synthesis Example A, except that crosslinked MA-PVA117(2.0)-SA(1.0) was used (hereinafter abbreviated as "collagen-MA-PVA117(2.0)-SA(1.0)-coated plate"). The amount of collagen immobilized per well (cell adhesion factor complexation density) was also measured in the same manner as in Synthesis Example A, and the cell adhesion factor complexation density was found to be 29.3 μg / well. The well plate was sealed in an aluminum-coated bag and sterilized by gamma ray irradiation (25 kGy) at Koga Isotope Co., Ltd.

[0115] <Gelatin Complexation> [Synthesis Example C] A gelatin-complexed MA-PVA117(2.0)-SA(3.4) coated well plate was obtained in the same manner as Synthesis Example A, except that gelatin was used instead of collagen (hereinafter abbreviated as "gelatin-MA-PVA117(2.0)-SA(3.4) coated plate"). The amount of gelatin immobilized per well (cell adhesion factor complexation density) was also measured in the same manner as Synthesis Example A and was found to be 16.6 μg / well. The well plate was sealed in an aluminum-coated bag and sterilized by gamma ray irradiation (25 kGy) at Koga Isotope Co., Ltd.

[0116] <Gelatin Complexation by Non-Covalent Bonding> [Synthesis Example 4] 0.15 g of MA-PVA117(2.0)-SA(3.4) was added to 99.85 mL of ion-exchanged water and dissolved at 80°C for 4 hours with stirring to prepare a 0.15 wt% aqueous solution. After cooling to 60°C, 10 mg of gelatin was added and dissolved, and the mixture was cooled to room temperature to obtain a PVA / gelatin mixture. 1 mL of the PVA / gelatin mixture was added to a 6-well well plate with a cell culture surface treatment, and the wells were coated by drying at room temperature for 72 hours or more. Then, 1 mL of a 0.1 wt% Omnirad 2959 methanol solution was added to the well and allowed to stand at room temperature for 30 minutes. The mixture was then illuminated with a metal halide lamp (103 mW / cm) manufactured by GS Yuasa. ２ ) and the irradiation energy amount was 520 mJ / cm ２The plate was then irradiated with ultraviolet (UV) rays for 100 s. The Omnirad 2959 methanol solution was removed, and 2 mL of ion-exchanged water was added and washed for 30 minutes. This procedure was repeated twice. The washing water was removed, and the plate was dried at room temperature for at least 24 hours to obtain a crosslinked MA-PVA117(2.0)-SA(3.4) coated well plate. The amount of gelatin immobilized per well (complexation density of cell adhesion factors) was measured using the same method as in Synthesis Example B and was found to be 53.8 μg / well. The well plate was sealed in an aluminum-coated bag and sterilized by gamma ray irradiation (25 kGy) at Koga Isotope Co., Ltd.

[0117] <Preparation of 293T Cells> [Preparation Example i] 293T cells were cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin) under 5% CO ２ The proliferated 293T cells were collected by treatment with trypsin / EDTA solution, washed three times with DMEM containing no fetal bovine serum or serum substitute (hereinafter abbreviated as "DMEM basal medium"; supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin), and finally cultured at 1 × 10 ５ 293T cells were dispersed in DMEM basal medium at a concentration of 100 cells / 5 mL.

[0118] <Preparation of Mouse ES Cells> [Preparation Example ii] Mouse ES cells were maintained and adapted to feeder-free culture according to the following method: They were maintained on feeder cells prepared by treating mouse embryonic fibroblasts (MEFs) with mitomycin C in a medium containing StemSure D-MEM (high glucose) (containing phenol red and sodium pyruvate), 15% StemSure Serum Replacement, 2 mM L-Glutamine, 1% Non-essential amino acid, penicillin-streptomycin (50 U / mL penicillin and 50 μg / mL streptomycin), 0.1 mM 2-mercaptoethanol, and 103 U / mL StemSure LIF.

[0119] The mouse ES cells maintained on these feeders were seeded onto gelatin-coated culture dishes and subjected to feeder-free culture in a medium containing a 1:1 mixture of DMEM / F-12, GlutaMAX supplement, and Neurobasal Medium, to which was added 0.5% N-2 supplement, 1% B-27 supplement, 25 μg / mL albumin, penicillin-streptomycin (50 U / mL penicillin and 50 μg / mL streptomycin), 103 U / mL StemSure LIF, 1 μM PD0325901, and 3 μM CHIR99021. The cells were passaged for at least five times to allow them to become fully acclimatized to feeder-free culture. The proliferated mouse ES cells were treated with trypsin / EDTA solution to recover, washed three times with DMEM basal medium, and finally collected at 1 × 10 ５ The cells were dispersed in DMEM basal medium at a concentration of 5 cells / 5 mL.

[0120] <Cell Culture Test Using Serum-Free Medium> [Example 1] 1 x 10 293T cells dispersed in DMEM basal medium were cultured on a collagen-MA-PVA117(2.0)-SA(3.4) coated plate prepared in Synthesis Example A. ５ cells / well and incubated in 5% CO ２ The cells were cultured at 37°C for 7 days without medium change, and then the 293T cells were harvested by trypsin / EDTA treatment and the number of cells per well was counted. The results are shown in Table 1.

[0121]

[0122] Examples 2 and 3 Evaluation of serum-free culture was carried out in the same manner as in Example 1, except that collagen-MA-PVA117(2.0)-SA(1.0)-coated plates and gelatin-MA-PVA117(2.0)-SA(3.4)-coated plates prepared in Synthesis Examples B and C, respectively, were used. The results are shown in Table 1.

[0123] Example 4: 1×10 mouse ES cells dispersed in DMEM basal medium were applied to the collagen-MA-PVA117(2.0)-SA(3.4) coated plate prepared in Synthesis Example A. ５cells / well and incubated in 5% CO ２ The cells were cultured at 37° C. The cells were cultured for 7 days while the DMEM basal medium was changed once every 3 days, and the mouse ES cells were recovered by trypsin / EDTA treatment and the number of cells per well was counted.

[0124] Comparative Example 1 Using a commercially available well plate (made of polystyrene) with a cell culture surface treatment, serum-free culture was evaluated in the same manner as in Example 1. The results are shown in Table 1.

[0125] Comparative Examples 2 and 3 Using commercially available collagen-coated well plates (made of polystyrene) or the crosslinked MA-PVA117(2.0)-SA(3.4)-coated well plates obtained in Synthesis Example A (no cell adhesion factor conjugated; sterilized by gamma ray irradiation (25 kGy) at Koga Isotope Co., Ltd. as in Synthesis Examples A to C), serum-free culture was evaluated in the same manner as in Example 1. The results are shown in Table 1.

[0126] Comparative Example 4 Using a commercially available collagen-coated well plate (made of polystyrene), serum-free culture was evaluated in the same manner as in Example 4. The results are shown in Table 1.

[0127] <Transfection and Protein Expression in Serum-Free Medium> [Example 5] A sterile PEI solution (PEI concentration: 1 mg / mL) at pH 7 was prepared using Polyethyleneimine (PEI) MAX according to the manufacturer's protocol. 50 μL of the sterile PEI solution was added to 10 μg of an expression vector encoding green fluorescent protein (GFP), and the resulting solution was adjusted to 300 μL with ion-exchanged water. The mixture was left at room temperature for 15 minutes, and then 5 mL of DMEM basal medium was added. Transient transfection was performed by adding this solution containing the GFP expression vector (5 mL / well) to 293T cells cultured for 7 days in DMEM basal medium, as in Example 1. Cells transfected with the GFP expression vector were cultured for 3 days in DMEM basal medium, and GFP expression was confirmed using a fluorescence microscope. Green fluorescence was detected in the 293T cells, confirming that GFP was expressed as a protein within the cells (Figure 1).

Claims

1. A method for culturing cells, comprising the step of adhering cells to a crosslinked body of a water-soluble polymer complexed with a cell adhesion factor using a serum-free medium and culturing the cells.

2. A method for producing a target substance, comprising a step of culturing cells by the method of claim 1.

3. The method of claim 1, wherein the serum-free medium is a protein-free medium.

4. The method according to claim 1, wherein the cell adhesion factor is covalently conjugated to the crosslinked water-soluble polymer.

5. The method according to claim 1, wherein the crosslinked water-soluble polymer is a crosslinked vinyl alcohol polymer.

6. The method according to claim 5, wherein the crosslinked vinyl alcohol polymer is crosslinked by a covalent bond.

7. The method according to claim 5, wherein the crosslinked vinyl alcohol polymer is a crosslinked vinyl alcohol polymer having an ethylenically unsaturated group and a carboxy group.

8. The method according to claim 7, wherein the carboxy group of the crosslinked body and the amino group of the cell adhesion factor are covalently bonded via an amide bond.

9. The method according to claim 7, wherein 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.

10. 8. The method according to claim 7, wherein the introduction rate of the ethylenically unsaturated group is 0.1 to 10 mol % of all structural units constituting the vinyl alcohol polymer.

11. 8. The method according to claim 7, wherein the introduction rate of the carboxyl group is 0.1 to 50 mol % of all structural units constituting the vinyl alcohol polymer.

12. The method according to claim 1, wherein the crosslinked body of the water-soluble polymer is an irregular particle, a spherical particle, a micromolded body, an arbitrarily shaped article formed by a 3D printer, a film, a thread, a hollow fiber, a porous monolith, or a coated article.

13. A method for culturing cells using the serum-free medium according to claim 1, which comprises allowing a physiologically active substance to act on the cells cultured in the serum-free medium.

14. A method for culturing cells using the serum-free medium according to any one of claims 1 to 13, comprising transfecting a gene vector into cells cultured in the serum-free medium to produce a physiologically active 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 a non-covalent bond.

16. A complex of gelatin or collagen and a crosslinked water-soluble polymer for use in cell culture.

17. The complex according to claim 16, wherein when the complex contains collagen, the collagen has a weight-average molecular weight of 100,000 to 400,000.

18. A complex of a cell adhesion factor and a crosslinked water-soluble polymer for use in the method according to any one of claims 1 to 13.