JP7323182B2 - Hydrophilic modified cell culture substratum with cell adhesion - Google Patents

Description

TECHNICAL FIELD The present invention relates to a hydrophilically modified cell culture substratum that is capable of cell culture and exhibits cell adhesiveness by sustaining the effect of hydrophilization treatment applied to the surface of the molded substratum for a long period of time.

Culture media with media for research and development of pharmaceuticals and agrochemicals, microbiological testing of food and drink, culture of bacteria and cells in health checkups, cell culture in biochemical research and medical practice, tissue culture in regenerative medicine, etc. wood is used.

Molded substrates molded into various shapes, such as cell culture plates, cell culture flasks, cell culture dishes, and cell culture tubes, are used for these cultures.

In general, the materials used for molding substrates include glass, which is easy to mold and has high transparency, as well as various plastics such as polystyrene and silicone. It is formed into various shapes by molding, vacuum forming, pressure forming, calendering, and the like.

Depending on the cells in the state of proliferation, cell culture is broadly divided into two types: a suspension culture system in which single cells or small clusters of cells are floating in the medium, and an adherent culture system in which cells are adhered to a container, etc. be done.

Normally, it is difficult to culture cells in an adherent culture system with molded substrates made of glass or plastic, so the surface on which cells are seeded needs to be surface-treated to suit each material.

The surface treatment includes surface modification treatment for hydrophilizing the surface, or coating with extracellular matrix proteins, fragments produced by digestion of extracellular matrix proteins, or polypeptides such as polylysine. Cell lines such as HeLa cells, which are human cervical cancer-derived cells, and V79 cells, which are Chinese hamster lung-derived fibroblasts, can generally be cultured in a culture vessel having a hydrophilized surface.

Extracellular matrix is an insoluble substance that exists outside cells such as intercellular spaces in each tissue of animals, and is composed of various proteins such as collagen, fibronectin, and laminin. Extracellular matrix is a biological component that is produced by cells, serves as a scaffold for cells, and plays an important role in adhesion, differentiation, proliferation, migration, and maintenance of cell functions.

Among the proteins that constitute extracellular matrices, human-derived proteins are generally difficult to obtain in sufficient quality and quantity for the purpose of coating culture vessels. Therefore, usually, even for culturing human-derived cells, proteins derived from bovine, porcine, etc. are used as extracellular matrix-derived proteins for coating culture vessels. However, in cell culture for transplantation in regenerative medicine and cell culture for detailed cell function studies in vivo, it is preferable not to use proteins and polypeptides derived from heterologous animals as much as possible.

Recent cell culture technology developments such as cell transplantation in regenerative medicine, organ-on-a-chip that reproduces organ functions to replace animal experiments in the drug discovery industry, and original cell function research in the in vivo environment Therefore, techniques for culturing in an environment more similar to the organs and tissues in the animal body are becoming important.

From silicone rubber, it is possible to create culture vessels with a hardness similar to that of various animal tissues, culture vessels with high air permeability, and culture vessels with high extensibility similar to those of the myocardium and blood vessels. . In addition, silicone rubber has the durability to withstand high-pressure sterilization. It is difficult to produce culture vessels having such various hardnesses, air permeability, extensibility, and durability only from glass and polystyrene materials.

Silicone rubber is attracting attention from fields such as regenerative medicine, the drug discovery industry, and basic research for the reasons described above. It is difficult to adhere cells to culture vessels. Generally, a culture vessel made of silicone rubber is coated with a protein or polypeptide that constitutes an extracellular matrix, such as collagen, for use in cell culture in an adherent culture system.

Cell culture for cell transplantation in regenerative medicine, cell culture for organ-on-a-chip that reproduces organ function to replace animal experiments in the drug discovery industry, and detailed cell function research in vivo In the research and development of cell culture and the like, there is a demand for a technique that realizes cell culture in an environment that is closer to the organs and tissues in the animal body.

In order to impart hydrophilicity to such silicone molding substrates, the silicone molding substrate is subjected to dry treatment or wet treatment, or silicone substrate forming treatment is performed by curing a hydrophilic compound-containing silicone raw material composition. Hydrophilic groups have been imparted to the silicone surface by surface modification treatment such as vapor deposition treatment.

Among these surface modification treatments, dry treatments include corona discharge treatment, plasma treatment, ultraviolet treatment, excimer treatment, etc., and are intended to impart hydrophilicity by generating hydrophilic groups such as hydroxyl groups on the surface. . Such dry processing is relatively simple. However, when a hydrophilic silicone molding base material obtained by dry treatment is stored, preserved, or used in the air or in water, the hydrophilic groups produced on the surface of the silicone base material degrade over time. In particular, it penetrates into the interior of the silicone rubber and loses its hydrophilicity in a relatively short period of about 3 to 6 months.

Among the surface modification treatments, the wet treatment involves chemically bonding a hydrophilic coating agent (e.g., a hydrophilic silane coupling agent) to the surface functional groups of the silicone molding substrate to chemically modify and introduce them. is. In such a wet treatment, the surface functional groups on the surface of the silicone molding substrate are sparsely present, resulting in uneven bonding of the hydrophilic coating agent. However, the contact angle with water is not so high, and the contact angle with water can only be reduced to about 30°. It is difficult to develop sufficient hydrophilicity due to the fact that there are very few types of hydrophilic coating agents that can be applied to silicone rubber. Moreover, when bonding a support or the like to this surface-treated surface, it is necessary to separately perform surface treatment or chemical modification for bonding, which impedes the desired hydrophilicity.

As another wet treatment, a polymer having a betaine structure, such as poly(meth)acrylate having an amino acid structure in the side chain or (meth)acrylamide having a sulfobetaine structure in the side chain, is applied to the surface of the silicone molding substrate. There is also a surface modification treatment that introduces chemical adsorption between charged groups. However, in such a surface modification treatment, since only physical adsorption or chemical adsorption is performed on the surface, even if it is uniformly adsorbed, it will run off relatively quickly when it comes into contact with water, an acidic test solution, or an alkaline test solution. Therefore, strong hydrophilicity cannot be expressed for a long period of time.

Among the surface modification treatments, a silicone base material forming treatment by curing a hydrophilic compound-containing silicone raw material composition includes mixing a hydrophilic compound, such as a hydrophilic oil, with a liquid or millable silicone raw material and curing the resulting silicone molding. There is a material modification treatment that imparts hydrophilicity by exposing a hydrophilic additive on the surface by forming a body. As an example of such a material modification treatment, Patent Document 1 describes (a) a step of preparing a master having a predetermined microstructure on the upper surface, and (b) a PDMS prepolymer on the microstructure-formed surface of the master. (c) oxygen plasma treatment of the microstructure-forming side of the molded PDMS sheet; (d) the PDMS sheet; and applying an organosilane solution to the microstructured side of the PDMS sheet. In general, in the material modification treatment, the degree of hydrophilicity is not so high and the contact angle with water cannot be reduced to about 30°, or it takes 10 minutes or more to develop hydrophilicity when in contact with water. It is difficult to express sufficient hydrophilicity, such as

Among the surface modification treatments, the surface vapor deposition treatment involves vapor-depositing silica or titanium oxide to cover and hide the silicone molecules on the surface of the silicone molding base material, but it does not contribute to improving the hydrophilicity so much.

In the conventional method of hydrophilizing a silicone molding base material, even if it is coated with collagen or the like, sufficient hydrophilicity cannot be imparted stably for a long period of time, resulting in insufficient cell culture and cell adhesion. rice field.

As an example of cell culture on a surface-modified silicone molding base material, Patent Document 2 discloses that a silicone molding base material is irradiated with an electron beam to make it hydrophilized and cell culture is performed. This technology is specialized for culturing single cells because the surface of the silicone molded base material is deformed into a concave shape, and is not suitable for general cell culture or microscopic observation.

From the viewpoint of a substrate for animal cell culture, silicone rubber has transparency and air permeability that are said to be effective for cell culture and cell observation. It has the advantage of being able to reproduce similar hardness, and the advantage of being able to reproduce the expansion and contraction of the myocardium and blood vessels due to its extensibility. However, despite the numerous advantages over glass and plastic, for the above reasons, the surface cannot be modified like glass and plastic culture substrates. Without sufficient peptide coating, cell culture cannot be performed on silicone rubber.

JP 2006-181407 A JP 2018-202352 A

The present invention has been made in order to solve the above-mentioned problems, and it is possible to easily impart sufficient hydrophilicity to the surface of a molding substrate selected from various rubbers, resins, and glass containing silicone, and to rapidly improve hydrophilicity. While maintaining a sufficiently low contact angle with water, cells can be cultured without coating extracellular matrix proteins or polypeptides, and can be stored, preserved, or used in air or water for a long period of time. It is an object of the present invention to provide a cell-adhesive, hydrophilically modified cell culture substrate capable of maintaining the culture performance of the cells and ensuring the hydrophilicity for a long period of half a year to several years.

In tissue culture and cell culture, the method of pre-coating the cell adhesion surface of a culture vessel with extracellular matrix-derived proteins such as collagen, fibronectin, and laminin improves the adhesion of cells to the vessel and reproduces the environment in the body. commonly used to While the extracellular matrix-derived protein coating has the advantage of making it easier for cells to adhere to each other, these proteins are often foreign substances or foreign substances from heterologous animals to cells. Therefore, it may not be preferable to use extracellular matrix-derived proteins in cells cultured for cell transplantation in regenerative medicine. In addition, it is assumed that the use of extracellular matrix-derived protein coats and polypeptides may adversely affect the evaluation of cell functions such as secretory proteins using cultured cells. Techniques that enable cell culture without extracellular matrix-derived protein coatings or polypeptides can solve these problems.

A cell-adhesive hydrophilically modified cell culture substrate that has been made to achieve the above object is a repeating unit in which a molding substrate made of any material selected from rubber, resin, and glass has a betaine structure. , an azide group, a sulfo group, a —Si(OR ^e1 ) ₃ group (where OR ^e1 is an alkyl group having 1 to 6 carbon atoms or a phenyl group), a hydroxyl group, and a hydroxyl group precursor which is an alkoxy group having 1 to 6 carbon atoms. and a repeating unit having an active functional group selected from groups , wherein the betaine structure and / or the active functional group in the copolymer is the corona discharge-treated surface of the molding substrate. , a plasma-treated surface, an ultraviolet-treated surface, an excimer-treated surface, and a silane coupling agent-treated surface, which reacts, bonds, or adsorbs with a surface functional group on at least one of the surfaces . More specifically, a molding substrate made of any material selected from rubber, resin, and glass comprises a repeating unit having a betaine structure and a repeating unit having an active functional group that is an azide group. wherein the betaine structure and/or the active functional groups in the copolymer are coated on the molding substrate with a corona discharge treated surface, a plasma treated surface, an ultraviolet treated surface, an excimer treated surface, and a surface treated with a silane coupling agent, reacting with, bonding with, or adsorbing to a surface functional group on at least one of the surfaces.

This cell-adhesive, hydrophilically modified cell culture substrate is characterized in that it does not require a coating of extracellular matrix proteins and/or polypeptides.

In this cell-adhesive hydrophilic modified cell culture substrate, for example, the main chain repeating the repeating units in the copolymer is a poly(meth)acrylic skeleton.

In this cell-adhesive hydrophilic modified cell culture substrate, the poly(meth)acryl skeleton in the copolymer is a poly(meth)acrylamide copolymer skeleton, a poly(meth)acrylate copolymer skeleton, or a poly (Meth)acrylamide and poly(meth)acrylate copolymer skeletons are preferred.

In this cell adhesive hydrophilic modified cell culture substrate, the betaine structure is selected from an anion group selected from a carboxylic acid group, a sulfonic acid group, and a phosphoric acid group, and an ammonium group, a sulfonium group, and a phosphonium group. It is more preferable to have a cationic group that is

In this cell-adhesive hydrophilically modified cell culture substrate, it is even more preferable that the betaine structure has the anionic group or the cationic group at the end of the side chain.

In this cell adhesive hydrophilic modified cell culture substrate, the active functional group is at least one functional group selected from an azide group, a sulfo group, a trialkoxysilyl group, and a hydroxyl group. good too.
In this cell-adhesive hydrophilic modified cell culture substrate, the betaine structure has either one of the anionic group and the cationic group at the end of the side chain and the other in the side chain. can be anything.
The cell-adhesive hydrophilic modified cell culture substrate may be such that the copolymers are random copolymers, block copolymers, alternating copolymers, and graft copolymers.

The cell-adhesive, hydrophilically modified cell culture substrate may comprise hydrocarbon aromatic groups, non-aromatic heterocyclic groups, aromatic heterocyclic groups, linear, branched and/or cyclic, saturated or unsaturated. At least one spacer group selected from a hydrocarbon group, an amide group, and an ester group of has the active functional group and is bonded to the repeating unit.

（式（１）及び（２）中、Ｒ^１及びＲ^２は水素原子又はメチル基であり、ｎ１～ｎ４は２～６の数である）で表されるもので前記ベタイン構造を有する繰返単位と、下記化学式（３）

（式（３）中、Ｒ^３は水素原子又はメチル基であり、ｎ５は２～６の数であり、ｎ６は０～１の数であり、Ｒ^４は前記活性官能基である）で表されるもので活性官能基を有する繰返単位とのランダム共重合体、ブロック共重合体、交互共重合体、又はグラフト共重合体であるというものである。 In this cell adhesive hydrophilic modified cell culture substrate, the copolymer is, for example, the following chemical formula (1) or (2)

(in formulas (1) and (2), R ¹ and R ² are hydrogen atoms or methyl groups, and n1 to n4 are numbers from 2 to 6), and the repeating having the betaine structure Unit and the following chemical formula (3)

(In formula (3), R ³ is a hydrogen atom or a methyl group, n5 is a number from 2 to 6, n6 is a number from 0 to 1, and R ⁴ is the active functional group). It is a random copolymer, block copolymer, alternating copolymer, or graft copolymer with repeating units having active functional groups.

The molding substrate is preferably made of rubber selected from silicone rubber, fluororubber, ethylene-propylene-diene terpolymer rubber, and urethane rubber.

It is preferable that the molding substrate is made of a resin selected from silicone resins, cycloolefin resins, polycarbonate resins, polyethylene terephthalate resins, and acrylic resins.

It is preferable that the molding substrate is made of the glass selected from quartz glass, borosilicate glass, and soda-lime glass.

In this cell adhesive hydrophilic modified cell culture substrate, the surface of the molded substrate is a corona discharge-treated surface, a plasma-treated surface, an ultraviolet-treated surface, an excimer-treated surface, and/or a silane coupling agent-treated surface. preferably

In this cell-adhesive hydrophilic modified cell culture substrate, for example, the surface functional group is at least one selected from a hydroxyl group, a carboxyl group, a carbonyl group, an amino group, an alkoxy group and an alkoxysilyl group. be.

In this cell-adhesive, hydrophilically modified cell culture substrate, the betaine structure is bound or adsorbed to the surface functional group by ionic bond, and the active functional group is bound by ionic bond or covalent bond. is preferred.

（式（９）中、分子量が最低でも３００００であり、ｍ３及びｍ４は、前記分子量と繰返単位の比を形成する任意の正数とし、ランダム共重合体、ブロック共重合体、交互共重合体、又はグラフト共重合体にする数）で表されるものであるというものである。 In this cell-adhesive hydrophilic modified cell culture substrate, the molding substrate is made of silicone rubber having any one selected from a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton, or It is made of silicone resin.
In this cell adhesive hydrophilic modified cell culture substrate, the copolymer is, for example, the following chemical formula (9)

(In formula (9), the molecular weight is at least 30000, m3 and m4 are arbitrary positive numbers that form the ratio of the molecular weight and the repeating unit, and random copolymers, block copolymers, alternating copolymers number of coalesced or graft copolymer).

The cell-adhesive hydrophilic modified cell culture substrate of the present invention is a copolymer having a repeating unit having a betaine structure in the side chain and a repeating unit having an active functional group in the side chain. , and glass, the surface of the molding substrate is provided with sufficient hydrophilicity.

This cell-adhesive, hydrophilic-modified cell culture substrate is hydrophilic, and since cells adhere to the main chain of the repeating unit of the copolymer, it is easy to grow cells. Moreover, cells can be grown without the need for coating with extracellular matrix proteins or polypeptides.

This cell-adhesive hydrophilically modified cell culture substrate reacts, bonds, or adsorbs especially to the surface functional groups on the molding substrate due to the intramolecular cation structure and anion structure derived from the betaine structure. It exhibits strong hydrophilicity while being attached to cell-adhesive, hydrophilic-modified cell culture substrates. On the other hand, this cell-adhesive, hydrophilically modified cell culture substrate reacts with another surface functional group of the same or different type that the active functional group reacts, binds, or adsorbs on the molded substrate, Bonding or adsorption, particularly reaction or bonding, makes it difficult to run off even when contacted with water, an acidic test solution or an alkaline test solution, and can exhibit hydrophilicity.

This cell-adhesive, hydrophilically modified cell culture substrate is capable of imparting binding properties and hydrophilicity in a so-called adsorption type such as physical adsorption or chemisorption, preferably chemisorption, and reaction or bonding, preferably covalent bonding. By imparting binding properties and hydrophilicity in the so-called bonding type, the problems associated with conventional techniques such as glass alone or plastic alone or coating such as a collagen substrate are solved, and sufficient hydrophilicity is expressed and hydrophilicity is stable for a long time. It maintains its properties, enables cell culture without coating with an extracellular matrix protein or polypeptide, is simple, has high quality, and can be produced easily and inexpensively.

In addition, since this cell-adhesive hydrophilically modified cell culture substrate quickly expresses hydrophilicity on the surface of the molded substrate made of a material selected from rubber, resin, and glass, it is When in contact with weakly acidic, neutral, or weakly alkaline culture fluids and media, the desired hydrophilicity can be expressed, and cell culture performance and cell adhesion performance are not impaired. The hydrophilicity can be maintained for half a year to several years while maintaining a sufficiently low contact angle with water of about 20° or less. Therefore, even if it is stored, preserved, or used in air or water for a long period of time, it does not lose its hydrophilicity and can maintain and ensure high quality, making it suitable for cell culture.

Furthermore, since this cell-adhesive hydrophilically modified cell culture substrate has low cytotoxicity and suppresses protein adsorption, various cells to be cultured such as hepatocytes, stem cells, endothelial cells, iPS cells, tumor cells, etc. It is expected to promote adhesion and hardly inhibit proliferation.

In addition, this cell-adhesive, hydrophilic-modified cell culture substrate can easily impart uniform and sufficient hydrophilicity to the surface of a molding substrate made of a material selected from rubber, resin, and glass, and can be used between lots. Mass processing and mass production are possible with no variation, high reproducibility and high yield.

A microscope enlarged photograph (b) after culturing HeLa cells on a silicone hydrophilic modified cell culture substrate to which the present invention is applied, and a microscope enlarged photograph after culturing HeLa cells on a silicone molding substrate to which the present invention is not applied. It is a photograph (a).

Modes for carrying out the present invention will be described in detail below, but the scope of the present invention is not limited to these modes.

The cell-adhesive hydrophilically modified cell culture substrate of the present invention is a cell-adhesive hydrophilic modified cell culture substrate obtained by hydrophilizing a molded substrate made of a material selected from rubber, resin and glass. This cell-adhesive, hydrophilically modified cell culture substratum has an anionic group and a cationic group as a betaine structure that reacts with, binds to, or adsorbs to the surface functional groups on the molded substratum. A molding substrate is coated with a copolymer having units and repeating units having active functional groups on their side chains that react, bond, or adsorb to surface functional groups on the molding substrate.

In this cell-adhesive, hydrophilic-modified cell culture substrate, the betaine structure imparts binding properties and hydrophilicity in a so-called adsorption type, such as physical adsorption or chemisorption, preferably chemisorption, and the surface of the molded substrate. It is intended to prevent surface functional groups and copolymers from penetrating into the interior of the molding substrate to maintain bonding and hydrophilicity. In addition, this cell-adhesive hydrophilically modified cell culture substrate imparts binding properties and hydrophilicity in a so-called bonding type such as reaction or bonding, preferably covalent bonding, through an active functional group, and the copolymer Prevents from entering the inside of the molded substrate to maintain binding and hydrophilicity, cells adhere to the copolymer main chain of the repeating structure of the copolymer and proliferate from there, extracellular matrix proteins and poly It is suitable for cell culture without inhibiting cell proliferation even without peptide coating.

Therefore, the cell-adhesive, hydrophilically modified cell culture substrate has a betaine structure or an active functional group by reacting, bonding, or adsorbing the betaine structure and the active functional group to the surface functional group on the molding substrate. There is a stronger interaction between the copolymer and the molding substrate than with only one of the functional groups. This makes it difficult for the copolymer to flow off from the molding substrate even when it comes into contact with water, an acidic test solution, or an alkaline test solution, thereby exhibiting strong hydrophilicity and continuing to exhibit strong hydrophilicity for a long period of time. Since it can be used for long-term cell culture, it is expected to perform subculturing, morphogenesis, cell differentiation, and the like.

In the copolymer, the repeating main chain repeating repeating units is, for example, a poly(meth)acrylic skeleton. A poly(meth)acrylic skeleton includes a polyacrylic skeleton and a polymethacrylic skeleton. More specifically, the poly(meth)acryl backbone is a poly(meth)acrylamide copolymer backbone, a poly(meth)acrylate copolymer backbone, or a poly(meth)acrylamide and poly(meth)acrylate copolymer backbone. Among them, when the copolymer has a poly(meth)acrylamide copolymer skeleton, it has excellent affinity with the molding substrate made of a material selected from rubber, resin, and glass, and also has affinity with cells. is particularly preferred.

The copolymer is not limited in the repeating form of repeating units, and may be, for example, any of random copolymers, block copolymers, alternating copolymers and graft copolymers of these repeating units. There is no restriction on the molar ratio between the repeating unit having the amphoteric ion in the side chain and the repeating unit having the active functional group in the side chain, but the molar ratio is preferably 1:1.

In the copolymer, the repeating unit having a betaine structure in the side chain is such that the anionic group and the cationic group react, bond, or adsorb to the surface functional groups on the molding substrate, especially hydroxyl groups on the molding substrate. They are physically adsorbed or chemically adsorbed by mutual electrostatic attraction or ionic attraction to such polar groups.

As the betaine structure, the side chain may have either an anionic group or a cationic group at the end of the side chain and the other in the side chain.

In the betaine structure, when the anionic group is at the end of the side chain, ^{the carboxylic acid group (-COO- group), sulfonic acid group (-SO3- group), phosphoric acid group (-R a1 -PO-} ₍ ^{OR a2} ) (OR ^a3 ); —R ^a1 — is a group up to the end of the side chain, OR ^a2 and OR ^a3 are an alkoxy group or phenoxy group having 1 to 6 carbon atoms or an O ^— anion, and at least one of them is an O ^— anion) When it is in the middle of the side chain, a substituted carboxylic acid group (—COO ^— group) branched from the side chain, a substituted sulfonic acid group (—SO ₃ ^— group) branched from the side chain, ^{Phosphate group (-R b1 -PO-(OR b2 ) ( OR a3 ) ;} O ² -anions).

In the betaine structure, the cationic group, when located at the end of the side chain, is an organic ammonium group such as a primary to quaternary ammonium group ((-NH ₃ ) ⁺ , (-N(R ^c1 )H ₂ ) ⁺ , (- N(R ^c2 ) ₂ H) ⁺ , (-N(R ^c3 ) ₃ ) ⁺ ; R ^c2 to R ^c3 are alkyl groups having 1 to 6 carbon atoms or phenyl groups), sulfonium groups, preferably organic sulfonium groups ((- S(R ^c4 ) ₂ ) ⁺ ; R ^c4 is an alkyl group having 1 to 6 carbon atoms or a phenyl group), a phosphonium group, preferably a quaternary phosphonium group ((-P(R ^c5 ) ₃ ) ⁺ ; R ^c5 is a carbon number 1 to 6 alkyl groups or phenyl groups), and when it is in the middle of the side chain, an organic ammonium group such as a quaternary ammonium group ((-R ^d1 -N(R ^d2 ) ₂ ( R ^d3 )) ⁺ ; R ^d2 is a group in the middle of the side chain, R ^d2 is an alkyl group having 1 to 6 carbon atoms or a phenyl group, R ^d3 is a group in the middle of the side chain to the end), a sulfonium group (-R ^d4 -S ⁺ (R ^d5 )-R ^d6 ); R ^c4 is a group in the middle of the side chain, R ^d5 is an alkyl group having 1 to 6 carbon atoms or a phenyl group, R ^d6 is a group in the middle of the side chain to the end ⁾ , a phosphonium group (-R ^{d7 -P +} (R ^d8 ) ₂ ^{-R d9} ); group from the middle of the side chain to the end)).

The betaine structure has a surface functional group that is a polar group exposed on the surface of the molding substrate, and the surface of the molding substrate has a corona discharge-treated surface, a plasma-treated surface, an ultraviolet-treated surface, an excimer-treated surface, and/or a silane coupling agent. reaction with surface functional groups such as hydroxyl groups, hydroxyl groups, carboxyl groups, and carbonyl groups generated by being a treated surface, or surface functional groups such as amino groups, alkoxy groups, and alkoxysilyl groups possessed by the molding substrate; Bonding or Adsorption In particular, surface functional groups and betaine structures are strongly interacting with the surface of the molding substrate through adsorption such as physical adsorption or chemical adsorption due to mutual electrostatic or ionic attraction.

で表されるものが挙げられる。 Preferred examples of the repeating unit having a betaine structure in the copolymer include those represented by the chemical formula (1) or (2). More specifically, the following chemical formulas (4) to ( 7)

Those represented by are mentioned.

Also, in the copolymer, the repeating units having active functional groups on their side chains are bonded to the surface functional groups on the molding substrate by reaction, bonding or adsorption, especially covalent bonding.

Active functional groups include an azide group (--N ₃ ), a sulfo group (--SO ₃ ), a trialkoxysilyl group (--Si(OR ^e1 ) ₃ ; OR ^e1 is an alkyl group having 1 to 6 carbon atoms or a phenyl group), At least any functional group selected from a hydroxyl group (--OH) or a hydroxyl group-blocking group that generates a hydroxyl group, such as a hydroxyl group precursor group such as an alkoxy group having 1 to 6 carbon atoms.

Among the active functional groups, an azide group (--N ₃ ) releases a nitrogen molecule by, for example, decomposition by ultraviolet light or light or by thermal decomposition to generate a nitrene group (--N: group), which is formed on a molding substrate. By reacting and/or ring-expanding with surface functional groups or unsaturated groups, alkyl groups, phenyl groups, or amino groups possessed by the main component of the molding substrate or other curing components, Form covalent bonds. Among the active functional groups, a trialkoxysilyl group forms a covalent bond, which is a silyl ether bond, by condensation reaction with a surface functional group such as a hydroxyl group on the molding substrate. Among the active functional groups, the hydroxyl group or hydroxyl-blocking group is a surface functional group on the molding substrate, such as a silanol group (--Si--OH) or a siloxy group (--Si--OR ^f1 group; R ^f1 has 1 to 6 carbon atoms. A covalent bond, which is an ether bond, is formed by a condensation reaction with an alkyl group or a phenyl group). These may be bound via ionic bonds.

In addition, among repeating units having an active functional group in the side chain in the copolymer, the side chain may be a hydrocarbon aromatic ring group such as phenyl or naphthyl, or a non-aromatic heterocyclic group such as piperazinyl, pyreridinyl, pyrozokidinyl or morpholinyl. cyclic groups, aromatic heterocyclic groups such as pyridyl, pyrazolyl, imidazolyl, triazolyl, pyrazinyl, triazonyl, methyl, ethyl, vinyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclobutyl, n-pentyl, isopentyl , neopentyl, cyclopentyl, n-hexyl, cyclohexyl, or linear, branched and/or cyclic saturated or unsaturated hydrocarbon groups such as benzyl or phenethyl, amide groups (-CO-N (R ^g1 )—; R ^g1 is an alkyl group having 1 to 6 carbon atoms or a phenyl group), any single spacer group selected from an ester group (—CO—O—), or at least any combination thereof The composite spacer group may have the active functional group.

The active functional group in the copolymer is a surface functional group that is a polar group exposed on the surface of the molding substrate, and the surface of the molding substrate is a corona discharge-treated surface, a plasma-treated surface, an ultraviolet-treated surface, an excimer-treated surface, and / Or surface functional groups such as hydroxyl groups, hydroxyl groups, carboxyl groups, and carbonyl groups generated by being treated with a silane coupling agent, or surfaces such as amino groups, alkoxy groups, and alkoxysilyl groups possessed by the molding substrate Molecules of the copolymer are attached to the surface of the molding substrate by reacting, bonding, or adsorbing with functional groups, especially chemically reacting with surface functional groups to form covalent bonds.

The molecular weight and molecular weight distribution of this copolymer are not particularly limited, but in the case of the following chemical formula (9), it is preferably a polymer with a molecular weight of 30,000 or more and a narrow molecular weight distribution.

（式（８）及び（９）中、Ｒ^５～Ｒ^８は水素原子又はメチル基、ｍ１及びｍ２並びにｍ３及びｍ４は、前記分子量と繰返単位の比を形成する任意の正数）で表されるものであることが好ましい。 Among them, the copolymer has the following chemical formula (8) or chemical formula (9)

(In formulas (8) and (9), R ⁵ to R ⁸ are hydrogen atoms or methyl groups, m1 and m2 and m3 and m4 are any positive numbers that form the ratio between the molecular weight and the repeating unit). It is preferable that

The molding substrate is preferably rubber, resin, or glass. More preferably, it is made of elastic or soft silicone rubber or hard silicone resin. It is preferable to have one selected from a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton, because it is readily available and easy to manufacture.

The cell-adhesive, hydrophilically-modified cell culture substratum exhibits excellent hydrophilicity with a water contact angle of 20° or less due to such a configuration. The untreated molded substrate has a water contact angle of about 40 to 108°, but the cell-adhesive hydrophilic modified cell culture substrate has a water contact angle of 20° or less immediately after preparation, and is Even after being stored in a clean room for half a year to a year or more, the water contact angle is 20° or less and high hydrophilicity can be maintained.

It was confirmed that the cell-adhesive, hydrophilic-modified cell culture substrate has low cytotoxicity against HeLa cells, which are human cervical cancer-derived cells, and V79 cells, which are Chinese hamster lung-derived fibroblasts. In addition, since adsorption of proteins such as albumin is less than half, it is suitable for cell culture vessels.

It has been confirmed that HeLa cells directly adhere to the hydrophilic surface of the cell-adhesive, hydrophilically modified cell culture substrate and proliferate. It is expected to be developed into medically useful iPS cells and the like.

Since the cell adhesive hydrophilically modified cell culture substrate is a sterilization-resistant substrate, it is exposed to various sterilization conditions such as autoclave sterilization, ethylene gas oxide sterilization, gamma ray sterilization, and electron beam sterilization, which are high-pressure steam sterilization treatments. However, high hydrophilicity can be maintained. For example, in autoclave sterilization at 121 ° C. and 2 atmospheres for 20 minutes, the water contact angle can be maintained at 20 ° or less even after 5 months. The angle is 20° or less and high hydrophilicity can be maintained. Therefore, when used for medical instruments, even if sterilization is performed, the effect of hydrophilicity can be exhibited for a sufficiently long period of time.

Such cell-adhesive, hydrophilic-modified cell culture substratum can be produced as follows.

First, a copolymer having a repeating unit having a betaine structure in the side chain represented by the chemical formula (1) and a repeating unit having an active functional group in the side chain represented by the chemical formula (3) is prepared as follows. is synthesized as

More specifically, the copolymer represented by the chemical formula (8) or (9) will be described as an example. Dimethylaminopropyl methacrylamide is reacted with propanesultone to synthesize a betaine structure-containing comonomer for forming a repeating unit represented by chemical formula (1).

The amino group of 4-aminobenzoic acid is diazotized and then azidated to an acid chloride, and then amidated by reacting with monoprotected piperidine in which one of the amino groups of piperidine is protected with a protecting group, followed by a protecting group. is removed to give mono-p-azide benzoic acid piperazine amide. Amidating methacrylic acid chloride and 6-aminocaproic acid, and further amidating with the free amino group of the mono-p-piperazinamide benzoate to form a repeating unit represented by the chemical formula (2) Synthesize active functional group-containing comonomers for

Alternatively, containing another active functional group for amidation with methacrylic acid chloride and the free amino group of the mono-p-azide piperazine amide to form a repeating unit as represented by the chemical formula (2) Synthesize the comonomer.

By copolymerizing a betaine structure-containing comonomer as a repeating unit as represented by the chemical formula (1) and an active functional group-containing comonomer as a repeating unit as represented by the chemical formula (3), the above A copolymer represented by the chemical formula (8) or (9) is obtained.

In addition, another comonomer having a different number of carbon atoms, for example, a betaine structure-containing comonomer for forming a repeating unit represented by chemical formula (1) or (2) or a repeating unit such as represented by chemical formula (3) A comonomer containing an active functional group can be synthesized in the same manner by adjusting the starting material.

The copolymer may be used after being diluted with various media such as water, water-soluble organic media such as alcohol and acetone, and water-insoluble organic media such as methylene chloride, chloroform and ether. High hydrophilicity can be exhibited when the content is 0.001 to 10 wt%, and more preferably 0.01 to 1.0 wt%.

A molding substrate made of a material selected from rubber, resin, and glass is subjected to corona discharge treatment, plasma treatment, ultraviolet treatment, excimer treatment, and/or silane coupling agent treatment, preferably excimer treatment, to form a surface of the molding substrate. Surface functional groups are newly generated in addition to those originally existing. Moreover, by using masking or the like, it is possible to generate surface functional groups on a part of the surface of the molding base material, and selectively develop hydrophilicity.

After that, the copolymer liquid is applied by a method such as spraying, coating, or immersion, and if necessary, the medium is removed by a method such as volatilization to obtain a molding substrate to which the copolymer is applied. At this stage, the betaine structure of the copolymer electrostatically interacts with the surface functional groups on the surface of the molding substrate, and the copolymer is adsorbed by physical adsorption or chemical adsorption. , while being strongly attached, exhibiting hydrophilicity.

Next, the betaine structure of the copolymer to which the copolymer is attached is irradiated with ultraviolet light or light irradiation, preferably with a wavelength of 220 to 410 nm, and with an arbitrary integrated light amount. formed, reacted with unsaturated groups, alkyl groups, phenyl groups, amino groups and/or ring expansion, resulting in surface functional groups on the molding substrate, or main components of the molding substrate and other curable components reacting and/or ring-expanding with unsaturated groups, alkyl groups, phenyl groups, or amino groups to form covalent bonds by reacting or bonding so that the copolymers are firmly attached by chemical bonds; while exhibiting hydrophilicity.

Thus, a cell-adhesive, hydrophilically modified cell culture substrate is obtained.

Although an example in which the active functional group is an azide group is shown, the active functional group is a sulfo group, a trialkoxysilyl group, a hydroxyl group, or a hydroxyl group-blocking group, and a silanol is a surface functional group on the molding substrate. A covalent bond, which is an ether bond, may be formed by a condensation reaction with a group or a siloxy group, and the copolymer may exhibit hydrophilicity while being firmly attached by the chemical bond.

Cell-adhesive, hydrophilically modified cell culture substrates are used for applications that exhibit hydrophilicity. For example, it is used for medical devices such as vascular catheters, cell culture vessels, and microfluidic chips so as to be compatible with the moisture of the living body.

Hereinafter, examples of cell-adhesive modified hydrophilic cell culture substrates to which the present invention is applied and comparative examples to which the present invention is not applied will be described in comparison.

First, a copolymer having a betaine structure-containing repeating unit and an active functional group-containing repeating unit, which is used for producing a cell adhesive modified hydrophilic cell culture substrate to which the present invention is applied, was prepared.

三口フラスコ（３００ｍｌ）にジメチルアミノプロピルメタクリルアミド１．７０ｇ（１０ｍｍｏｌ）と脱水アセトン１２０ｍｌを入れ、室温で撹拌した。１，３－プロパンサルトン１．８３ｇ（１５ｍｍｏｌ）の脱水アセトン溶液２０ｍｌを滴下し、室温で４８時間撹拌した。アセトンを留去し、得られた固体にアセトン２０ｍｌを加えて洗浄した。得られた固体をメタノールに溶かし、ヒドロキノンを加えて溶媒を留去し、固体を取り出して減圧乾燥したところ、収率９８％で無色固体のメタクリルアミドプロピルスルホベタインが得られ、ｍ／ｚ：２９３（Ｍ＋Ｈ^＋）であり、その構造が支持された。 (Synthesis Example 1: Synthesis of copolymer [chemical formula (9)])
(1-1) Synthesis of methacrylamidopropylsulfobetaine

1.70 g (10 mmol) of dimethylaminopropyl methacrylamide and 120 ml of dehydrated acetone were placed in a three-necked flask (300 ml) and stirred at room temperature. A solution of 1.83 g (15 mmol) of 1,3-propanesultone in 20 ml of dehydrated acetone was added dropwise, and the mixture was stirred at room temperature for 48 hours. Acetone was distilled off, and 20 ml of acetone was added to the obtained solid for washing. The obtained solid was dissolved in methanol, hydroquinone was added, the solvent was distilled off, and the solid was taken out and dried under reduced pressure to obtain methacrylamidopropylsulfobetaine as a colorless solid with a yield of 98%, m/z: 293. (M+H ⁺ ) and the structure was supported.

三口フラスコ（３００ｍｌ）にピペラジン１２．９ｇ（１５０ｍｍｏｌ）、水酸化ナトリウム１．２０ｇ（３０ｍｍｏｌ）、ジオキサン７５ｍｌ、水７５ｍｌを入れ、０℃で撹拌した。ジ－ｔ－ブチルジカーボネート６．５４ｇ（３０ｍｍｏｌ）のジオキサン溶液５０ｍｌを滴下し、さらに室温で１２時間撹拌した。反応液を５ｗｔ％炭酸ナトリウム水溶液２００ｍｌに注ぎ、クロロホルム２００ｍＬで抽出した。クロロホルム、ジオキサン、及び未反応ピペラジンを留去し、減圧乾燥したところ、無色固体のモノＢｏｃピペラジンを粗収率８０％で得ることができ、そのまま次の反応に用いた。 (1-2) Synthesis of methacrylamide piperazine phenylazide monomer
(1-2(i)) Synthesis of mono-Boc piperazine

12.9 g (150 mmol) of piperazine, 1.20 g (30 mmol) of sodium hydroxide, 75 ml of dioxane and 75 ml of water were placed in a three-necked flask (300 ml) and stirred at 0°C. 50 ml of a dioxane solution containing 6.54 g (30 mmol) of di-t-butyl dicarbonate was added dropwise, and the mixture was further stirred at room temperature for 12 hours. The reaction solution was poured into 200 ml of a 5 wt % sodium carbonate aqueous solution and extracted with 200 mL of chloroform. Chloroform, dioxane and unreacted piperazine were distilled off and dried under reduced pressure to obtain mono-Boc piperazine as a colorless solid with a crude yield of 80%, which was directly used in the next reaction.

三口フラスコ（１Ｌ）に４－アミノ安息香酸６．８６ｇ（５０ｍｍｏｌ）、エタノール５００ｍｌ、濃塩酸２５０ｍｌを入れ、０℃で撹拌した。亜硝酸ナトリウム５．１８ｇ（７５ｍｍｏｌ）の水溶液１００ｍｌを滴下し、０℃で１時間撹拌してジアゾニウム塩溶液を調整した。三口フラスコ（２Ｌ）にアジ化ナトリウム３２．５ｇ（５００ｍｍｏｌ）、エタノール２５０ｍｌ、水２５０ｍｌを入れ、０℃で撹拌した。この溶液に調整したジアゾニウム塩溶液を滴下し、室温で２４時間撹拌した。エタノールを留去した反応液をクロロホルム５００ｍｌで二回抽出した。クロロホルム、エタノールを留去し、減圧乾燥したところ、淡黄色固体のｐ－アジ化安息香酸を粗収率１００％で得ることができ、そのまま次の反応に用いた。 (1-2(ii)) Synthesis of p-benzoic acid azide

6.86 g (50 mmol) of 4-aminobenzoic acid, 500 ml of ethanol and 250 ml of concentrated hydrochloric acid were placed in a three-necked flask (1 L) and stirred at 0°C. 100 ml of an aqueous solution of 5.18 g (75 mmol) of sodium nitrite was added dropwise and stirred at 0° C. for 1 hour to prepare a diazonium salt solution. 32.5 g (500 mmol) of sodium azide, 250 ml of ethanol and 250 ml of water were placed in a three-necked flask (2 L) and stirred at 0°C. The prepared diazonium salt solution was added dropwise to this solution, and the mixture was stirred at room temperature for 24 hours. The reaction solution from which ethanol was distilled off was extracted twice with 500 ml of chloroform. Chloroform and ethanol were distilled off and the residue was dried under reduced pressure to obtain p-benzoic acid azide as a pale yellow solid in a crude yield of 100%, which was directly used in the next reaction.

ナスフラスコ（５００ｍｌ）に粗アジ化安息香酸３．２６ｇ（２０ｍｍｏｌ）、オキザリルクロライド５．０８ｇ（４０ｍｍｏｌ）、ベンゼン３００ｍＬを入れ、室温で撹拌した。ＤＭＦ４滴を加え、室温で６時間撹拌した。ベンゼン、オキザリルクロライドを留去し、減圧乾燥したところ、褐色液体のｐ－アジ化安息香酸クロライドを粗収率１００％で得ることができ、そのまま次の反応に用いた。 (1-2(iii)) Synthesis of p-benzoyl chloride azide

3.26 g (20 mmol) of crude benzoic acid azide, 5.08 g (40 mmol) of oxalyl chloride and 300 mL of benzene were placed in an eggplant flask (500 ml) and stirred at room temperature. Four drops of DMF were added and stirred at room temperature for 6 hours. Benzene and oxalyl chloride were distilled off and the residue was dried under reduced pressure to obtain brown liquid p-benzoic acid chloride in a crude yield of 100%, which was directly used in the next reaction.

三口フラスコ（５００ｍｌ）に粗モノＢｏｃピペラジン４．９５ｇ（９０％、２４ｍｍｏｌ）、トリエチルアミン６．０６ｇ（６０ｍｍｏｌ）、ＴＨＦ３００ｍｌを入れ、０℃で撹拌した。粗アジ化安息香酸クロライド３．６３ｇ（２０ｍｍｏｌ）のＴＨＦ溶液５０ｍｌを滴下し、さらに室温で１２時間撹拌した。ＴＨＦを留去し、得られた残渣を５ｗｔ％塩酸２００ｍｌに注ぎ、クロロホルム２００ｍｌで抽出した。クロロホルムを留去し、残渣をゲル浸透クロマトグラフィー（ＧＰＣ）にて精製したところ、淡褐色固体のｐ－アジ化安息香酸Ｂｏｃピペラジンアミドを収率９７％で得た。 (1-2(iv)) Synthesis of p-benzoic acid Boc piperazine amide

A three-neck flask (500 ml) was charged with 4.95 g (90%, 24 mmol) of crude mono-Boc piperazine, 6.06 g (60 mmol) of triethylamine and 300 ml of THF and stirred at 0°C. 50 ml of a THF solution containing 3.63 g (20 mmol) of crude azide benzoyl chloride was added dropwise, and the mixture was further stirred at room temperature for 12 hours. THF was distilled off, and the resulting residue was poured into 200 ml of 5 wt % hydrochloric acid and extracted with 200 ml of chloroform. Chloroform was distilled off, and the residue was purified by gel permeation chromatography (GPC) to obtain p-benzoic acid Boc piperazine amide as a pale brown solid with a yield of 97%.

ナスフラスコ（２００ｍｌ）にアジ化安息香酸Ｂｏｃピペラジンアミド３．３１ｇ（１０ｍｍｏｌ）を入れ、０℃で撹拌した。トリフルオロ酢酸２０ｍｌを加え、室温で３０分撹拌した。トリフルオロ酢酸を留去し、得られた残渣に５ｗｔ％炭酸ナトリウム水溶液１００ｍｌを注ぎ、クロロホルム１００ｍｌで二回抽出した。クロロホルムと副成したピペラジンとを留去し、減圧乾燥したところ、褐色固体のモノ－ｐ－アジ化安息香酸ピペラジンアミドを粗収率９０％で得ることができ、そのまま次の反応に用いた。 (1-2(v)) Synthesis of mono-p-benzoic acid piperazine amide mono-p-azide

3.31 g (10 mmol) of Boc piperazine amide benzoate azide was placed in an eggplant flask (200 ml) and stirred at 0°C. 20 ml of trifluoroacetic acid was added and stirred at room temperature for 30 minutes. Trifluoroacetic acid was distilled off, 100 ml of a 5 wt % sodium carbonate aqueous solution was poured into the resulting residue, and the mixture was extracted twice with 100 ml of chloroform. Chloroform and by-product piperazine were distilled off and dried under reduced pressure to obtain mono-p-piperazinamide azide as a brown solid in a crude yield of 90%, which was directly used in the next reaction.

三口フラスコ（１００ｍｌ）にメタクリルアミドプロピルスルホベタイン１．４６ｇ（５ｍｍｏｌ）、トリエチルアミン１．０１ｇ（１０ｍｍｏｌ）、ＴＨＦ６０ｍｌを入れ、０℃で撹拌した。メタクリル酸クロライド６２７ｍｇ（６ｍｍｏｌ）のＴＨＦ溶液１５ｍｌを滴下し、さらに室温で１２時間撹拌した。ＴＨＦを留去し、得られた残渣を５ｗｔ％炭酸ナトリウム水溶液１００ｍｌに注ぎ、クロロホルム１００ｍｌで抽出した。クロロホルムを留去し、再結晶（ヘキサン：クロロホルム＝２５：５ｍＬ）すると、タール状不純物が除去でき、淡褐色固体のメタクリルアミドピペラジン－ｐ－フェニルアジドアミドモノマーが、収率５４％で得られた。 (1-2(vi)) Synthesis of methacrylamidopiperazine-p-phenylazidamide monomer

1.46 g (5 mmol) of methacrylamidopropylsulfobetaine, 1.01 g (10 mmol) of triethylamine and 60 ml of THF were placed in a three-necked flask (100 ml) and stirred at 0°C. 15 ml of a THF solution containing 627 mg (6 mmol) of methacrylic acid chloride was added dropwise, and the mixture was further stirred at room temperature for 12 hours. THF was distilled off, and the resulting residue was poured into 100 ml of a 5 wt % sodium carbonate aqueous solution and extracted with 100 ml of chloroform. By distilling off chloroform and recrystallizing (hexane: chloroform = 25: 5 mL), tar-like impurities could be removed, and methacrylamide piperazine-p-phenylazidamide monomer as a pale brown solid was obtained with a yield of 54%. .

ナスフラスコ（１００ｍｌ）にメタクリルアミドプロピルスルホベタイン１．４６ｇ（５ｍｍｏｌ）、メタクリルアミドピペラジン－ｐ－フェニルアジドアミドモノマー１５．０ｍｇ（０．０５ｍｍｏｌ）、過硫酸アンモニウム１１．４ｍｇ、水５ｍｌを入れ、アルミホイルで蓋をして均一溶液にした。７０℃のウォーターバスにナスフラスコを１時間浸けて重合させた。反応液に水５ｍｌを加えて撹拌し、溶液をメタノール５０ｍｌに注いだ。生成した沈殿物を集め、メタノールで洗浄し、減圧乾燥したところ、淡黄色固体のランダム共重合体[化学式（９）]が、収率９２％で得られ、ＧＰＣで測定したところ数平均分子量９２０００であり、その構造が支持された。 (1-3) Copolymerization to copolymer [chemical formula (9)]

1.46 g (5 mmol) of methacrylamidopropyl sulfobetaine, 15.0 mg (0.05 mmol) of methacrylamidopiperazine-p-phenylazidamide monomer, 11.4 mg of ammonium persulfate, and 5 ml of water were placed in an eggplant flask (100 ml), and wrapped with aluminum foil. to make a homogeneous solution. The eggplant flask was immersed in a water bath at 70° C. for 1 hour to polymerize. 5 ml of water was added to the reaction solution and stirred, and the solution was poured into 50 ml of methanol. The resulting precipitate was collected, washed with methanol, and dried under reduced pressure to give a pale yellow solid random copolymer [chemical formula (9)] with a yield of 92%. and its structure was supported.

(Example 1)
Using the copolymer [chemical formula (9)] prepared in Synthesis Example 1, various substrates were subjected to hydrophilic modification treatment. A crosslinked silicone rubber sheet was used as the base material. The copolymer [chemical formula (9)] was used by preparing a 0.1 wt % aqueous solution. The substrate was plasma-treated or excimer-treated under predetermined conditions, and then immersed in a 0.1 wt % copolymer aqueous solution at room temperature for 10 minutes. Then, after taking out the base material, it was irradiated with ultraviolet rays to obtain a hydrophilic modified cell culture base material.

(Example 2)
A hydrophilic modified cell culture substrate was obtained in the same manner as in Example 1, except that polycarbonate (manufactured by Mitsubishi Gas Chemical Company: Iupilon Film FE-2000) was used as the substrate.

(Example 3)
A hydrophilic modified cell culture substrate was obtained in the same manner as in Example 1, except that soda lime glass (manufactured by Matsunami Glass Industry: S9213) was used as the substrate.

(Comparative example 1)
A hydrophilic modified cell culture substrate was produced in the same manner as in Example 1, except that a phosphorylcholine polymer having a number average molecular weight (i) of 95,000 or (ii) of 253,000 was used.

(Measurement of contact angle)
Hydrophilicity was evaluated by the contact angle of water of the resulting hydrophilically modified cell culture substrate. 10 seconds after dropping one drop of water onto the resulting hydrophilically modified cell culture substrate, the contact angle with water was measured using an automatic contact angle meter (manufactured by Kyowa Interface Science Co., Ltd.; DM-501). Measurements were taken at 5 to 7 points per test piece, and the average value was obtained. Table 1 shows the results.

As is clear from Table 1, it was found that the hydrophilicity-improving treatment reduced the contact angle and improved the hydrophilicity. Moreover, from Examples 1, 2 and 3, it was found that the hydrophilic property was improved regardless of the type of substrate.

Next, a cell test was performed on the hydrophilic modified cell culture substrate of Example 1. For comparison, Comparative Example 1 was also tested in the same manner. The obtained results are summarized in Table 2.

(Cytotoxicity test)
A cytotoxicity test (colony formation method by extraction method) of the hydrophilically modified cell culture substrate was performed according to ISO 10993-5:2009, Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity. Cells used were V79 cells and HeLa cells.

(cell culture test)
A cell culture test of the hydrophilic modified cell culture substrate was performed. First, a hydrophilic modified cell culture substrate was fixed on a polystyrene dish, and a culture solution was dispensed into the dish. After that, HeLa cells were seeded on the surface of the hydrophilically modified cell culture substrate and cultured for 3 days in a carbon dioxide incubator at a temperature of 37° C. and a CO ₂ concentration of 5%. Fig. 1 shows an enlarged microscope photograph (b) after culturing HeLa cells on the hydrophilic modified cell culture substrate of Example 1, and an enlarged microscope image after culturing HeLa cells on a silicone molding substrate to which the present invention is not applied. This is shown by photo (a).

As is clear from Table 2 and FIG. 1, it was confirmed that there was no cytotoxicity in both Example 1 and Comparative Example 1. In cell culture, cell proliferation was confirmed in Example 1, whereas cell proliferation could not be confirmed in Comparative Example. As a factor for this, it is generally necessary to form an extracellular matrix, which is essential for cell adhesion, on a substrate during cell culture. Therefore, it is conceivable that Comparative Example 1 could not be cell cultured. On the other hand, in Example 1, cells could be cultured although no extracellular matrix was formed. From this, it was confirmed that the present invention enables cell culture without coating extracellular matrix proteins or polypeptides.

Next, the following various sterilization treatments were performed on the obtained hydrophilic modified cell culture substratum, and resistance to various sterilization treatments was confirmed. The test was carried out in Example 1. The obtained results are summarized in Table 3.

(Autoclave sterilization test)
The autoclave sterilization treatment was carried out at 121° C. and 2 atm for 20 minutes in a high-pressure steam sterilizer (ASV-2402 manufactured by Sakura Seiki Co., Ltd.), and then the contact angle of water was measured. In addition, to confirm long-term storage properties, the sterilized hydrophilic modified cell culture substrate was put in a petri dish and stored for 6 months at room temperature and a humidity of 60%, and then the contact angle of water was measured. .

(γ-ray/electron beam irradiation sterilization test)
Radiation resistance was confirmed by γ-ray and electron beam irradiation to the hydrophilic modified cell culture substrate. The hydrophilic modified cell culture substrate was irradiated with radiation at 20 to 50 kGy using a radiation generator. After that, the contact angle with water was measured. In addition, to confirm long-term storage properties, the sterilized hydrophilic modified cell culture substrate was put in a petri dish and stored for 6 months at room temperature and a humidity of 60%, and then the contact angle of water was measured. .

As is clear from Table 3, the contact angle slightly increased before and after each test, but hydrophilicity was maintained. Also, even after 6 months had passed, the hydrophilicity was maintained, although a slight increase in the contact angle was observed. From this, it was confirmed that the present invention is resistant to various sterilization treatments and can maintain hydrophilicity over a long period of time.

Next, a protein test on the hydrophilically modified cell culture substrate of Example 1 was performed. The results obtained are shown in Table 4.

(Evaluation of protein adsorption)
A protein adsorption test of the hydrophilic modified cell culture substrate was performed. A 30×30 cm hydrophilic modified cell culture substrate was soaked in the albumin solution for 2 hours. After immersion, the hydrophilically modified cell culture substrate was taken out, fluorescently labeled with Cy3 dye-labeled carboxylic acid, and low molecular weight components were washed with phosphate buffered saline (PBS) to prepare a test piece. The fluorescence intensity of the obtained test piece was measured with a spectrophotometer, and the degree of albumin adsorption was calculated from the integrated volume. For comparison, a similar test was also conducted on an untreated silicone rubber sheet.

As is clear from Table 4, with respect to protein adsorption, the untreated silicone rubber sheet had an albumin adsorption volume of 2.5×10 ⁵ , whereas the albumin adsorption volume of Example 1 was 1.2×10 ⁵ . , which was about half the adsorption volume of Comparative Example 1. From this, it was confirmed that the present invention can reduce the amount of protein adsorption.

From the above results, it can be seen that the present invention can easily impart sufficient hydrophilicity to the surface of a molding base material, quickly develop the hydrophilicity, and maintain a sufficiently low contact angle with water, while maintaining a sufficiently low contact angle with water. Cells can be cultured without coating the outer matrix or polypeptide, and the culture performance can be maintained even when stored, preserved or used in the air or water for a long period of time. It was confirmed that it can be used as a plasma cell culture substrate.

The cell-adhesive, hydrophilic-modified cell culture substrate of the present invention can be used for medical devices such as vascular catheters that require long-term maintenance of hydrophilicity, cell culture vessels, microchannel chips, and organ-on. - Used for a-chip and the like.

Claims (17)

A molding substrate made of any material selected from rubber, resin, and glass is coated with a copolymer containing a repeating unit having a betaine structure and a repeating unit having an active functional group that is an azide group. and the betaine structure and/ or the active functional group in the copolymer is selected from a corona discharge-treated surface, a plasma-treated surface, an ultraviolet-treated surface, an excimer-treated surface, and a silane coupling agent-treated surface of the molding substrate. A cell-adhesive, hydrophilically modified cell culture substrate characterized by reacting, bonding, or adsorbing with surface functional groups on at least one of the surfaces thereof . 2. The cell-adhesive hydrophilically modified cell culture substrate according to claim 1, wherein cell culture is possible without coating the surface of the substrate with proteins and/or polypeptides. Hydrophilic modifier of cell adhesion according to any one of claims 1 and 2, wherein the main chain repeating the repeating unit in the copolymer is a poly(meth)acrylic skeleton. Cell culture substrate. In the copolymer, the poly(meth)acryl skeleton is a poly(meth)acrylamide copolymer skeleton, a poly(meth)acrylate copolymer skeleton, or a poly(meth)acrylamide and poly(meth)acrylate copolymer skeleton. The cell-adhesive hydrophilic modified cell culture substrate according to claim 3, characterized in that: Claims 1-, wherein the betaine structure has an anionic group selected from a carboxylic acid group, a sulfonic acid group, and a phosphoric acid group, and a cationic group selected from an ammonium group, a sulfonium group, and a phosphonium group. 5. The cell-adhesive hydrophilic modified cell culture substrate according to any one of 4. 6. The cell-adhesive hydrophilic modified cell culture substrate according to claim 5, wherein the betaine structure has the anionic group or the cationic group at the end of a side chain. 7. The cell-adhesive hydrophilicity according to claim 6, wherein the betaine structure has one of the anionic group and the cationic group at the end of the side chain and the other in the side chain. modified cell culture substrates. The cell -adhesive hydrophilic according to any one of claims 1 to 7, wherein the copolymer is a random copolymer, a block copolymer, an alternating copolymer, and a graft copolymer. Modified cell culture substrate. at least one selected from a hydrocarbon aromatic ring group, a non-aromatic heterocyclic group, an aromatic heterocyclic group, a linear, branched and/or cyclic saturated or unsaturated hydrocarbon group, an amide group, and an ester group 9. The hydrophilic modifier of cell adhesion according to any one of claims 1 to 8 , wherein any spacer group has the active functional group and is bound to the repeating unit. Cell culture substrate. The copolymer has the following chemical formula (1) or (2)

(in formulas (1) and (2), R ¹ and R ² are hydrogen atoms or methyl groups, and n1 to n4 are numbers from 2 to 6), and the repeating having the betaine structure Unit and the following chemical formula (3)

(In formula (3), R ³ is a hydrogen atom or a methyl group, n5 is a number from 2 to 6, n6 is a number from 0 to 1, and R ⁴ is the active functional group). and is a random copolymer, block copolymer, alternating copolymer, or graft copolymer with a repeating unit having an active functional group. The cell-adhesive hydrophilic modified cell culture substrate described. 11. The molding base material according to any one of claims 1 to 10 , characterized in that said molding base material is made of said rubber selected from silicone rubber, fluororubber, ethylene-propylene-diene terpolymer rubber, and urethane rubber. of hydrophilically modified cell culture substrates. The hydrophilic modifier according to any one of claims 1 to 10 , wherein the molding substrate is made of the resin selected from silicone resin, cycloolefin resin, polycarbonate resin, polyethylene terephthalate resin, and acrylic resin. Plasma cell culture substrate. The hydrophilic modified cell culture substrate according to any one of claims 1 to 10 , wherein the molding substrate is made of glass selected from quartz glass, borosilicate glass, and soda lime glass. 14. The cell adhesion according to any one of claims 1 to 13, wherein the surface functional group is at least one selected from a hydroxyl group, a carboxyl group, a carbonyl group, an amino group, an alkoxy group, and an alkoxysilyl group. hydrophilic modified cell culture substrate. 15. The betaine structure according to any one of claims 1 to 14, wherein the betaine structure is bound or adsorbed to the surface functional group by an ionic bond, and the active functional group is bound by an ionic bond or a covalent bond. cell-adhesive, hydrophilically modified cell culture substrates. Any of claims 1 to 15, wherein the molding base material is made of silicone rubber or silicone resin and has any one selected from a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton. 3. The cell-adhesive, hydrophilically modified cell culture substrate according to any one of . The copolymer has the following chemical formula (9)

(In formula (9), the molecular weight is at least 30000, m3 and m4 are arbitrary positive numbers that form the ratio of the molecular weight and the repeating unit, and random copolymers, block copolymers, alternating copolymers 17. The cell-adhesive hydrophilic modified cell culture substrate according to any one of claims 1 to 16, which is represented by the number of coalescing or graft copolymers) .