JP2021158968A - Cell adhesiveness hydrophilic modification cell culture material - Google Patents

Abstract

[Problem] To easily impart sufficient hydrophilicity to the surface of a molding base material such as rubber, resin, or glass, rapidly develop the hydrophilicity, and maintain a sufficiently low contact angle with water. , cells can be cultured without coating with proteins or polypeptides, the culture performance can be maintained even when stored, preserved or used in the air or water for a long period of time, and the hydrophilicity can be secured for a long period of half a year to several years. A hydrophilically modified cell culture substrate is provided. A cell-adhesive, hydrophilic modified cell culture substrate has a repeating unit having a betaine structure and an active functional group in a molding substrate made of any material selected from rubber, resin, and glass. It is coated with a copolymer containing repeating units, and the betaine structure or the active functional groups in the copolymer react, bond or adsorb to the surface functional groups on the molding substrate. [Selection figure] None

Description

The present invention relates to a hydrophilic modified cell culture substrate which is capable of cell culture and exhibits cell adhesion by sustaining the effect of hydrophilization treatment applied to the surface of the molded substrate for a long time.

Culture medium-based culture medium for research and development of pharmaceuticals and pesticides, microbiological examination of foods and drinks, culture of bacteria and cells in health examinations, cell culture in biochemical research and medical sites, tissue culture in regenerative medicine, etc. The material is used.

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

Generally, as the material of the molding base material, various plastics such as polystyrene and silicone are widely used in addition to glass which is easy to mold and has high transparency, and the molding method includes compression molding, injection molding, extrusion molding, and blow. It is molded into various shapes by molding, vacuum molding, pneumatic molding, calendar molding, etc.

The form of cell culture is roughly divided into two types, a suspension culture system in which a single cell or a small cell mass is floating in a medium, and an adhesion culture system in which cells are adhered to a container, etc., depending on the proliferating cells. Will be done.

Normally, it is difficult to culture cells in an adhesive culture system with a molded base material that is simply molded from glass or plastic, so that the surface on which cells are seeded needs to be surface-treated according to each material.

The surface treatment is coated with a surface modification treatment for hydrophilizing the surface, or a fragment produced by digestion and decomposition of extracellular matrix protein or extracellular matrix protein, or a polypeptide such as polylysine. Established cells 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 whose surface is hydrophilic.

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

In general, among the proteins constituting the extracellular matrix, human-derived proteins are difficult to obtain in sufficient quality and sufficient quantity for the purpose of coating the culture vessel. Therefore, usually, even for culturing human-derived cells, a protein derived from bovine or porcine is used as a protein derived from extracellular matrix for coating a culture vessel. However, it is preferable not to use proteins and polypeptides derived from heterologous animals as much as possible in cell cultures for transplantation in regenerative medicine and cell cultures for detailed cell function studies in vivo.

In recent cell culture technology development, 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 research on the original cell functions in the environment in vivo. , The technique of culturing in an environment closer to the organs and tissues in the animal body is becoming important.

From silicone rubber, it is possible to make a culture vessel that is close to the hardness of various tissues of animals, to make a culture vessel with high air permeability, and to make a culture vessel with high extensibility close to the myocardium and vascular intestinal tract. .. In addition, silicone rubber has durability that can withstand high-pressure sterilization. It is difficult to make a culture vessel having such various hardness, breathability, extensibility, and durability only from glass material and polystyrene material.

Silicone rubber is attracting attention from fields such as regenerative medicine, drug discovery industry, and basic research for the above reasons, but silicone rubber is based on silicone rubber because of its water and oil repellency and poor hydrophilicity. It is difficult to attach cells to the culture vessel. Usually, a culture vessel formed of silicone rubber is used by coating the surface with a protein or polypeptide constituting an extracellular matrix such as collagen in order to carry out cell culture of an adhesive 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, for detailed cell function research in vivo In research and development such as cell culture, there is a demand for a technique for realizing cell culture in an environment closer to the organs and tissues in the animal body.

In order to impart hydrophilicity to such a silicone molded base material, a dry treatment or a wet treatment on the silicone molded base material, or a silicone base material forming treatment by curing a silicone raw material composition containing a hydrophilic compound, a surface A hydrophilic group was imparted to the silicone surface by a surface modification treatment such as a vapor deposition treatment.

Among these surface modification treatments, the dry treatment includes corona discharge treatment, plasma treatment, ultraviolet treatment, excimer treatment, etc., and imparts hydrophilicity by generating hydrophilic groups such as hydroxyl groups on the surface. .. Such a dry process is relatively simple. However, when the hydrophilic silicone molded base material obtained by the dry treatment is stored, stored or used in the air or water, the hydrophilic groups generated on the surface of the silicone base material become silicone over time. In particular, it sneaks into the inside of the silicone rubber and loses its hydrophilicity in a relatively short period of about 3 to 6 months.

Further, among the surface modification treatments, the wet treatment involves chemically binding a hydrophilic coating agent (for example, a hydrophilic silane coupling agent) to the surface functional group of the silicone molded base material, chemically modifying the surface functional group, and introducing the treatment. Is. In such a wet treatment, the surface functional groups on the surface of the silicone molded substrate are sparsely present, resulting in uneven bonding of the hydrophilic coating agent, and the hydrophilicity can be maintained for about 3 to 6 months, but the degree of hydrophilicity is high. However, it is not so high and the contact angle with water can be lowered only to about 30 °, and the hydrophilic coating agent molecules bonded on the surface gradually sneak into the inside from the surface of the silicone molded base material, and the silicone molecules are easily exposed. Therefore, it is difficult to develop sufficient hydrophilicity because the hydrophilicity is lowered or the types of hydrophilic coating agents applicable to silicone rubber are extremely small. Further, when joining a support or the like to this surface-treated surface, it is necessary to separately perform surface treatment or chemical modification for joining, which hinders the hydrophilicity of the folding angle.

As another wet treatment, a polymer having a betaine structure, for example, a poly (meth) acrylate having an amino acid structure in the side chain or a (meth) acrylamide having a sulfobetaine structure in the side chain is applied to the surface of a silicone molded substrate with anions / cations or positive or negative. There is also a surface modification treatment in which the charging groups are chemically adsorbed and introduced. However, in such a surface modification treatment, since it is only physically or chemically adsorbed on the surface, even if it is uniformly adsorbed, it flows off relatively quickly due to contact with water, an acidic test solution, or an alkaline test solution. Therefore, strong hydrophilicity cannot be developed for a long period of time.

In addition, among the surface modification treatments, as a silicone base material forming treatment by curing a hydrophilic compound-containing silicone raw material composition, a hydrophilic compound such as a hydrophilic oil is mixed with a liquid to mirable silicone raw material and cured to form a silicone. There is a material modification treatment in which a hydrophilic additive is exposed on the surface of the body to impart hydrophilicity by forming the body. As an example of such a material modification treatment, Patent Document 1 states that (a) a step of preparing a master having a predetermined microstructure on the upper surface and (b) a PDMS prepolymer on the microstructure forming surface side of the master. A step of injecting a mixture of a curing agent and a polyether-modified surfactant, (c) a step of treating the microstructure-forming surface side of the molded PDMS sheet with oxygen plasma, and (d) the PDMS sheet. A method for producing a PDMS-made sheet having permanent hydrophilicity, which comprises a step of applying an organosilane solution to the microstructure-forming surface side of the above, is disclosed. Generally, 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 develop sufficient hydrophilicity, such as.

Further, among the surface modification treatments, the surface vapor deposition treatment is a treatment in which silica or titanium oxide is vapor-deposited and the silicone molecules on the surface of the silicone molded base material are coated and concealed, but it does not contribute to the improvement of hydrophilicity to some extent.

In the conventional method of hydrophilizing a silicone molded base material, even if it is coated with collagen or the like, it is not possible to sufficiently impart cell culture and cell adhesion because it is not possible to impart sufficient hydrophilicity for a long period of time. rice field.

As an example of cell culture on a surface-modified silicone-molded substrate, Patent Document 2 states that the silicone-molded substrate is hydrophilized by electron beam irradiation and cell-cultured. Since the surface of the silicone-molded substrate is deformed into a concave shape in this technology, it is specialized for single cell culture, and is not suitable for general cell culture and microscopic observation.

From the viewpoint of a base material for culturing animal cells, silicone rubber has transparency and breathability, which is said to be effective for cell culturing and cell observation, and can be used in tissues from which cells are derived by organ-on-a-chip or the like. It has the advantage of being able to reproduce close hardness and the advantage of being able to reproduce the expansion and contraction of the myocardium and vascular intestinal tract due to its extensibility. However, despite its many advantages over glass and plastic, for the above reasons, surface modifications such as those made of glass and plastic culture media have not been possible, and at present, extracellular matrix proteins and polys are not available. Cell culture cannot be performed on silicone rubber unless the peptide is sufficiently coated.

Japanese Unexamined Patent Publication No. 2006-181407 Japanese Unexamined Patent Publication No. 2018-20352

The present invention has been made to solve the above-mentioned problems, and it is possible to easily impart sufficient hydrophilicity to the surface of a molded base material selected from various rubbers / resins including silicone and glass, and the hydrophilicity can be quickly increased. It can be expressed and cell-cultured without coating with extracellular matrix proteins or polypeptides while maintaining a sufficiently low contact angle with water, and can be stored, stored or used in the air or water for a long period of time. It is an object of the present invention to provide a cell-adhesive hydrophilic modified cell culture substrate capable of maintaining culture performance and ensuring hydrophilicity for a long period of six months to several years.

In tissue culture and cell culture, the method of precoating extracellular matrix-derived proteins such as collagen, fibronectin, and laminin on the cell adhesion surface of the culture vessel improves the adhesion of cells to the vessel and reproduces the internal environment. It is commonly used to do this. While the coat of extracellular matrix-derived proteins has the advantage of facilitating cell adhesion, these proteins are often foreign or foreign to cells from heterologous animals. 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.

The cell-adhesive hydrophilic modified cell culture substrate made to achieve the above object is a repeating unit in which a molded substrate made of any material selected from rubber, resin, and glass has a betaine structure. And coated with a copolymer containing a repeating unit having an active functional group, the betaine structure in the copolymer or the active functional group reacts with, binds to, or reacts with a surface functional group on the molded substrate. It is characterized by being adsorbed.

This cell-adherent hydrophilic modified cell culture substrate is characterized by not requiring coating of extracellular matrix proteins and / or polypeptides.

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

In this cell-adhesive hydrophilic modified cell culture substrate, the poly (meth) acrylic skeleton in the copolymer is a poly (meth) acrylamide copolymer skeleton, a poly (meth) acrylate copolymer skeleton, or a poly. It is preferably a (meth) acrylamide and poly (meth) acrylate copolymer skeleton.

In this cell-adhesive hydrophilic modified cell culture substrate, the betaine structure is selected from an anionic 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 to be used.

It is even more preferable that the betaine structure of the cell-adhesive hydrophilic modified cell culture substrate has the anion group or the cation 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 azido group, a sulfo group, a trialkoxysilyl group, and a hydroxyl group. May be good.

This cell-adhesive hydrophilic modified cell culture substrate is saturated or unsaturated with hydrocarbon aromatic ring groups, non-aromatic heterocyclic groups, aromatic heterocyclic groups, linear, branched chain and / or cyclic. At least one of the spacer groups selected from the hydrocarbon group, the amide group, and the ester group of the above may be bonded to the repeating unit while having the active functional group.

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

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

It is preferable that the molded base material is made of the rubber selected from silicone rubber, fluororubber, ethylene-propylene-diene ternary copolymer rubber, and urethane rubber.

It is preferable that the molded base material is made of the resin selected from silicone resin, cycloolefin resin, polycarbonate resin, polyethylene terephthalate resin, and acrylic resin.

It is preferable that the molding base material 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. Is preferable

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 hydrophilic modified cell culture substrate, the betaine structure is bonded or adsorbed to the surface functional group by an ionic bond, and the active functional group is bonded by an ionic bond or a covalent bond. Is preferable.

The cell-adhesive hydrophilic modified cell culture base material is made of silicone rubber or has a molded base material selected from a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton. It is made of silicone resin.

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 is a rubber or resin. , By coating the molding base material made of a material selected from glass, sufficient hydrophilicity is imparted to the surface of the molding base material.

This cell-adhesive hydrophilic modified cell culture substrate is hydrophilic and the cells adhere to the main chain of the repeating unit of the copolymer, so that the cells can easily proliferate. Moreover, cells can be proliferated without the need for coating with extracellular matrix proteins or polypeptides.

This cell-adhesive hydrophilic modified cell culture substrate reacts, binds, or adsorbs, especially adsorbs, to surface functional groups on the molded substrate due to the intramolecular cation and anion structures derived from the betaine structure. It is attached to a cell-adhesive hydrophilic modified cell culture substrate and exhibits strong hydrophilicity. On the other hand, this cell-adherent hydrophilic modified cell culture substrate reacts with another surface functional group of the same kind or different kind as the one in which the active functional group is reacted, bound, or adsorbed on the molded substrate. Bonding or adsorption In particular, by reacting or binding, it is difficult for the cells to run off even when they come into contact with water, an acidic test solution or an alkaline test solution, and hydrophilicity can be exhibited.

This cell-adhesive hydrophilic modified cell culture substrate is subjected to so-called adsorption-type binding and hydrophilicity such as physical adsorption or chemisorption, preferably chemical adsorption, and reaction or binding, preferably covalent bonding. By imparting bondability and hydrophilicity in the so-called binding type, the problems caused by coating with glass alone, plastic alone, collagen substrate, etc. as in the prior art can be solved, and sufficient hydrophilicity is exhibited and long-term stable hydrophilicity is achieved. It exhibits sexual maintenance, allows cell culture without coating with extracellular matrix proteins or polypeptides, is simple and of high quality, and can be manufactured easily and inexpensively.

Further, since this cell-adhesive hydrophilic modified cell culture base material rapidly develops hydrophilicity on the surface of a molded base material made of a material selected from rubber, resin, and glass, water, a buffer, or a buffer solution, or Upon contact with a weakly acidic, neutral, or weakly alkaline culture medium or medium, the desired hydrophilicity can be exhibited, and the cell culture performance and cell adhesion performance are not impaired. Its hydrophilicity can be maintained for half a year to several years while the contact angle with water is sufficiently low, about 20 ° or less. Therefore, even if it is stored, stored or used in the air or water for a long period of time, the hydrophilicity is not impaired, high quality can be maintained and guaranteed, and it is suitable for cell culture.

Furthermore, this cell-adherent hydrophilic modified cell culture substrate has low cell toxicity and suppresses protein adsorption, so that various cells to be cultured such as hepatocytes, stem cells, endothelial cells, iPS cells, and tumor cells It is expected that it promotes adhesion and does not easily inhibit proliferation.

Moreover, this cell-adhesive hydrophilic modified cell culture base material can easily impart uniform and sufficient hydrophilicity to the surface of a molded base material 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 and high yield with good reproducibility.

Microscopic magnified photograph (b) after culturing HeLa cells on a silicone hydrophilic modified cell culture substrate to which the present invention is applied, and microscopic enlargement after culturing HeLa cells on a silicone molded substrate to which the present invention is not applied. It is a photograph (a).

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

The cell-adhesive hydrophilic modified cell culture base material of the present invention is a cell-adhesive hydrophilic modified cell culture base material obtained by hydrophilizing a molded base material made of a material selected from rubber, resin, and glass. This cell-adhesive hydrophilic modified cell culture substrate repeatedly has amphoteric ions of an anion group and a cation group in the side chain as a betaine structure that reacts with, binds to, or adsorbs a surface functional group on the molded substrate. The molded substrate is coated with a copolymer having a unit and a repeating unit having an active functional group in the side chain that reacts, binds to, or adsorbs to a surface functional group on the molded substrate.

In this cell-adhesive hydrophilic modified cell culture base material, the betaine structure imparts so-called adsorption-type binding and hydrophilicity such as physical adsorption or chemisorption, preferably chemical adsorption, and the surface of the molded base material. It prevents surface functional groups and copolymers from sneaking into the molded base material and maintains bondability and hydrophilicity. In addition, this cell-adhesive hydrophilic modified cell culture substrate imparts so-called binding property and hydrophilicity such as reaction or bond preferably covalent bond via an active functional group, and is a copolymer. Keeps binding and hydrophilicity by preventing cells from sneaking into the molded substrate, cells adhere to the copolymerized main chain of the repeating structure of the copolymer and proliferate from there, and extracellular matrix proteins and poly It does not inhibit cell proliferation even without coating with a peptide, and is suitable for cell culture.

Therefore, this cell-adherent hydrophilic modified cell culture substrate has a betaine structure or activity due to the betaine structure and the active functional group reacting, binding, or adsorbing to the surface functional group on the molded substrate. The copolymer and the molded substrate interact more strongly than those having only one of the functional groups. As a result, the copolymer does not easily flow down from the molded substrate even when it comes into contact with water, an acidic test solution or an alkaline test solution, and exhibits strong hydrophilicity and continues to develop strong hydrophilicity for a long period of time. Since it can be cultured and cells can be cultured for a long period of time, it is expected that subculture, morphogenesis, cell differentiation, etc. will be performed.

In the copolymer, the repeating main chain that repeats the repeating unit is, for example, a poly (meth) acrylic skeleton. The poly (meth) acrylic skeleton includes a polyacrylic skeleton and a polymethacrylic skeleton. More specifically, the poly (meth) acrylic 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. Among them, when the copolymer has a poly (meth) acrylamide copolymer skeleton, it has an excellent affinity with a molded base material made of a material selected from rubber, resin, and glass, and also has an affinity with cells. Is particularly preferable.

The copolymer is not limited in the repeating format of the repeating unit, and may be any of, for example, a random copolymer, a block copolymer, an alternating copolymer, and a graft copolymer of these repeating units. The molar ratio of the repeating unit having the zwitterion in the side chain and the repeating unit having the active functional group in the side chain is not limited, but a molar ratio of 1: 1 is preferable.

In the copolymer, the repeating unit having a betaine structure in the side chain is such that an anionic group and a cation group react, bond, or adsorb to a surface functional group on the molding substrate, particularly like a hydroxyl group on the molding substrate. Adsorption such as physical adsorption or chemical adsorption is performed on the polar groups by mutual electrostatic attraction or ion attraction.

As a betaine structure, the side chain may have either one of an anion group and a cation group at the end of the side chain and the other in the side chain.

During betaine structure, the anionic group is, if the side chain terminal carboxylic acid group (-COO ^- group), a sulfonic acid group (-SO ₃ ^- group), a 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) They include anionic groups selected from, when in the side chain middle, substituted carboxylic acid groups branched from the side chain (-COO ^- group), substituted sulfonic acid groups branched from the side-chain (-SO ₃ ^- group), Phosphate group (-R ^b1- PO- (OR ^b2 ) (OR ^a3 ); R ^b1 is a group up to the middle of the ^{side chain, OR b2} is a group from the middle to the end of the side chain or O ^- anion, OR ^b3 is Anion groups selected from O ^- anion) can be mentioned.

In the betaine structure, when the cation group is at the end of the side chain, an organic ammonium group such as a primary to quaternary ammonium group ((-NH ₃ ) ⁺ , (-N (R ^c1 ) H ₂ ) ⁺ , (-N (R c1) H 2) +, (- N (R ^c2 ) ₂ H) ⁺ , (-N (R ^c3 ) ₃ ) ⁺ ; R ^{c2 to} R ^c3 are alkyl or phenyl groups with 1 to 6 carbon atoms), sulfonium groups, preferably organic sulfonium groups ((--) S (R ^c4 ) ₂ ) ⁺ ; R ^c4 is an alkyl group or phenyl group having 1 to 6 carbon atoms), a phosphonium group, preferably a quaternary phosphonium group ((-P (R ^c5 ) ₃ ) ⁺ ; R ^c5 is a carbon number. An organic ammonium group ((-R ^d1 −N (R ^d2 )) ₂ ((-R d1 −N (R d2)) 2 ( ^{R d3)) +;} R ^d2 represents a group having the halfway of the side ^{chains, R d2} alkyl group or a phenyl group having 1 to 6 carbon ^atoms, groups in the middle of ^{R d3} is the side chain to the end), a sulfonium group (-R ^d4- S ⁺ (R ^d5 ) -R ^d6 ); R ^c4 is a group up to the middle of the side chain, R ^{d5 is} an alkyl group or phenyl group having 1 to 6 carbon atoms, and R ^d6 is a group from the middle to the end of the side chain. ^{), Phosnium} group (-R ^d7- P ⁺ (R ^d8 ) _2- R d9); R ^d7 is a group up to the middle of the side chain, R ^d8 is an alkyl group or phenyl group having 1 to 6 carbon atoms, and R ^d9 is. A cation group selected from the group from the middle to the end of the side chain) can be mentioned.

The betaine structure is a surface functional group that is a polar group exposed on the surface of the molded 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. Reaction with surface functional groups such as hydroxyl groups, hydroxyl groups, carboxyl groups and carbonyl groups generated by the 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, adsorption such as physical adsorption or chemical adsorption is performed by mutual electrostatic attraction or ion attraction of the surface functional group and the betaine structure, and strongly interacts with the surface of the molded base material.

で表されるものが挙げられる。 In the copolymer, the repeating unit having a betaine structure includes those represented by the above chemical formulas (1) or (2) as a preferable example, and more specifically, the following chemical formulas (4) to (2). 7)

The one represented by is mentioned.

Further, in the copolymer, the repeating unit having an active functional group in the side chain is bonded to the surface functional group on the molded substrate by reaction, bond, or adsorption, particularly covalent bond.

The active functional group is an azide group (-N ₃ ), a sulfo group (-SO ₃ ), a trialkoxysilyl group (-Si (OR ^e1 ) ₃ ; OR ^e1 is an alkyl group or a phenyl group having 1 to 6 carbon atoms), Examples thereof include at least one functional group selected from a hydroxyl group precursor group such as a hydroxyl group (−OH) or a hydroxyl group forming a hydroxyl group, for example, an alkoxy group having 1 to 6 carbon atoms.

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

Further, in the repeating unit having an active functional group in the copolymer in the side chain, the side chain is a hydrocarbon aromatic ring group such as phenyl or naphthyl, or a non-aromatic complex such as piperazinyl, pyreridinyl, pyrozoxinyl or morpholinyl. Aromatic heterocyclic groups such as ring groups, 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 chain and / or cyclically saturated or unsaturated hydrocarbon groups such as benzyl or phenethyl, amide groups (-CO-N). (R ^g1 )-; R ^g1 is an alkyl group or phenyl group having 1 to 6 carbon atoms), any single spacer group selected from an ester group (-CO-O-), or a combination of at least one of them. The composite spacer group may have the active functional group.

The active functional groups in the copolymer are surface functional groups that are polar groups exposed on the surface of the molded substrate, corona discharge-treated surfaces, plasma-treated surfaces, ultraviolet-treated surfaces, excima-treated surfaces, and the surface of the molded substrate. / Or a surface functional group such as a hydroxyl group, a hydroxyl group, a carboxyl group or a carbonyl group generated by the surface treated with a silane coupling agent, or a surface such as an amino group, an alkoxy group or an alkoxysilyl group contained in a molding base material. Reaction, bond, or adsorption with a functional group A covalent bond is formed by chemically reacting with a surface functional group in particular, and a copolymer molecule is attached to the surface of the molded substrate.

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 preferable that the polymer has 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, and m1 and m2 and m3 and m4 are arbitrary positive numbers that form the ratio of the molecular weight to the repeating unit). It is preferable that the product is used.

The molded base material is preferably rubber, resin, or glass. More preferably, it is made of elastic or soft silicone rubber, or hard silicone resin. It is preferable that it has any of a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton because it is easily available and easy to manufacture.

The cell-adhesive hydrophilic modified cell culture substrate exhibits excellent hydrophilicity with a water contact angle of 20 ° or less by having such a structure. The untreated molded substrate has a water contact angle of about 40 to 108 °, whereas the cell-adherent hydrophilic modified cell culture substrate has a water contact angle of 20 ° or less immediately after preparation, and is at room temperature or lower. Even if it is stored in a clean room for half a year to one year or more, the water contact angle is 20 ° or less and high hydrophilicity can be maintained.

The cell-adhesive hydrophilic modified cell culture substrate was confirmed to have low cell toxicity to HeLa cells, which are cells derived from human cervical cancer, and V79 cells, which are fibroblasts derived from Chinese hamster lungs. In addition, since the 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 and proliferate on the hydrophilic surface of the cell-adhesive hydrophilic modified cell culture substrate, and the development and regeneration of a new cell culture substrate that does not require extracellular matrix. It is expected to be applied to iPS cells and the like that are useful in medicine.

Since the cell-adhesive hydrophilic modified cell culture substrate is a sterilization-resistant substrate, it is exposed to various sterilization conditions such as autoclave sterilization, ethylene gas oxide sterilization, γ-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 atm for 20 minutes, the water contact angle can be maintained at 20 ° or less even after 5 months have passed. The angle is 20 ° or less and high hydrophilicity can be maintained. Therefore, when used in medical devices, even if sterilized, the effect of hydrophilicity can be exhibited for a sufficiently long period of time.

Such a cell-adhesive hydrophilic modified cell culture substrate can be prepared as follows.

First, the 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 described below. It is synthesized as follows.

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

The amino group of 4-aminobenzoic acid is diazotized and then azide to acid chloride, and then one of the amino groups of piperidine is reacted with monoprotected piperidine protected by a protecting group to amidate, and then the protective group. To obtain the mono-p-azilated benzoate piperazinamide. Chloride methacrylate and 6-aminocaproic acid are amidated and further amidated with the free amino group of the mono-p-azilated benzoic acid piperazinamide to form a repeating unit as represented by the chemical formula (2). To synthesize an active functional group-containing comonomer for this purpose.

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

When 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) are copolymerized, the above-mentioned A copolymer represented by the chemical formula (8) or (9) can be obtained.

It should be noted that another comonomer having a different number of carbon atoms, for example, a betaine structure-containing comonomer for forming a repeating unit represented by the chemical formula (1) or (2) or a repeating unit represented by the chemical formula (3). The active functional group-containing comonomer can be synthesized in the same manner by adjusting the starting material.

The copolymer may be diluted with water, a water-soluble organic medium such as alcohol or acetone, or a water-insoluble organic medium such as methylene chloride, chloroform or ether, and the copolymer concentration may be high. High hydrophilicity can be exhibited when the content is 0.001 to 10 wt%, and more preferably 0.01 to 1.0 wt%.

A molded base material 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, on the surface of the molded base material. In addition to the original one, a new surface functional group is generated. Further, by using masking or the like, it is possible to generate a surface functional group on a part of the surface of the molded base material, and it is possible to selectively develop hydrophilicity.

Then, the copolymer liquid is applied by a method such as spraying, coating, or dipping, and if necessary, the medium is removed by a method such as volatilization to obtain a molded base material to which the copolymer is attached. At this stage, the betaine structure of the copolymer electrostatically interacts with the surface functional groups on the surface of the molded substrate, and the copolymer is adsorbed by physical adsorption or chemisorption. , While being firmly attached, it exhibits hydrophilicity.

Next, the betaine structure of the copolymer to which the copolymer is attached is irradiated with ultraviolet rays or light irradiation treatment, preferably with a wavelength of 220 to 410 nm, and the integrated light amount is arbitrary ultraviolet rays, whereby the azide group is decomposed to form a nitrene group. It occurs and reacts with unsaturated groups, alkyl groups, phenyl groups, amino groups and / or expands the ring to have surface functional groups on the molded substrate, or the main components of the molded substrate and other curable components. By reacting with an unsaturated group, an alkyl group, a phenyl group, or an amino group and / or expanding the ring to react or bond, a covalent bond is formed, and the copolymer is firmly attached by a chemical bond. At the same time, it exhibits hydrophilicity.

In this way, a cell-adhesive hydrophilic 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 block group, and silanol which is a surface functional group on a molded 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 be strongly attached by a chemical bond while exhibiting hydrophilicity.

Cell-adhesive hydrophilic modified cell culture substrates are used for applications that exhibit hydrophilicity. For example, it is used for medical instruments such as vascular catheters, cell culture containers, microchannel chips, etc. so as to be easily compatible with the water content of the living body.

Hereinafter, examples of a cell-adhesive hydrophilic modified cell culture substrate to which the present invention is applied and comparative examples to which the present invention is not applied will be described while comparing them.

First, a copolymer having a betaine structure-containing repeating unit and an active functional group-containing repeating unit used for preparing a cell-adhesive hydrophilic modified 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 methacrylamide propyl sulfobetaine

1.70 g (10 mmol) of dimethylaminopropylmethacrylamide and 120 ml of dehydrated acetone were placed in a three-necked flask (300 ml), and the mixture was stirred at room temperature. 20 ml of a dehydrated acetone solution of 1.83 g (15 mmol) of 1,3-propanesaltone 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 to distill off the solvent, and the solid was taken out and dried under reduced pressure to obtain methacrylamidepropylsulfobetaine as a colorless solid in a yield of 98%, m / z: 293. (M + H ⁺ ), and its 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 phenyl azide 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 the mixture was stirred at 0 ° C. 50 ml of a dioxane solution of 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 a colorless solid monoBoc piperazine with a crude yield of 80%, which was used as it was 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-azinate benzoic acid

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 the mixture was stirred at 0 ° C. 100 ml of an aqueous solution of 5.18 g (75 mmol) of sodium nitrite was added dropwise, and the mixture was 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 the mixture was 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 dried under reduced pressure to obtain a pale yellow solid p-azilated benzoic acid in a crude yield of 100%, which was used as it was 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-azidated benzoic acid chloride

3.26 g (20 mmol) of crude azinated benzoic acid, 5.08 g (40 mmol) of oxalyl chloride, and 300 mL of benzene were placed in an eggplant flask (500 ml), and the mixture was stirred at room temperature. 4 drops of DMF were added and the mixture was stirred at room temperature for 6 hours. When benzene and oxalyl chloride were distilled off and dried under reduced pressure, p-azilated benzoic acid chloride in a brown liquid could be obtained in a crude yield of 100%, and was used as it was 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-azinate benzoic acid Boc piperazine amide

4.95 g (90%, 24 mmol) of crude monoBoc piperazine, 6.06 g (60 mmol) of triethylamine, and 300 ml of THF were placed in a three-necked flask (500 ml), and the mixture was stirred at 0 ° C. 50 ml of a THF solution of 3.63 g (20 mmol) of crude azide benzoic acid chloride was added dropwise, and the mixture was further stirred at room temperature for 12 hours. THF was distilled off, and the obtained residue was poured into 200 ml of 5 wt% hydrochloric acid and extracted with 200 ml of chloroform. After distilling off chloroform and purifying the residue by gel permeation chromatography (GPC), a light brown solid p-azinate benzoic acid Boc piperazine amide was obtained in 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-azidated benzoic acid piperazine amide

3.31 g (10 mmol) of Boc piperazine amide azide benzoic acid was placed in an eggplant flask (200 ml), and the mixture was stirred at 0 ° C. 20 ml of trifluoroacetic acid was added, and the mixture was 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 obtained residue, and the mixture was extracted twice with 100 ml of chloroform. Chloroform and piperazine as a by-product were distilled off and dried under reduced pressure to obtain a brown solid mono-p-azinate benzoic acid piperazine amide in a crude yield of 90%, which was used as it was 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 methacrylamide piperazine-p-phenyl azide amide monomer

1.46 g (5 mmol) of methacrylamide propyl sulfobetaine, 1.01 g (10 mmol) of triethylamine, and 60 ml of THF were placed in a three-necked flask (100 ml), and the mixture was stirred at 0 ° C. 15 ml of a THF solution of 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 obtained residue was poured into 100 ml of a 5 wt% sodium carbonate aqueous solution and extracted with 100 ml of chloroform. Chloroform was distilled off and recrystallized (hexane: chloroform = 25: 5 mL) to remove tar-like impurities, and a light brown solid methacrylamide piperazine-p-phenyl azide amide monomer was obtained in 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 with copolymer [Chemical formula (9)]

Put 1.46 g (5 mmol) of methacrylamide propyl sulfobetaine, 15.0 mg (0.05 mmol) of methacrylamide piperazine-p-phenyl azideamide monomer, 11.4 mg of ammonium persulfate, and 5 ml of water in an eggplant flask (100 ml), and aluminum foil. It was covered with and made into a uniform solution. The eggplant flask was immersed in a water bath at 70 ° C. for 1 hour for polymerization. 5 ml of water was added to the reaction mixture, the mixture was stirred, and the solution was poured into 50 ml of methanol. When the produced precipitate was collected, washed with methanol and dried under reduced pressure, a pale yellow solid random copolymer [chemical formula (9)] was obtained in a yield of 92%, and the number average molecular weight was 92000 as measured by GPC. 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. A copolymer [chemical formula (9)] was prepared and used in a 0.1 wt% aqueous solution. The base material was subjected to plasma treatment or excimer treatment 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 substrate, it was irradiated with ultraviolet rays to obtain a hydrophilic modified cell culture substrate.

(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, Ltd .: Iupiron 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 Co., Ltd .: S9213) was used as the substrate.

(Comparative Example 1)
A hydrophilic modified cell culture substrate was prepared in the same manner as in Example 1 except that a phosphorylcholine polymer having a number average molecular weight of (i) 95000 or (ii) 253000 was used.

(Measurement of contact angle)
The hydrophilicity of the obtained hydrophilic modified cell culture substrate was evaluated by the contact angle of water. One drop of water was dropped onto the obtained hydrophilic modified cell culture substrate, and 10 seconds later, the contact angle with water was measured with an automatic contact angle meter (manufactured by Kyowa Interface Science Co., Ltd .; DM-501). Five to seven points were measured for each test piece, and the average value was calculated. The results are shown in Table 1.

As is clear from Table 1, the contact angle was reduced by performing the hydrophilic modification treatment, and it was found that the hydrophilicity was modified. Further, from Examples 1, 2 and 3, it was found that the hydrophilicity was modified regardless of the type of the base material.

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

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

(Cell culture test)
A cell culture test of a hydrophilic modified cell culture substrate was carried out. First, a hydrophilic modified cell culture substrate was fixed on a polystyrene dish, and the culture solution was dispensed into the dish. Then, HeLa cells were seeded on the surface of the hydrophilic modified cell culture substrate, and cultured in a carbon dioxide incubator at a temperature of 37 ° C. and a CO ₂ concentration of 5% for 3 days. FIG. 1 shows a microscopic magnified photograph (b) after culturing HeLa cells on the hydrophilic modified cell culture substrate of Example 1 and a microscopic enlargement after culturing HeLa cells on a silicone molded substrate to which the present invention is not applied. It is shown by the photograph (a).

As is clear from Table 2 and FIG. 1, it was confirmed that neither Example 1 nor Comparative Example 1 had cytotoxicity. In cell culture, cell proliferation could be confirmed in Example 1, whereas proliferation could not be confirmed in Comparative Example. As a factor for this, it is generally necessary to form an extracellular matrix on the substrate, which is essential for cell adhesion, during cell culture. Therefore, it is considered that the cell culture of Comparative Example 1 could not be performed. On the other hand, in Example 1, although the extracellular matrix was not formed in the same manner, cell culture was possible. From this, it was confirmed that the present invention enables cell culture without coating with extracellular matrix protein or polypeptide.

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

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

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

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

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

(Evaluation of protein adsorption)
A protein adsorption test of a hydrophilic modified cell culture substrate was carried out. A 30 × 30 cm hydrophilic modified cell culture substrate was immersed in albumin solution for 2 hours. After immersion, the hydrophilic modified cell culture substrate was taken out, fluorescently labeled with Cy3 dye-labeled carboxylic acid, and the 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. A similar test was performed on an untreated silicone rubber sheet for comparison.

Table 4 As is apparent from the adsorption of protein, whereas in the silicone rubber sheet of raw albumin adsorption capacity was 2.5 × 10 ^5, Example 1, the albumin adsorption capacity is 1.2 × 10 ⁵ It 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 adsorbed.

From the above results, the present invention can easily impart sufficient hydrophilicity to the surface of the molded substrate, rapidly develop hydrophilicity, and maintain the contact angle with water at a sufficiently low cell. Cell culture can be performed without coating with an outer matrix or polypeptide, culture performance can be maintained even when stored, stored or used in the air or water for a long period of time, and cell-adhesive hydrophilicity modification that can ensure hydrophilicity for a long period of time. It was confirmed that it can be used as a substrate for culturing cells.

The cell-adhesive hydrophilic modified cell culture substrate of the present invention includes medical instruments such as vascular catheters that require long-term maintenance while exhibiting hydrophilicity, cell culture vessels, microchannel chips, organ-on. It is used for -a-chip and the like.

Claims (16)

A molded base material 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, and the copolymer. A cell-adhesive hydrophilic modified cell culture substrate, characterized in that the betaine structure inside or the active functional group reacts with, binds to, or adsorbs a surface functional group on the molded substrate. The cell-adhesive hydrophilic modified cell culture substrate according to claim 1, wherein cell culture is possible without coating the surface of the substrate with a protein and / or polypeptide. The cell-adhesive hydrophilic modification according to any one of claims 1 and 2, wherein in the copolymer, the main chain repeating the repeating unit is a poly (meth) acrylic skeleton. Cell culture substrate. In the copolymer, the poly (meth) acrylic 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 to 1, wherein the betaine structure has an anion group selected from a carboxylic acid group, a sulfonic acid group, and a phosphoric acid group, and a cation group selected from an ammonium group, a sulfonium group, and a phosphonium group. 4. The cell-adhesive hydrophilic modified cell culture substrate according to any one of 4. The cell-adhesive hydrophilic modified cell culture substrate according to claim 5, wherein the betaine structure has the anion group or the cation group at the end of the side chain. The cell adhesion according to any one of claims 1 to 6, wherein 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. Hydrophilic modified cell culture substrate. At least selected from hydrocarbon aromatic ring groups, non-aromatic heterocyclic groups, aromatic heterocyclic groups, linear, branched chain and / or cyclically saturated or unsaturated hydrocarbon groups, amide groups, and ester groups. The cell-adhesive hydrophilic modification according to any one of claims 1 to 7, wherein any of the spacer groups 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 the formulas (1) and (2), R ¹ and R ² are hydrogen atoms or methyl groups, and n1 to n4 are numbers 2 to 6), and the repeat having the betaine structure. Unit and the following chemical formula (3)

(In the formula (3), R ³ is a hydrogen atom or a methyl group, n5 is a number of 2 to 6, n6 is a number of 0 to 1, and R ⁴ is the active functional group). One of claims 1 to 8, wherein the copolymer is a random copolymer, a block copolymer, an alternating copolymer, or a graft copolymer with a repeating unit having an active functional group. The cell-adhesive hydrophilic modified cell culture substrate according to the description. The invention according to any one of claims 1 to 9, wherein the molded base material is made of the rubber selected from silicone rubber, fluorine rubber, ethylene-propylene-diene ternary copolymer rubber, and urethane rubber. Hydrophilic modified cell culture substrate. The hydrophilic modification according to any one of claims 1 to 9, wherein the molded base material is made of the resin selected from a silicone resin, a cycloolefin resin, a polycarbonate resin, a polyethylene terephthalate resin, and an acrylic resin. Plastic cell culture substrate. The hydrophilic modified cell culture substrate according to any one of claims 1 to 9, wherein the molded substrate is made of the glass selected from quartz glass, borosilicate glass, and soda-lime glass. One of claims 1 to 12, wherein 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. The cell-adhesive hydrophilic modified cell culture substrate according to the description. 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. Sexual hydrophilic modified cell culture substrate. The invention according to any one of claims 1 to 14, wherein the betaine structure is bonded or adsorbed to the surface functional group by an ionic bond, and the active functional group is bonded by an ionic bond or a covalent bond. Cell-adhesive hydrophilic modified cell culture substrate. Claims 1 to 15 wherein the molded base material is made of a silicone rubber or a silicone resin having any one selected from a polydimethylsiloxane skeleton, a polymethylphenylsiloxane skeleton, and a polymethylhydrogensiloxane skeleton. The cell-adhesive hydrophilic modified cell culture substrate according to any one of.

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