JP6960726B2 - Random copolymer of N-vinylimidazolidine-2-one compound and (meth) acrylic acid ester derivative and cell culture material using it - Google Patents

Description

The present invention relates to a random copolymer of an N-vinylimidazolidine-2-one compound and a (meth) acrylic acid ester compound, which is expected to be applied as a material for cell culture or a temperature-responsive polymer material.

In recent years, there has been active development of temperature-responsive materials synthesized using stimulus-responsive materials, particularly N-isopropylacrylamide (NIPAAM). In the case of NIPAAM homopolymers, the lower critical solution temperature (LCST) is 32 ° C. near body temperature, and at lower temperatures the NIPAAM homopolymers are soluble in water, but at higher temperatures they are insoluble in water. It has been known. Copolymers of NIPAAM and various comonomer have been synthesized, and their application to drug release control materials, chromatographic separation gels, cell culture beds, and the like is being investigated. In addition, there are reports that LCST is regulated and the affinity for a specific protein is enhanced by controlling the type and composition of comonomer (see Non-Patent Documents 1 to 4).

On the other hand, Patent Documents 1 and 2 describe N-vinylimidazolidine-2-one derivatives and polymers thereof. Specifically, poly (1-vinylimidazolidine-2-one), poly (1-methyl-3-vinylimidazolidine-2-one), poly (1-ethyl-3-vinylimidazolidine-2-one) And synthetic examples of poly (1-butyl-3-vinylimidazolidine-2-one), or bactericidal compositions using poly (1-methyl-3-vinylimidazolidine-2-one) are shown. However, a description of the synthesis of a random copolymer of an N-vinylimidazolidine-2-one compound and a (meth) acrylic acid ester derivative according to the present invention, and a cell culture material and a temperature-responsive material using the same. There is no.

Japanese Unexamined Patent Publication No. 2001-172262.

Japanese Unexamined Patent Publication No. 2001-172333.

Langmuir, Vol. 14, pp. 4657-4662 (1998).

Macromolecules, Vol. 33, pp. 8312-8316 (2000).

Biomacromolecules, Vol. 5, pp. 505-510 (2004).

Langmuir, Vol. 28, pp. 16623-16637 (2012).

Provided is a random copolymer of an N-vinylimidazolidine-2-one compound and a (meth) acrylic acid ester derivative, which can be applied as a material for cell culture.

As a result of diligent studies to solve the above problems, the present inventors have made a coating agent component of a random copolymer of an N-vinylimidazolidine-2-one compound having a specific structure and a (meth) acrylic acid ester derivative. It has been found that the surface of the carrier can be easily treated and the carrier obtained by treating with a coating agent can be used as a material for cell culture, and the present invention has been completed.

That is, the present invention
The following general formula (1)

（式中、Ｒ^１は水素原子、炭素数１〜１２のアルキル基または炭素数１〜１２のアシル基を表す。）で表される繰り返し単位と、下記一般式（２）

(In the formula, R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or an acyl group having 1 to 12 carbon atoms), and a repeating unit represented by the following general formula (2).

（式中、Ｒ^２は水素原子またはメチル基を表す。Ｒ^３は炭素数１〜１２のアルキル基、または炭素数１〜６のアルキル基もしくは炭素数１〜６のアルコキシ基で置換されていてもよいフェニル基を表す。）で表される繰り返し単位からなるランダム共重合体（以下、本発明のランダム共重合体と称する。）に関するものであり、下記一般式（３）

(In the formula, R ² represents a hydrogen atom or a methyl group. R ³ is substituted with an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. It relates to a random copolymer composed of a repeating unit represented by (representing a good phenyl group) (hereinafter, referred to as a random copolymer of the present invention), and relates to the following general formula (3).

（式中、Ｒ^１は水素原子、炭素数１〜１２のアルキル基または炭素数１〜１２のアシル基を表す。）で表されるＮ−ビニルイミダゾリジン−２−オン化合物、および下記一般式（４）

(In the formula, R ¹ represents a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or an acyl group having 1 to 12 carbon atoms.) An N-vinylimidazolidine-2-one compound represented by a hydrogen atom, and the following general formula. (4)

（式中、Ｒ^２は水素原子またはメチル基を表す。Ｒ^３は炭素数１〜１２のアルキル基、または炭素数１〜６のアルキル基もしくは炭素数１〜６のアルコキシ基で置換されていてもよいフェニル基を表す。）で表される（メタ）アクリル酸エステル誘導体をラジカル重合させるＮ−ビニルイミダゾリジン−２−オン化合物と（メタ）アクリル酸エステル誘導体とのランダム共重合体の製造法に関するものである。さらに本発明のランダム共重合体を含むコート剤、および該コート剤で担体表面を処理して得られる細胞培養用材料に関するものである。

(In the formula, R ² represents a hydrogen atom or a methyl group. R ³ is substituted with an alkyl group having 1 to 12 carbon atoms, or an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. A method for producing a random copolymer of an N-vinylimidazolidine-2-one compound and a (meth) acrylic acid ester derivative, which radically polymerizes a (meth) acrylic acid ester derivative represented by (). It is about. Further, the present invention relates to a coating agent containing the random copolymer of the present invention, and a cell culture material obtained by treating the surface of a carrier with the coating agent.

In the repeating unit and the formula (3) of N- vinyl-2-one compounds of the general formula (1), the alkyl group having 1 to 12 carbon atoms represented by R ¹ has a branched and / or cyclic structure Also, methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, cyclobutyl group, pentyl group, neopentyl group, isopentyl group, tert-pentyl group. Group, 1-methylbutyl group, 1-ethylpropyl group, cyclobutylmethyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group, heptyl Group, 1-methylhexyl group, 1-ethylpentyl group, cyclohexylmethyl group, cycloheptyl group, octyl group, 1-methylheptyl group, 2-ethylhexyl group, nonyl group, decyl group, 3,7-dimethyloctyl group, Examples thereof include an undecyl group and a dodecyl group. Acyl group having 1 to 12 carbon atoms represented by R ¹ may have a branched and / or cyclic structures, a formyl group, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, pentanoyl group, 3-methylbutyryl group, 2 , 2-Dimethylpropanoyl group, hexanoyl group, heptanoyl group, cyclohexanecarbonyl group, octanoyl group, nonanoyl group, decanoyl group, dodecanoyl group and the like can be exemplified. ^{R 1} is a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a tert-butyl group, an acetyl group, a propionyl group, and a butyryl group because of its ease of synthesis and suitability for cell culture. Alternatively, it is preferably any of an isobutyryl group, more preferably an ethyl group, a propyl group, an isopropyl group, an acetyl group or a propionyl group.

In the repeating unit of the general formula (2) and the (meth) acrylic acid ester derivative of the general formula (4), the alkyl group having 1 to 12 carbon atoms represented ^{by R 3 may have a branched and / or cyclic structure.} Methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, cyclobutyl group, pentyl group, neopentyl group, isopentyl group, tert-pentyl group, 1 -Methylbutyl group, 1-ethylpropyl group, cyclobutylmethyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group, heptyl group, 1 -Methylhexyl group, 1-ethylpentyl group, cyclohexylmethyl group, cycloheptyl group, octyl group, 1-methylheptyl group, 2-ethylhexyl group, nonyl group, decyl group, 3,7-dimethyloctyl group, undecyl group, Examples thereof include a dodecyl group. Examples of the phenyl group represented by R ³ which may be substituted with an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms include a phenyl group, a 2-methylphenyl group, a 4-methylphenyl group, and 2, 4-Dimethylphenyl group, 2,4,6-trimethylphenyl group, 4-ethylphenyl group, 4-propylphenyl group, 4-isopropylphenyl group, 3-methyl-4-isopropylphenyl group, 4-butylphenyl group, 4-tert-Butylphenyl group, 4-pentylphenyl group, 4- (2-methylbutyl) phenyl group, 4- (1,1-dimethylpropyl) phenyl group, 4-hexylphenyl group, 2-tert-butyl-4 , 6-Dimethylphenyl group, 2,4-di-tert-butylphenyl group, 2,6-di-tert-butyl-4-methylphenyl group, 2-methoxyphenyl group, 4-methoxyphenyl group, 2-methoxy -4-Methylphenyl group, 2,6-dimethoxy-4-methylphenyl group, 4-ethoxyphenyl group, 4-propoxyphenyl group, 4-butoxyphenyl group, 4-amyloxyphenyl group, 4-hexyloxyphenyl group Etc. can be exemplified. Ease of synthesis, and from the viewpoint of excellent coatability, ^{R 3} is a methyl group, an ethyl group, a propyl group, an isopropyl group, butyl group, isobutyl group, sec- butyl group, tert- butyl group, a pentyl group, a neopentyl group, Isopentyl group, tert-pentyl group, 1-methylbutyl group, 1-ethylpropyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group, Alternatively, a phenyl group is preferable.

Specifically, as the (meth) acrylic acid ester derivative (4), methyl acrylate, ethyl acrylate, propyl acrylate, isopropyl acrylate, butyl acrylate, isobutyl acrylate, sec-butyl acrylate, tert-acrylate. Butyl, pentyl acrylate, neopentyl acrylate, isopentyl acrylate, tert-pentyl acrylate, 1-methylbutyl acrylate, 1-ethylpropyl acrylate, cyclopentyl acrylate, hexyl acrylate, 1-methylpentyl acrylate, acrylate 4-Methylpentyl, 1-ethylbutyl acrylate, 2-ethylbutyl acrylate, cyclohexyl acrylate, phenyl acrylate, methyl methacrylate, ethyl methacrylate, propyl methacrylate, isopropyl methacrylate, butyl methacrylate, isobutyl methacrylate, methacryl Sec-butyl acid, tert-butyl methacrylate, pentyl methacrylate, neopentyl methacrylate, isopentyl methacrylate, tert-pentyl methacrylate, 1-methylbutyl methacrylate, 1-ethylpropyl methacrylate, cyclopentyl methacrylate, hexyl methacrylate, Examples thereof include 1-methylpentyl methacrylate, 4-methylpentyl methacrylate, 1-ethylbutyl methacrylate, 2-ethylbutyl methacrylate, cyclohexyl methacrylate, and phenyl methacrylate.

When producing the random copolymer of the present invention, radical polymerization can be used to easily and efficiently obtain the polymer. As a more specific radical polymerization method, known methods such as bulk polymerization, solution polymerization, and emulsion polymerization using free radical polymerization can be mentioned. Any amount of radical initiator can be added to initiate radical polymerization more efficiently. Examples of radical initiators preferably used in the reaction include inorganic peroxides such as potassium persulfate and ammonium persulfate, which are called polymerization accelerators N, N, N', N'-tetramethylethylenediamine, N, N-. Rapid polymerization at low temperatures is possible when used in combination with amine compounds such as dimethylparatoluidine. Further, as radical initiators, organic peroxides such as dilauroyl peroxide, benzoyl peroxide, di-tert-butyl peroxide, tert-butyl hydroperoxide and cumene hydroperoxide, and α, α-azobisisobutyronitrile (AIBN) And azo compounds such as azobiscyclohexanecarbonitrile can be exemplified.

The random copolymer of the present invention can be produced by mixing the N-vinylimidazolidine-2-one compound (3) and the (meth) acrylic acid ester derivative (4) in an arbitrary ratio and performing radical polymerization. .. Usually, the random copolymer can be produced with good reproducibility by polymerizing in an atmosphere of an inert gas such as nitrogen gas or argon gas or under deoxidation, and the polymerization reaction proceeds smoothly when a solvent is used. Solvents include, for example, water, methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexyl alcohol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, etc. Carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, ethyl acetate, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide (DMF), N, N-dimethylacetamide, Ammonia, trimethylamine, diethylamine, triethylamine, diisopropylamine, butylamine, dibutylamine, tributylamine, pyridine, picolin and the like can be used, but the present invention is not limited thereto. In addition, two or more of these solvents can be mixed and used. The polymerization reaction usually proceeds smoothly in the range of 0 ° C. to 100 ° C.

The ratio of the repeating unit (1) to the repeating unit (2) in the random copolymer of the present invention is not particularly limited as long as the coating on the carrier surface and cell culture are possible, but generally, the repeating unit (1): The repeating unit (2) = 1: 9 to 9: 1 (weight ratio), preferably 2: 8 to 8: 2. This ratio can be obtained as a number average value from the integrated value of the spectrum derived from each repeating unit by measuring a nuclear magnetic resonance spectrum such as ^{1 H-NMR, and can be obtained by converting it into a weight ratio.}

The molecular weight of the random copolymer of the present invention can be used depending on the measuring method such as weight average molecular weight, number average molecular weight, and viscosity average molecular weight. The weight average molecular weight (Mw) is preferably 1000 to 1000000, and more preferably 5000 to 500000 from the viewpoint of controlling the properties of the polymer and processability. The molecular weight distribution (PD) is not particularly limited, but is preferably in the range of about 1 to 20, and more preferably in the range of 1 to 5 from the viewpoint of polymer uniformity. Examples of the method for calculating the molecular weight include known methods such as gel permeation chromatography (GPC) method, viscosity method, and light scattering method in which a standard sample such as polystyrene or polyethylene glycol is converted as a reference.

A coating agent can be prepared by dissolving the random copolymer of the present invention in a solvent. The solvent used for the coating agent is not particularly limited as long as the solvent can be removed after dissolving the random copolymer of the present invention and applying it to the surface of a carrier or the like, but water, methanol, ethanol, propanol, isopropyl alcohol, butanol, etc. Hexanol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, Dioxane, acetonitrile, dimethylsulfoxide, DMF, N, N-dimethylacetamide, N-methylpyrrolidin-2-one, ethylene glycol, 2-methoxyethanol, 2-butoxyethanol and the like can be used, but are limited thereto. It's not a thing. In addition, two or more of these solvents can be mixed and used.

The concentration of the coating agent is not particularly limited as long as it does not interfere with the coating, but it is generally preferable to adjust the concentration of the random copolymer of the present invention to 0.01 to 20% by weight.

The surface of a carrier having various shapes such as a petri dish made of various materials such as glass and resin, a bag, a sponge-like porous base material, a granular porous base material, and a fiber base material such as a non-woven fabric or a woven fabric is provided in the present invention. A material for cell culture can be prepared by treating with a coating agent containing a random copolymer. The method for treating the coating agent is not particularly limited, and a known method can be used. For example, a dip method, a spray method, a casting method, a spin casting method, an inkjet printing method, or the like can be used.

By treating the surface of carriers of various materials and shapes with a coating agent containing the random copolymer of the present invention, the influence of the surface of these carriers on cells can be reduced, and cell culture, storage of cultured cells, and culture can be performed. Since it can improve the convenience of handling such as cell recovery and transfer, it is expected to be applied as a biocompatible material and a material for cell culture.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The molecular weight of the obtained polymer was determined from the results of GPC. The GPC system is a GL-7400 manufactured by GL Science Co., Ltd. (detector: GL-7456, column (4): TSKgel Super H5000, H4000 × 2, H2000, column temperature: 40 ° C., developing solvent: 0.01 M LiCl DMF solution. , Standard polystyrene conversion) was used. The structure and copolymerization ratio of the random copolymer were determined from the results of ^{1 H-NMR measurement using AVANCE III-400 manufactured by Bruker-Biospin.}

Reference example 1

２００ｍＬナスフラスコにイミダゾリジン−２−オン１０．３ｇ（１２０ｍｍｏｌ）およびプロピオン酸無水物１００ｍＬを加えて、１５０℃で１時間撹拌した。室温まで放冷して析出した固体を回収し、少量の冷エタノールで洗浄することにより、無色固体の１−プロピオニルイミダゾリジン−２−オン１３．０ｇ（収率：７６．２％）を得た。^１Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，ｐｐｍ），δ：１．６６（３Ｈ，ｔ，Ｊ＝７．４Ｈｚ），２．９３（２Ｈ，ｑ，Ｊ＝７．４Ｈｚ），３．５０（２Ｈ，ｔ，Ｊ＝８．１Ｈｚ），３．９６（２Ｈ，ｔ，Ｊ＝８．１Ｈｚ），５．６８（１Ｈ，ｂｓ）．ＥＩ−ＭＳ，ｍ／ｚ：１４２（Ｍ）^＋，１２７（Ｍ−ＣＨ_３）^＋，１１３（Ｍ−Ｃ_２Ｈ_５）^＋，１００．ＩＲ（ｎｅａｔ，ｃｍ^−１），ν：３２３０（ｗ），３１２９（ｗ），２９８３（ｗ），２８９８（ｗ），１７３４（ｍ），１６７０（ｍ），１３７７（ｍ），１２６５（ｍ），１１５９（ｍ），１０６２（ｍ），１０２６（ｍ），９４１（ｍ）。

10.3 g (120 mmol) of imidazolidine-2-one and 100 mL of propionic anhydride were added to a 200 mL eggplant flask, and the mixture was stirred at 150 ° C. for 1 hour. The solid was allowed to cool to room temperature, and the precipitated solid was recovered and washed with a small amount of cold ethanol to obtain 13.0 g (yield: 76.2%) of 1-propionylimidazolidine-2-one as a colorless solid. .. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.66 (3H, t, J = 7.4 Hz), 2.93 (2H, q, J = 7.4 Hz), 3.50 (2H) , T, J = 8.1Hz), 3.96 (2H, t, J = 8.1Hz), 5.68 (1H, bs). EI-MS, m / z: 142 (M) ⁺ , 127 (M-CH ₃ ) ⁺ , 113 (MC ₂ H ₅ ) ⁺ , 100. IR (neat, cm ^-1 ), ν: 3230 (w), 3129 (w), 2983 (w), 2898 (w), 1734 (m), 1670 (m), 1377 (m), 1265 (m) , 1159 (m), 1062 (m), 1026 (m), 941 (m).

アルゴン雰囲気下、１００ｍＬのシュレンク管に１−プロピオニルイミダゾリジン−２−オン２．８６ｇ（２０．１ｍｍｏｌ）、ヨウ化銅（Ｉ）０．１９ｇ（１．０ｍｍｏｌ）、Ｎ，Ｎ’−ジメチルエチレンジアミン０．２１ｍＬ（２．０ｍｍｏｌ），炭酸カリウム５．５３ｇ（４０．０ｍｍｏｌ）および１Ｍの臭化ビニルのＴＨＦ溶液４０．０ｍＬ（４０．０ｍｍｏｌ）を加えて、７０℃で７時間撹拌した。反応液をセライトで濾過して濾液を濃縮した。アルミナカラムクロマトグラフィー（展開液：クロロホルム）で精製することにより、無色固体の１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ）２．８３ｇ（収率：８３．６％）を得た。^１Ｈ−ＮＭＲ（４００ＭＨｚ，ＣＤＣｌ_３，ｐｐｍ），δ：１．１７（３Ｈ，ｔ，Ｊ＝７．４Ｈｚ），２．９６（２Ｈ，ｑ，Ｊ＝７．４Ｈｚ），３．５７（２Ｈ，ｔ，Ｊ＝８．２Ｈｚ），３．９３（２Ｈ，ｔ，Ｊ＝８．２Ｈｚ），４．３３（１Ｈ，ｄｄ，Ｊ＝１．１，１５．９Ｈｚ），４．４６（１Ｈ，ｄｄ，Ｊ＝１．１，９．０Ｈｚ），６．９９（１Ｈ，ｄｄ，Ｊ＝９．０，１５．９Ｈｚ）．ＥＩ−ＭＳ，ｍ／ｚ：１６８（Ｍ）^＋，１３９（Ｍ−Ｃ_２Ｈ_５）^＋，１１１（Ｍ−ＣＯＣ_２Ｈ_５）^＋．ＩＲ（ｎｅａｔ，ｃｍ^−１）：ν：３１１１（ｗ），２９７９（ｗ），１７１６（ｍ），１６９１（ｍ），１６３３（ｍ），１４０２（ｍ），１３６０（ｍ），１３６０（ｍ），１２３０（ｍ），１０４９（ｍ）。

1.86 g (20.1 mmol) of 1-propionyl imidazolidine-2-one, 0.19 g (1.0 mmol) of copper (I) iodide, N, N'-dimethylethylenediamine 0 in a 100 mL Schlenk tube under an argon atmosphere. .21 mL (2.0 mmol), 5.53 g (40.0 mmol) of potassium carbonate and 40.0 mL (40.0 mmol) of 1 M solution of vinyl bromide in THF were added, and the mixture was stirred at 70 ° C. for 7 hours. The reaction mixture was filtered through Celite to concentrate the filtrate. Purification by alumina column chromatography (developing solution: chloroform) gave 2.83 g (yield: 83.6%) of 1-propionyl-3-vinylimidazolidine-2-one (VIm) as a colorless solid. .. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.17 (3H, t, J = 7.4 Hz), 2.96 (2H, q, J = 7.4 Hz), 3.57 (2H) , T, J = 8.2Hz), 3.93 (2H, t, J = 8.2Hz), 4.33 (1H, dd, J = 1.1, 15.9Hz), 4.46 (1H, 1H, dd, J = 1.1, 9.0Hz), 6.99 (1H, dd, J = 9.0, 15.9Hz). EI-MS, m / z: 168 (M) ⁺ , 139 (MC ₂ H ₅ ) ⁺ , 111 (M-COC ₂ H ₅ ) ⁺ . IR (neat, cm ^-1 ): ν: 3111 (w), 2979 (w), 1716 (m), 1691 (m), 1633 (m), 1402 (m), 1360 (m), 1360 (m) , 1230 (m), 1049 (m).

実施例１〜３
参考例１で得られた１−プロピオニル−３−ビニルイミダゾリジン−２−オン（ＶＩｍ）とブチルメタクリレート（ＢＭＡ）をナスフラスコに所定量秤量し、これらのトータルモノマーの１／５０当量のＡＩＢＮを加えて、２ｍｏｌ／Ｌとなるように溶媒を加えた。凍結脱気を行い、封管した後、６０℃で２２時間撹拌した。過剰量のジエチルエーテルに投入して析出した目的物を得た。共重合組成比ｘ／ｙは^１Ｈ−ＮＭＲの積分値から求めた。結果を表１に示した。

Examples 1-3
1-Propionyl-3-vinylimidazolidine-2-one (VIm) and butyl methacrylate (BMA) obtained in Reference Example 1 are weighed in a predetermined amount in an eggplant flask, and 1/50 equivalent of AIBN of these total monomers is obtained. In addition, a solvent was added so as to have a concentration of 2 mol / L. After freezing and degassing and sealing the tube, the mixture was stirred at 60 ° C. for 22 hours. It was added to an excess amount of diethyl ether to obtain a precipitated target product. The copolymer composition ratio x / y was determined from the integrated value ^{of 1 1 H-NMR.} The results are shown in Table 1.

実施例１〜３で得られたランダム共重合体の^１Ｈ−ＮＭＲスペクトルチャートを図１〜３に示した。ＶＩｍ由来の重合体に帰属される２．８ｐｐｍ付近のピークの積分値とＢＭＡ由来の重合体に帰属される０．９ｐｐｍ付近のピークの積分値の比から組成比を求めた。
実施例４
実施例１で得られたランダム共重合体０．０２ｇをＤＭＦ２ｍＬに溶解して１重量％ＤＭＦ溶液を調製し、コート剤とした。スピンコート法にて直径１５ｍｍ丸型カバーグラス（松浪硝子工業社製）にコート剤を塗布した後、乾燥させた。

^{The 1} H-NMR spectrum charts of the random copolymers obtained in Examples 1 to 3 are shown in FIGS. 1 to 3. The composition ratio was determined from the ratio of the integrated value of the peak around 2.8 ppm attributed to the polymer derived from VIm and the integrated value of the peak around 0.9 ppm attributed to the polymer derived from BMA.
Example 4
0.02 g of the random copolymer obtained in Example 1 was dissolved in 2 mL of DMF to prepare a 1 wt% DMF solution, which was used as a coating agent. A coating agent was applied to a round cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 15 mm by a spin coating method, and then dried.

Subsequently, the cover glass was fitted into a 24-well plate manufactured by Corning Inc. and used as a base material for cell culture evaluation. Chinese hamster ovary (CHO) cells were ^{seeded on the cover glass at a density of 1.9 × 10 4} cells / well. As the medium, Ham's F-12 medium containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well. The cells were cultured at 37 ° C. in a 5% carbon dioxide atmosphere, and observed with a 10 × 10 times inverted phase-contrast microscope (IX73 manufactured by Olympus Corporation) at predetermined time intervals. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 650 pieces / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 5
0.02 g of the random copolymer obtained in Example 2 was dissolved in 2 mL of DMF to prepare a 1 wt% DMF solution, which was used as a coating agent. A coating agent was applied to a round cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 15 mm by a spin coating method, and then dried.

Subsequently, the cover glass was fitted into a 24-well plate manufactured by Corning Inc. and used as a base material for cell culture evaluation. Chinese hamster ovary cells (CHO cells) were seeded at a density of ^{1.9 × 10 4 cells / well.} As the medium, Ham's F-12 medium containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well. The cells were cultured at 37 ° C. in a 5% carbon dioxide atmosphere, and observed with a 10 × 10 times inverted phase-contrast microscope (IX73 manufactured by Olympus Corporation) at predetermined time intervals. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 670 pieces / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

Example 6
0.02 g of the random copolymer obtained in Example 3 was dissolved in 2 mL of DMF to prepare a 1 wt% DMF solution, which was used as a coating agent. A coating agent was applied to a round cover glass (manufactured by Matsunami Glass Ind. Co., Ltd.) having a diameter of 15 mm by a spin coating method, and then dried.

Subsequently, the cover glass was fitted into a 24-well plate manufactured by Corning Inc. and used as a base material for cell culture evaluation. Chinese hamster ovary cells (CHO cells) were seeded at a density of ^{1.9 × 10 4 cells / well.} As the medium, Ham's F-12 medium containing 10 vol% fetal bovine serum was used, and 1.0 mL was added per well. The cells were cultured at 37 ° C. in a 5% carbon dioxide atmosphere, and observed with a 10 × 10 times inverted phase-contrast microscope (IX73 manufactured by Olympus Corporation) at predetermined time intervals. The initial cell density of 100 / mm ^2, cell adhesion density 3 days reached 680 / mm ^2, a cover glass treated with coating agent acted effectively as a material for cell culture.

The random copolymer of the present invention reduces the influence of these carrier surfaces on cells by treating the surface of carriers of various materials and shapes, and can be used for cell culture, preservation of cultured cells, recovery of cultured cells, and the like. Since it can improve the convenience of handling such as transfer, it is expected to be applied as a biocompatible material and a material for cell culture.

¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 1. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 2. ¹ H-NMR spectrum chart (CDCl ₃ solution) of the random copolymer obtained in Example 3.

Claims (4)

The following general formula (1)

(In the formula, R1 represents an alkyl group having 2 to 3 carbon atoms or an acyl group having 2 to 3 carbon atoms. ) And the repeating unit represented by the following general formula (2).

(In the formula, R2 represents a hydrogen atom or a methyl group. R3 represents an alkyl group or a phenyl group having 1 to 6 carbon atoms.) A random copolymer containing a repeating unit represented by a repeating unit. The following general formula (3)

(In the formula, R1 represents an alkyl group having 2 to 3 carbon atoms or an acyl group having 2 to 3 carbon atoms. ) An N-vinylimidazolidine-2-one compound represented by the formula, and the following general formula (4).

The random according to claim 1, wherein the acrylic acid ester derivative represented by (R2 represents a hydrogen atom or a methyl group; R3 represents an alkyl group or a phenyl group having 1 to 6 carbon atoms) is radically polymerized. Method for producing a copolymer. A coating agent containing the random copolymer according to claim 1. A material for cell culture obtained by treating the surface of a carrier with the coating agent according to claim 3.

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