Water-insoluble polymer compounds and surface treatment agents containing them

Abstract

To provide a surface treatment agent containing a cell-adhesive polymer.SOLUTION: Provided is a water-insoluble polymer compound containing the structural components of (A) a monomer having an acidic functional group, (B) a monomer having an HLB value (Griffin's method) within a range of 0-5.0, and (C) a monomer having an HLB value (Griffin's method) within a range of 5.0-9.0, where the ratio of (C) is 10-50 mol% in (A), (B) and (C).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a water-insoluble polymeric compound. [Background technology]

[0002] Biopharmaceuticals made from cells are expected to become widespread due to their high efficacy, and attention is being focused on technology for mass-cultivating raw cells.

[0003] Many useful cells need to adhere to some kind of substrate during culture, and a culture substrate with excellent cell adhesiveness is required. Plasma treatment is a common method for achieving cell adhesiveness, but it is not sufficient to achieve this, so a new technology for imparting cell adhesiveness was needed to improve cell culture efficiency. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 57-186491 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a water-insoluble polymer compound having cell adhesive properties. [Means for solving the problem]

[0006] In view of the above, the present inventors have conducted extensive research and discovered that a water-insoluble polymeric compound comprising the following components: (A) a monomer having an acidic functional group; (B) a monomer having an HLB value (Griffin method) in the range of 0 to 5; and (C) a monomer having an HLB value (Griffin method) in the range of 5 to 9, wherein the ratio of (C) to (A), (B), and (C) is 10 to 50 mol %, can be dissolved in an organic solvent that is not highly aggressive to typical plastic substrates, and can impart cell adhesiveness to a substrate by coating with the compound, thereby completing the present invention. Specifically, the present invention encompasses the following aspects. <1> A water-insoluble polymer compound comprising the following components (A), (B), and (C), wherein the ratio of (C) to (A), (B), and (C) is 10 to 50 mol %: (A) Monomer with an acidic functional group. (B) Monomers with HLB values ​​(Griffin method) in the range of 0 to 5.0. (C) Monomers with an HLB value (Griffin method) in the range of 5.0 to 9.0. <2> The molar ratio A / B of components (A) and (B) is 0.1 to 2.0 (mol / mol). <1> The water-insoluble polymer compound described above. <3> The functional group exhibiting acidity in component (A) is selected from a hydroxy group, a carboxy group, a sulfonic acid group, and a phosphate group. <1> or <2> 1. The water-insoluble polymer compound according to claim 1. <4> The acid dissociation constant pKa of the acidic functional group of component (A) is -5.0 to 6.0 <1> ~ <3> The water-insoluble polymer compound according to any one of claims 1 to 10. <5> <1> ~ <4> A surface treatment agent comprising the water-insoluble polymer compound according to any one of claims 1 to 4. <6> <5> A film obtained by applying the surface treatment agent described above to a substrate. <7> The base material is plastic <6> The membrane according to claim 1. <8> <6> or <7> A cell culture substrate having a surface coated with the membrane described in 1. [Effects of the Invention]

[0007] A water-insoluble polymer compound containing the components (A) a monomer having an acidic functional group, (B) a monomer having an HLB value (Griffin method) in the range of 0 to 5.0, and (C) a monomer having an HLB value (Griffin method) in the range of 5.0 to 9.0, wherein the ratio of (C) to (A), (B), and (C) is 10 to 50 mol%, can be dissolved in an organic solvent that is not highly invasive to general plastic substrates, and can be coated onto a substrate to impart cell adhesiveness. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, an embodiment for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The present embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be carried out by appropriately modifying it within the scope of its spirit.

[0009] The cell adhesive polymer of the present invention is a water-insoluble polymeric compound characterized in that it contains the following constituent components: (A) a monomer having an acidic functional group, (B) a monomer having an HLB value (Griffin method) in the range of 0 to 5.0, and (C) a monomer having an HLB value (Griffin method) in the range of 5.0 to 9.0, and that the ratio of (C) to (A), (B), and (C) is 10 to 50 mol %.

[0010] The acidic functional group of component (A) of the present invention is a functional group that ionizes in water to exhibit anionic properties. Examples of the functional group include, but are not limited to, a hydroxy group, a carboxy group, a sulfonic acid group, and a phosphate group. Among these, a carboxy group is preferred because it facilitates acidity control. These monomers may contain two or more functional groups, or may contain other functional groups, such as an aldehyde group, a carbonyl group, a nitro group, an amino group, an ether group, an ester group, or an amide group.

[0011] The acid dissociation constant herein refers to the acid dissociation constant pKa in water. The acid dissociation constant pKa herein is determined based on the side chain structure of the polymer structure. To confer cell adhesiveness, electrostatic bonding between cells and the substrate must be promoted. The acid dissociation constant pKa of the acidic functional group of component (A) of the present invention is not particularly limited, but is, for example, -5.0 to 6.0, preferably -3.0 to 5.0, and more preferably 1 to 4.5. If the acid dissociation constant pKa is less than -5.0, copolymerization with component (B) may be difficult, while if the acid dissociation constant pKa exceeds 6.0, the effect of promoting electrostatic bonding may be weakened. Examples of acid dissociation constants include 4.76 for the side chain structure of acrylic acid (-COOH), 4.20 for the side chain structure of carboxystyrene (-CHCOOH), and -2.8 for the side chain structure of styrenesulfonic acid (-CHSOH).

[0012] The polymer compound of the present invention is water-insoluble. In this specification, "water-insoluble" means that the amount of the polymer compound dissolved in 100 g of water at 20°C is 100 mg or less.

[0013] The structure of component (A) of the present invention is not particularly limited, but examples include acrylic acid, carboxystyrene (the carboxy group may be in the ortho-, meta-, or para-position), styrenesulfonic acid (the sulfonic acid group may be in the ortho-, meta-, or para-position), and derivatives thereof. Among these, p-carboxystyrene, acrylic acid, and p-styrenesulfonic acid are preferred because they have a side chain structure with a more preferred pKa, and p-carboxystyrene is even more preferred.

[0014] In this specification, the HLB value (Hydrophile-Lipophile Balance) is a value that indicates the degree of affinity for water and oil, as described in W.C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). Methods for determining the HLB value by calculation include the Atlas method, the Griffin method, the Davis method, and the Kawakami method. In the present invention, the value calculated by the Griffin method was used, and the value was calculated using the following formula based on the formula weight of the hydrophilic moiety in the repeating unit and the total formula weight of the repeating units. HLB value = 20 × (hydrophilic moiety formula weight) ÷ (total formula weight) In the Griffin method, the HLB value ranges from 0 to 20, with values ​​closer to 0 indicating higher hydrophobicity and closer to 20 indicating higher hydrophilicity. Examples of the hydrophilic moiety in the repeating unit of each block mentioned above include sulfo group (-SO3-), phospho group (-PO3-), carboxy group (-COOH), ester group (-COO-), amide group (-CONH-), imide group (-CON-), aldehyde group (-CHO), carbonyl group (-CO-), hydroxy group (-OH), amino group (-NH2), acetyl group (-COCH3), ethyleneamine group (-CH2CH2N-), ethyleneoxy group (-CH2CHO-), alkali metal ion, alkaline earth metal ion, ammonium ion, halide ion, and acetate ion. When calculating the hydrophilic moiety in a repeating unit, atoms constituting the hydrophilic moiety must not overlap with atoms constituting other hydrophilic moieties. An example of calculating the HLB value in a repeating unit is shown below. For example, in the case of styrene (molecular weight: 104.15), there is no hydrophilic portion and the molecular weight of the hydrophilic portion is 0, so the HLB value is 0.0. In the case of carboxystyrene (molecular weight: 148.15), the hydrophilic portion is 1 part carboxy group and the molecular weight of the hydrophilic portion is 45.02, so the HLB value is 6.1. In the case of n-butyl acrylate (molecular weight: 128.2), the hydrophilic portion is 1 part ester portion and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 6.9.

[0015] To impart cell adhesiveness, it is necessary to promote hydrophobic bonding between the cells and the substrate, and the component (B) of the present invention is a monomer having an HLB value in the range of 0 to 5.0, preferably 0 to 3.0. If the HLB value exceeds 5.0, the effect of promoting hydrophobic bonding is weakened.

[0016] Although the component (B) of the present invention is not particularly limited, examples thereof include styrene, vinylnaphthalene, vinylanthracene, and derivatives thereof. Among these, styrene is preferred because it is easily copolymerizable with the components (A) and (C).

[0017] In order to dissolve in organic solvents that are less aggressive to general plastic substrates, the component (C) of the present invention is a monomer having an HLB value in the range of 5.0 to 9.0, preferably 5.0 to 8.0. If the HLB value is less than 5.0, the solubility in organic solvents decreases, and if the HLB value exceeds 9.0, the monomer becomes more soluble in water.

[0018] Although the component (C) of the present invention is not particularly limited, examples thereof include acrylic acid esters and methacrylic acid esters. Among these, n-butyl (meth)acrylate is preferred because it is easily copolymerizable with the components (A) and (B).

[0019] The ratio of (C) among the components (A), (B), and (C) of the present invention is 10 to 50 mol %. If it is less than 10 mol %, the solubility in organic solvents decreases, and when used as a surface treatment agent, coating unevenness may occur. If it exceeds 50 mol %, it becomes difficult to exhibit sufficient cell adhesiveness.

[0020] The molar ratio A / B (mol / mol) of the components (A) and (B) of the present invention is not particularly limited, but is, for example, 0.1 to 2.0, preferably 0.2 to 1.0, and more preferably 0.5 to 0.9. The balance between A and B is important for the expression of cell adhesiveness, and if the ratio is outside the range of 0.1 to 2.0, sufficient cell adhesiveness may not be obtained.

[0021] The polymer compound of the present invention may contain components other than components (A), (B), and (C), and may be a random copolymer in which the components are arranged randomly, or a block copolymer in which polymers composed of the respective components are linked together.

[0022] The polymer compound of the present invention is not particularly limited, but has a number average molecular weight Mn of 5,000 to 1,000,000, preferably 10,000 to 300,000. If it is less than 5,000, it becomes easily soluble in water, and if it exceeds 1,000,000, its solubility in solvents decreases.

[0023] The polymerization method for the polymer compound of the present invention is not particularly limited, and examples thereof include addition polymerization, polycondensation, ionic polymerization, ring-opening polymerization, living radical polymerization, and coordination polymerization.

[0024] The method for producing the polymer compound of the present invention is not particularly limited, and examples thereof include bulk polymerization, solution condensation, suspension polymerization, and emulsion polymerization.

[0025] The polymer compound of the present invention can be used as a surface treatment agent by dissolving it in a solvent. The solvent for the surface treatment agent is not particularly limited, but it is preferable to select a solvent that does not dissolve the substrate to be coated. Examples of the solvent include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, acetone, methyl ethyl ketone, diethyl ether, tetrahydrofuran, dimethylformamide, n-hexane, benzene, toluene, 1,4-dioxane, 2-methoxyethanol, 1-methoxy-2-propanol, and mixtures selected from these. The concentration of the polymer compound in the surface treatment agent is not particularly limited, but is, for example, 0.01 to 10 wt %. Furthermore, the surface treatment agent may contain compounds other than the polymer compound of the present invention.

[0026] The surface treatment agent of the present invention can be applied to a substrate and the solvent removed by drying or the like to form a film made of the polymer compound of the present invention on the surface of the substrate. The type of substrate is not particularly limited, and examples include plastics (e.g., polyethylene, polypropylene, polyolefin, acrylic polymers, methacrylic acid-based polymers, silicone rubber, polystyrene, polyethylene terephthalate, polycarbonate, etc.), metals, ceramics, and glass. The shape of the substrate is also not particularly limited, and examples include plate-like, film-like, bead-like, and fibrous shapes, as well as holes, grooves, and protrusions provided in a plate-like substrate. The method for forming the film is not particularly limited, and various commonly known methods can be used, such as brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, and blade coating. The film thickness is not particularly limited, and examples thereof are 1 nm to 100 μm. Substrates coated with the membrane of the present invention can be used as cell culture substrates. Cells that can be used with the cell culture substrate of the present invention are not particularly limited, but examples include mesenchymal stem cells, Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells. Furthermore, epithelial and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes involved in the metabolism of the body, as well as stem cells present in various tissues and cells induced to differentiate therefrom. Other examples include cells contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, as well as microorganisms, viruses, and protozoa present in the body or in the environment. [Example]

[0027] Examples of the present invention will be described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used. <Block copolymer composition> Proton nuclear magnetic resonance spectroscopy ( 1 The values ​​were determined by H-NMR spectroscopy. <Molecular weight and molecular weight distribution of block copolymer> The weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC system used was a Tosoh Corporation HLC-8320GPC. Two Tosoh Corporation TSKgel Super AWM-H columns were used. The column temperature was set to 40°C, and the eluent was 10 mM sodium trifluoroacetate containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg / mL. A molecular weight calibration curve was prepared using polymethyl methacrylate (Polymer Laboratories) of known molecular weight. <Measurement of cell number and cell viability> 10 μL of the cell suspension was added to a cell counting slide (Thermo Fisher Scientific, product name: Countess Cell Counting Chamber Slide), and the cell number and cell viability were measured using an automatic cell counter (Thermo Fisher Scientific, product name: CountessR II).

[0028] Example 1 A 200 mL two-neck flask was charged with 0.889 g (6 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A), 0.833 g (8 mmol) of styrene (St, HLB = 0) as component (B), and 0.769 g (6 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.288 g of a white solid polymer compound 1, poly(CSt / St / BA). The resulting polymer compound 1 had a composition of CSt:St:BA = 28:49:23 (mol%), a molar ratio A / B of 0.57, and a number-average molecular weight Mn of 9.1 × 10 4 The molecular weight distribution Mw / Mn was 1.9.

[0029] 0.05 g of polymer compound 1 was weighed out and dissolved in 9.95 g of 2-butanol. 100 μL of this solution was dropped onto a 60 mm IWAKI polystyrene dish for suspension culture, and spin-coated at 3000 rpm for 60 seconds to prepare a cell culture substrate coated with polymer compound 1. 1.0 × 10 human bone marrow-derived mesenchymal stem cells (manufactured by Lonza Japan, Product Code: PT-2501) were added to this cell culture substrate. 5 The cells were seeded and cultured at 37°C with a CO2 concentration of 5%. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS / DMEM) containing 10 vol% fetal bovine serum (Colombia). After 6 days of culture, the cell number was counted and found to be 4.0 × 10 5 It was cells.

[0030] Example 2 A 200 mL two-neck flask was charged with 0.889 g (6 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A), 0.625 g (6 mmol) of styrene (St, HLB = 0) as component (B), and 1.282 g (10 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.128 g of a white solid polymer compound 2 (poly(CSt / St / BA)). The resulting polymer compound 2 had a composition of CSt:St:BA = 25:29:46 (mol%), a molar ratio A / B of 0.86, and a number-average molecular weight Mn of 9.4 × 10 4 The molecular weight distribution Mw / Mn was 1.9.

[0031] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 2 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted and found to be 3.6 × 10 5 It was cells.

[0032] Example 3 A 200 mL two-neck flask was charged with 0.593 g (4 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A), 0.729 g (7 mmol) of styrene (St, HLB = 0) as component (B), and 0.385 g (3 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.089 g of a white solid polymer compound 3, poly(CSt / St / BA). The resulting polymer compound 3 had a composition of CSt:St:BA = 31:51:18 (mol%), a molar ratio A / B of 0.61, and a number-average molecular weight Mn of 9.0 × 10 4 The molecular weight distribution Mw / Mn was 1.9.

[0033] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 3 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted, and it was found to be 3.4 × 10 5 It was cells.

[0034] Example 4 A 200 mL two-neck flask was charged with 1.482 g (10 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A), 0.521 g (5 mmol) of styrene (St, HLB = 0) as component (B), and 1.482 g (10 mmol) of n-butyl methacrylate (BMA, HLB = 6.2) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added, and the mixture was purged with nitrogen gas and heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.301 g of a white solid polymer compound 7, poly(CSt / St / BMA). The composition of the obtained polymer compound 7 was CSt:St:BMA=38:20:42 (mol%), the molar ratio A / B was 1.90, and the number average molecular weight Mn was 9.0×10 4 The molecular weight distribution Mw / Mn was 2.0.

[0035] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 7 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted and found to be 3.8 × 10 5 It was cells.

[0036] Example 5 A 200 mL two-neck flask was charged with 0.360 g (5 mmol) of acrylic acid (AA, pKa = 4.76) as component (A), 1.250 g (12 mmol) of styrene (St, HLB = 0) as component (B), and 0.641 g (5 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction mixture was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.084 g of a white solid polymer compound 8 (poly(AA / St / BA)). The resulting polymer compound 8 had a composition of AA:St:BA = 25:59:16 (mol%), a molar ratio A / B of 0.42, and a number-average molecular weight Mn of 5.0 × 10 4 The molecular weight distribution Mw / Mn was 2.3.

[0037] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 8 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted, and it was found to be 3.6 × 10 5 It was cells.

[0038] Example 6 Sodium p-styrenesulfonate was dissolved in ion-exchanged water and passed through a column packed with H-type cation exchange resin. The solution was then concentrated and dried using a rotary evaporator at 20°C to obtain p-styrenesulfonic acid as a white powder.

[0039] A 200 mL two-neck flask was charged with 1.289 g (7 mmol) of p-styrenesulfonic acid (SSA, pKa = -2.80) as component (A), 1.042 g (10 mmol) of styrene (St, HLB = 0) as component (B), and 1.538 g (12 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of ethanol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.497 g of a white solid polymer compound 9 (poly(SSA / St / BA)). The resulting polymer compound 9 had a composition of SSA:St:BA = 24:31:45 (mol%), a molar ratio A / B of 0.77, and a number-average molecular weight Mn of 3.3 × 10. 4 The molecular weight distribution Mw / Mn was 2.6.

[0040] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 9 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted and found to be 3.5 × 10 5 It was cells.

[0041] Comparative Example 1 A 200 mL two-neck flask was charged with 2.371 g (16 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A), 0.417 g (4 mmol) of styrene (St, HLB = 0) as component (B), and 0.769 g (6 mmol) of n-butyl acrylate (BA, HLB = 6.9) as component (C). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.224 g of polymer compound 4 (poly(CSt / St / BA)) as a white solid. The resulting polymer compound 4 had a composition of CSt:St:BA = 79:7:14 (mol%), a molar ratio A / B of 11.29, and a number-average molecular weight Mn of 9.4 × 10 4 The molecular weight distribution Mw / Mn was 1.9.

[0042] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 4 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted and found to be 1.2 × 10 5 It was cells.

[0043] Comparative Example 2 A 200 mL two-neck flask was charged with 1.458 g (14 mmol) of styrene (St, HLB value = 0) as component (B) and 0.769 g (6 mmol) of n-butyl acrylate (BA, HLB value = 6.9) as component (C), followed by the addition of 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol. After purging with nitrogen gas, the mixture was heated and stirred at 64°C for 24 hours. Purification was carried out in the same manner as in Example 1 to obtain 1.215 g of polymer compound 5 poly(St / BA). The resulting polymer compound 5 had a composition of St:BA = 60:40 (mol%), a molar ratio A / B of 0.0, and a number-average molecular weight Mn of 8.3 × 10 4 The molecular weight distribution Mw / Mn was 2.0.

[0044] A cell culture substrate was prepared in the same manner as in Example 1, except that polymer compound 5 was used, and human bone marrow-derived mesenchymal stem cells were cultured. After 6 days of culture, the cell number was counted and found to be 2.3 × 10 5 It was cells.

[0045] Comparative Example 3 A 200 mL two-neck flask was charged with 2.222 g (15 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A) and 0.521 g (5 mmol) of styrene (St, HLB = 0) as component (B). 16 mg (100 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were then added. After purging with nitrogen gas, the mixture was heated and stirred at 64°C for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 1.530 g of polymer compound 6 poly(CSt / St) as a white solid. The resulting polymer compound 6 had a composition of CSt:St = 80:20 (mol%), a molar ratio A / B of 4.00, and a number-average molecular weight Mn of 8.9 × 10 4 The molecular weight distribution Mw / Mn was 1.9.

[0046] 0.05 g of polymer compound 6 was weighed out and an attempt was made to dissolve it in 9.95 g of 2-butanol, but it failed. 100 μL of this suspension was dropped onto a 60 mm IWAKI polystyrene dish for suspension culture and spin-coated at 3000 rpm for 60 seconds, but uneven coating appeared on the surface of the culture substrate, and a uniform coating was not achieved. Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that this cell culture substrate was used. After 6 days of culture, the number of cells was counted, and it was found to be 3.7 × 10 5 It was cells.

[0047] Comparative Example 4 Human bone marrow-derived mesenchymal stem cells (Lonza Japan, Product Code: PT-2501) were cultured at 1.0 × 10 cells / well in a 60 mm IWAKI polystyrene dish for suspension culture. 5 The cells were seeded and cultured at 37°C with a CO2 concentration of 5%. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS / DMEM) containing 10 vol% fetal bovine serum (Colombia). After 6 days of culture, the cell number was counted and found to be 2.0 × 10 5 It was cells.

[0048] Comparative Example 5 Human bone marrow-derived mesenchymal stem cells (Lonza Japan, Product Code: PT-2501) were cultured at 1.0 × 10 5 The cells were seeded and cultured at 37°C with a CO2 concentration of 5%. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS / DMEM) containing 10 vol% fetal bovine serum (Colombia). After 6 days of culture, the cell number was counted and found to be 3.0 × 10 5 It was cells.

[0049] [Table 1]

Claims

1. A cell culture substrate having a surface coated with a film obtained by applying a surface treatment agent containing a water-insoluble polymer compound to a substrate, the surface treatment agent comprising the following components (A), (B), and (C), wherein the ratio of (C) to (A), (B), and (C) is 10 to 50 mol %, A cell culture substrate in which the molar ratio A / B of components (A) and (B) is 0.1 to 2.0 (mol / mol): (A) Carboxystyrene or styrene sulfonic acid (B) Styrene (C) an acrylic acid ester or a methacrylic acid ester.

2. 2. The cell culture substrate according to claim 1, wherein the substrate is made of plastic.

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