Surface treatment agent for cell culture

Abstract

To provide a surface treatment agent for cell culture.SOLUTION: A cell culture substrate coated with a surface treatment agent containing a catechol group-containing polymer expresses high clonogenicity under serum-free culture conditions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a surface treatment agent for cell culture. [Background technology]

[0002] Cell-based biopharmaceuticals are expected to become widespread due to their high efficacy, and attention is focused on technologies for efficiently culturing these raw materials. Human mesenchymal stem cells are used as raw materials, and cell adhesion to the substrate and the action of growth factors and other hormones in the culture medium are crucial. Generally, serum culture media using fetal bovine serum are used. However, when using serum culture media, there is a risk that components in the serum may become antigens if they contaminate the cell product, and furthermore, when using animal-derived serum, there is a possibility of lot-to-lot variability. Therefore, serum-free culture media, which have been replaced with appropriate nutritional and hormonal components, are now commercially available. Because they allow for stable cell culture, the use of serum-free culture is preferred in the manufacture of biopharmaceuticals.

[0003] When using serum-free culture media, cell adhesion to the substrate is weakened, so culture substrates with specific surface treatments are used. For example, by applying a strong plasma treatment to the surface of a polystyrene substrate, the substrate surface is modified with hydroxyl groups, carboxyl groups, etc., improving cell adhesion to the substrate, and this method has been applied to culture in serum-free media (Non-Patent Literature 1). However, there is a problem that sufficient surface modification cannot be achieved even with plasma treatment, and a surface modification method different from conventional methods was needed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Pardo, AMP et al. Corning(R) CellBIND(R) Surface: An Improved Surface for Enhanced Cell Attachment. Corning Life Sciences Technical Monograph CLS-AN-0057 Rev1; 2005: Article can be downloaded from Technical Information / Cell Culture / Culture Surfaces at www.corning.com / lifesciences [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a surface treatment agent containing a cell-adhesion polymer. [Means for solving the problem]

[0006] In view of the above points, the inventors of the present invention conducted extensive research and found that a cell culture substrate coated with a surface treatment agent containing a catechol group polymer can exhibit high cell proliferation under serum-free culture conditions, thus completing the present invention. That is, the present invention encompasses the following aspects. <1> A surface treatment agent comprising a catechol group-containing polymer, wherein the catechol group content of the polymer is 5 to 60 wt%. <2> <1> A surface treatment agent in which the structure of the catechol group-containing polymer described above is (meth)acrylate or (meth)acrylamide. <3> <1> or <2> A surface treatment agent in which the polymer described is a copolymer with the following component (A). (A) Monomers with an HLB value (Griffin method) in the range of 0 to 15.0. <4> <3> A surface treatment agent in which the polymer described is a block copolymer. <5> <3> from <4> A surface treatment agent having a polymer concentration of 0.1 to 10 wt% as described above. <6><4> to <5>, wherein the solubility parameter of the solvent of the surface treatment agent is 9.0 to 14.0 (cal / cm 3 ) 0.5 A surface treatment agent, which is as defined above. <7>The surface treatment agent according to <6>, wherein the solvent of the surface treatment agent is an alcoholic solvent. <8>A film obtained by coating the surface treatment agent according to any one of <6> to <7>. <9>A cell culture substrate having a surface coated with the film according to <8>. <10>A method for producing a surface treatment agent, comprising a step of dissolving a catechol group-containing polymer in a deoxygenated solvent. <11>A cell culture method using the cell culture substrate according to <9>. <12>The cell culture method according to <11>, which is serum-free culture. Effects of the Invention

[0007] A cell culture substrate coated with a surface treatment agent containing a catechol group-containing polymer, characterized in that the content of catechol groups in the polymer is 5 to 60 wt%, can exhibit high cell proliferative activity under serum-free culture conditions Mode for Carrying Out the Invention

[0008] Hereinafter, modes for carrying out the present invention (hereinafter simply referred to as "the present embodiment") will be described in detail. The following present embodiment is an illustration for describing the present invention, and is not intended to limit the present invention to the following contents. The present invention can be carried out with appropriate modifications within the scope of its gist.

[0009] The present invention relates to a surface treatment agent comprising a polymer having a catechol group weight ratio of 5 to 60 wt%, and a cell culture substrate coated with the surface treatment agent. In the present invention, a catechol group is a functional group in which two hydroxyl groups are bonded adjacent to the ortho position of a benzene ring. The polymer constituting the surface treatment agent of the present invention contains this catechol group, and the weight ratio of the catechol group is 5 to 60 wt%. In order to achieve both the film-forming properties of the surface treatment agent and the cell proliferation properties of the cell culture substrate coated with the surface treatment agent in the present invention, the weight ratio of the catechol group in the polymer is preferably 20 to 60 wt%, and more preferably 30 to 60 wt%.

[0010] The monomers that can be used to obtain the polymer containing catechol groups of the present invention are not particularly limited, but examples include 3,4-dihydroxyphenyl)methyl(meth)acrylate, 2-(3,4-dihydroxyphenyl)ethyl(meth)acrylate, 3-(3,4-dihydroxyphenyl)propyl(meth)acrylate, 4-(3,4-dihydroxyphenyl)butyl(meth)acrylate, N-(3,4-dihydroxyphenyl)methyl(meth)acrylamide, N-[2-(3,4-dihydroxyphenyl)ethyl](meth)acrylamide (hereinafter referred to as "dopamine(meth)acrylamide" or "DMA"), N-[3-(3,4-dihydroxyphenyl)propyl](meth)acrylamide, N-[4-(3,4-dihydroxyphenyl)butyl](meth)acrylamide, etc. Furthermore, monomers that do not contain catechol groups may be copolymerized as long as the weight ratio of catechol groups in the polymer is 5 to 60 wt%. There are no particular limitations on the copolymerization method; it may be a random copolymer in which the constituent components are arranged randomly, or a block copolymer in which polymers made of each constituent component are linked together. Examples of monomers to be copolymerized include 2-methoxyethyl acrylate and n-butyl acrylate, in order to improve coating properties on the substrate and solubility in organic solvents.

[0011] The structure of the polymer containing the catechol group of the present invention is not particularly limited, but it is preferably composed of (meth)acrylate or (meth)acrylamide because it is relatively easy to manufacture.

[0012] In this specification, the HLB value (Hydrophile-Lipophile Balance) is a value representing the degree of affinity to water and oil, as described in WCGriffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It takes a value from 0 to 20, with values ​​closer to 0 indicating higher hydrophobicity and values ​​closer to 20 indicating higher hydrophilicity. Methods for determining the HLB by calculation include the Atlas method, Griffin method, Davis method, and Kawakami method. In this invention, the value calculated by the Griffin method was used, and it was determined using the following formula based on the formula weight of the hydrophilic portion in the repeating unit and the total formula weight of the repeating unit. HLB value = 20 × (molecular weight of hydrophilic part) ÷ (total molecular weight) Examples of the hydrophilic portion in the repeating units of each block mentioned above include the sulfone portion (-SO3-), phosphono portion (-PO3-), carboxyl portion (-COOH), ester portion (-COO-), amide portion (-CONH-), imide portion (-CON-), aldehyde portion (-CHO), carbonyl portion (-CO-), hydroxyl portion (-OH), amino portion (-NH2), acetyl portion (-COCH3), ethyleneamine portion (-CH2CH2N-), ethyleneoxy portion (-CH2CH2O-), alkali metal ions, alkaline earth metal ions, ammonium ions, halide ions, and acetate ions.

[0013] In calculating the hydrophilic portion within a repeating unit, atoms constituting the hydrophilic portion must not overlap with atoms constituting other hydrophilic portions. An example of calculating the HLB value within a repeating unit is shown below. For example, in the case of n-butyl methacrylate (molecular weight: 142.20), the hydrophilic portion consists of 1 part ester portion, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 6.2.

[0014] The polymer of the present invention may include monomer components as component (A) having an HLB value (Griffin method) in the range of 0 to 15.0. Preferably, the HLB value is in the range of 0 to 14, and more preferably in the range of 0 to 10, in order to obtain a stable film that does not peel off in water when applied to a substrate. Examples of repeating units contained in component (A) include styrene (HLB value = 0.0) and its derivatives, alkyl (meth)acrylates such as methyl methacrylate (HLB value = 8.8), ethyl acrylate (HLB value = 8.8), ethyl methacrylate (HLB value = 7.7), n-propyl acrylate (HLB value = 7.7), n-propyl methacrylate (HLB value = 6.9), n-butyl acrylate (HLB value = 6.9), n-butyl methacrylate (HLB value = 6.2), and 2-methoxyethyl acrylate (HLB value = 13.5). If the HLB value is 15 or higher, the film is likely to peel off in water when applied to a substrate, making it impossible to obtain a stable film.

[0015] The polymer concentration in the surface treatment agent of the present invention is not particularly limited, and any concentration can be selected according to the film formation method and target film thickness. For example, it is 0.1 to 10 wt%, preferably 0.2 to 5.0 wt%, and more preferably 0.5 to 3.0 wt%. Compounds other than the polymer of the present invention may also be included.

[0016] In this specification, the solubility parameter is the affinity parameter defined in the regular solution theory described in RFFedors, Polymer Engineering & Science, 14, 147 (1974), and the value used is in accordance with Fedors' estimation method. Fedors' estimation method is a method of estimation from molecular structure and was obtained using the following formula. δ = [ΣE / ΣV] 1 / 2 E represents the cohesive energy and V represents the molar volume; the values ​​proposed by Fedors are used for each molecular structure. For example, in the case of 2-methoxyethanol, CH3-(E=1125kcal / mol, V=16.1cm 3 ( / mol) is 1 part, -CH2- (E=1180kcal / mol, V=33.5cm³) 3 / mol) is 2 parts, -O-(E=800 kcal / mol, V=3.8 cm 3 / mol) is 1 part, -OH (E=7120 kcal / mol, V=10.0 cm 3 / mol) is 1 part, so the solubility parameter is 12.0.

[0017] The solvent used for the surface treatment agent of the present invention is not particularly limited, but for example, the solubility parameter is 9.0 to 14.0 (cal / cm 3 ) 0.5 , preferably 10.0 to 13.0 (cal / cm 3 ) 0.5 , more preferably 11.0 to 12.5 (cal / cm 3 ) 0.5 . When the solubility parameter is less than 9.0 (cal / cm 3 ) 0.5 or exceeds 14.0 (cal / cm 3 ) 0.5 , the catechol group-containing polymer may not dissolve. The type of solvent is not particularly limited, and it may be a single solvent or a mixed solvent. Examples of single solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, 2-methoxyethanol, 1-methoxy-2-propanol, acetone, and 1,4-dioxane; alcohol solvents such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, t-butanol, 2-methoxyethanol, and 1-methoxy-2-propanol are preferred. As for mixed solvents, it is preferable that the solubility parameter in terms of molar ratio is 9.0 to 14.0 (cal / cm 3 ) 0.5 , and examples include a mixed solvent of water and acetone, and a mixed solvent of methanol and acetone.

[0018] The method for producing the surface treatment agent of the present invention is not particularly limited, but since catechol groups are easily oxidized, it is preferable to include a step of dissolving the catechol group-containing polymer in an oxygen-free solvent. Furthermore, it is preferable to use pressure filtration to suppress changes in the concentration of the surface treatment agent. Moreover, since the catechol group-containing polymer is hydrophilic, it is preferable to use a hydrophilic filter for the pressure filtration.

[0019] The surface treatment agent of the present invention can be applied to a substrate and dried to form a film made of the polymer of the present invention on the surface of a cell culture substrate. There are no particular limitations on the type of cell culture substrate, and examples include polyethylene, polypropylene, polyolefin, acrylic polymer, methacrylic acid polymer, silicone rubber, polystyrene, polyethylene terephthalate, polycarbonate, metal, ceramics, and glass. There are also no particular limitations on the shape of the cell culture substrate, and examples include plate-like, film-like, bead-like, and fibrous shapes, as well as holes, grooves, and protrusions provided on plate-like substrates. There are no particular limitations on the method of forming the film, and various commonly known methods such as brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, and blade coating can be used. There are no particular limitations on the film thickness, and examples include 1 nm to 100 μm, preferably 5 nm to 10 μm, and more preferably 8 nm to 1 μm.

[0020] A substrate coated with the film of the present invention can be used as a cell culture substrate. The cells applicable to 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 fetal kidney-derived HEK293 cells, human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that make up various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glial cells, and fibroblasts that make up the nervous system, hepatocytes, non-parenchymal hepatocytes, and adipocytes that are involved in the metabolism of the body, and cells with differentiation potential such as stem cells present in various tissues, and cells differentiated from them. Other examples include cells contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, and microorganisms, viruses, protozoa, etc. that exist in the body or environment.

[0021] The cell culture substrate of the present invention can be used for cell culture in serum-free medium. There are no particular limitations on the medium used for serum-free culture, but examples include Eagle's Minimum Essential Medium (EMEM), Dulbecco-Voigt Modified Eagle's Minimum Essential Medium (DMEM), Iskov Modified Dulbecco's Medium (IMDM), MCDB medium, RPMI1640 medium, etc. Amino acids, vitamins, and / or metals may be added to these basic media as needed. Furthermore, CiMS TM -Commercial serum-free culture media such as BM (Cell Science Institute Co., Ltd.) can be used. [Examples]

[0022] <Synthesis of monomers> 4.0 g of sodium bicarbonate, 10.0 g of sodium tetraborate, and 100 mL of pure water were added to a 500 mL three-necked flask and stirred. Then, 5.0 g (26.4 mmol) of dopamine hydrochloride and 4.7 mL (29.1 mmol) of methacrylic anhydride dissolved in 25 mL of tetrahydrofuran were added. Further, 1 M sodium hydroxide was added to adjust the pH of the solution to 8 or higher. After purging with nitrogen gas, the mixture was stirred at room temperature for 18 hours. The reaction mixture was transferred to a 300 mL separatory funnel, and the liquid-liquid separation was performed twice with 50 mL of ethyl acetate. The collected aqueous layer was then filtered by suction to remove solids. 2 M hydrochloric acid was added to the aqueous solution after suction filtration to adjust the pH to 2 or lower, and the liquid-liquid separation was performed three times with 50 mL of ethyl acetate. The organic layer was collected, and magnesium sulfate was added and stirred. Magnesium sulfate was removed by suction filtration, and the solution was concentrated to approximately 25 mL using an evaporator. The concentrated solution was added dropwise to hexane, and the precipitate was collected and dried under reduced pressure to obtain dopamine methacrylamide (DMA), a white powder monomer.

[0023] <Weight ratio of catechol groups> The results were obtained by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectral analysis using a nuclear magnetic resonance spectrometer (manufactured by JEOL Ltd., product name JNM-ECZ400S / L1).

[0024] <Measurement of cell count> 10 μL of the cell suspension was added to a cell counting slide (Thermo Fisher Scientific, Inc., trade name Countess Cell Counting Chamber Slid), and the cell count was measured using an automated cell counter (Thermo Fisher Scientific, Inc., trade name Countess(R) II).

[0025] (Example 1) 1.77 g (8.0 mmol) of DMA, 13 mg (80 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added to a 50 mL three-necked flask. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a white solid polymer compound 1, polyDMA. The weight ratio of catechol groups in polymer compound 1 was 49 wt%.

[0026] 0.05 g of polymer compound 1 and 9.95 g of 2-methoxyethanol were added to a glass container, and after stirring, the polymer was allowed to stand overnight to dissolve, thereby preparing polymer solution 1. 100 μL of polymer solution 1 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060, made of polystyrene), and cell culture substrate 1 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0027] In cell culture substrate 1, human bone marrow-derived mesenchymal stem cells (manufactured by Lonza Japan Co., Ltd., Product Code: PT-2501) are placed in a quantity of 1.0 × 10⁻¹. 5 Cells were seeded and cultured at 37°C with a CO2 concentration of 5%. CiMS was used as the culture medium. TM CiMS™-BM (manufactured by Cell Science Institute Co., Ltd.) with added sAF was used. After culturing for 3 days, cells were harvested using trypsin and the cell count was measured, resulting in 5.18 × 10⁶ cells. 5 They were cells.

[0028] (Example 2) Cell culture substrate 2 was prepared by applying polymer solution 1 to a film cut from a culture bag (A-1000Nl, manufactured by Nipro Corporation, made of polyethylene) using a bar coater (manufactured by OSG System Products Co., Ltd., OSP-05) and air-drying it.

[0029] A cell culture substrate 2, cut into a 50mm diameter circle, is placed in an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and human bone marrow-derived mesenchymal stem cells (Lonza Japan Co., Ltd., Product Code: PT-2501) are placed in a 1.0 × 10⁶ container. 5Cells were seeded and cultured at 37°C with a CO2 concentration of 5%. CiMS was used as the culture medium. TM CiMS with -sAF added TM -BM (manufactured by Cell Science Institute Co., Ltd.) was used. After culturing for 5 days, cells were harvested using trypsin and the cell count was measured, resulting in 3.27 × 10⁶ cells. 5 They were cells.

[0030] (Example 3) In a 50 mL three-necked flask, 0.61 g (2.8 mmol) of DMA, 1.91 g (14.7 mmol) of 2-methoxyethyl acrylate (MEA), 42 mg (300 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a brown solid polymer compound 2poly(DMA-co-MEA). The composition of polymer compound 2 was DMA / MEA = 10 / 90 [mol%], and the weight ratio of catechol groups was 8 wt%.

[0031] 0.05 g of polymer compound 2 and 9.95 g of 2-methoxyethanol were added to a glass container, and after stirring, the polymer was allowed to stand overnight to dissolve, thereby preparing polymer solution 2. 100 μL of polymer solution 2 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 3 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0032] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 3 was used. The number of cells after 3 days of culture was 3.22 × 10⁶. 5 They were cells.

[0033] (Example 4) A cell culture substrate 4 was prepared by applying polymer solution 2 to a film cut from a culture bag (A-1000Nl, manufactured by Nipro Corporation) using a bar coater (OSG System Products Co., Ltd., OSP-05) and air-drying it.

[0034] A cell culture substrate 4, cut into a 50 mm diameter circle, was placed in an untreated dish for suspension cells (AGC Technoglass Co., Ltd., 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 2. After 5 days of culture, the cell count was 3.78 × 10⁶. 5 They were cells.

[0035] (Comparative Example 1) Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that an untreated dish for suspension cells (AGC Technoglass Co., Ltd., 1010-060) was used as the cell culture substrate. After 3 days of culture, the cell count was 2.11 × 10⁶. 5 They were cells.

[0036] (Comparative Example 2) A culture bag (A-1000Nl, manufactured by Nipro Corporation) was cut into a 50 mm diameter circle and placed in an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060). Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 2. After 5 days of culture, the cell count was 2.20 × 10⁶. 5 They were cells.

[0037] (Reference example 1) Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that CellBIND (registered trademark, manufactured by Corning Incorporated) and a 60 mm dish were used as the cell culture substrate. After 3 days of culture, the cell count was 3.99 × 10⁶. 5 They were cells.

[0038] [Table 1]

[0039] [Table 2] (Example 5) In a 50 mL three-necked flask, 2.21 g (10.0 mmol) of DMA, 20.2 mg (50 μmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 6 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 72 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain pale yellow solid polyDMA. In a 50 mL three-necked flask, 0.500 g of pale yellow solid polyDMA, 0.195 g (1.52 mmol) of n-butyl acrylate (BA), 0.5 mg (3 μmol) of azobisisobutyronitrile, and 4 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 72 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a pale yellow solid block copolymer, polymer compound 3poly(DMA-b-BA). The composition of polymer compound 3 was DMA / BA = 73 / 27 [mol%], and the weight ratio of catechol groups was 40 wt%.

[0040] 0.05 g of polymer compound 3 and 9.95 g of 2-methoxyethanol, from which oxygen had been removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer and prepare polymer solution 3. 100 μL of polymer solution 3 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 5 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0041] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 5 was used. The number of cells after 3 days of culture was 4.52 × 10⁶. 5 They were cells.

[0042] (Example 6) In a 50 mL three-necked flask, 2.21 g (10.0 mmol) of DMA, 20.2 mg (50 μmol) of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 6 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60°C for 72 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain pale yellow solid polyDMA. In a 50 mL three-necked flask, 0.500 g of pale yellow solid polyDMA, 0.289 g (2.26 mmol) of BA, 0.5 mg (3 μmol) of azobisisobutyronitrile, and 4 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60°C for 72 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a pale yellow solid block copolymer, polymer compound 4poly(DMA-b-BA). The composition of polymer compound 4 was DMA / BA = 57 / 43 [mol%], and the weight ratio of catechol groups was 35 wt%.

[0043] 0.05 g of polymer compound 4 and 9.95 g of 2-methoxyethanol, from which oxygen had been removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer and prepare polymer solution 4. 100 μL of polymer solution 4 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 6 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0044] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 6 was used. The number of cells after 3 days of culture was 3.95 × 10⁶. 5 They were cells.

[0045] (Example 7) 2.00 g (9.0 mmol) of DMA, 0.50 g (3.9 mmol) of BA, 42 mg (300 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added to a 50 mL three-necked flask. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a light brown solid polymer compound 5poly(DMA-co-BA). The composition of polymer compound 5 was DMA / BA = 70 / 30 [mol%], and the weight ratio of catechol groups was 40 wt%.

[0046] 0.05 g of polymer compound 5 and 9.95 g of 2-methoxyethanol, from which oxygen was removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer, and polymer solution 5 was prepared. 100 μL of polymer solution 7 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 7 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0047] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 7 was used. The number of cells after 3 days of culture was 3.75 × 10⁶. 5 They were cells.

[0048] (Example 8) 1.50 g (7.2 mmol) of DMA, 1.00 g (7.8 mmol) of BA, 42 mg (300 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added to a 50 mL three-necked flask. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a light brown solid polymer compound 6poly(DMA-co-BA). The composition of polymer compound 6 was DMA / BA = 46 / 54 [mol%], and the weight ratio of catechol groups was 30 wt%.

[0049] 0.05 g of polymer compound 6 and 9.95 g of 2-methoxyethanol, from which oxygen was removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer, and polymer solution 6 was prepared. 100 μL of polymer solution 8 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 8 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0050] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 8 was used. The number of cells after 3 days of culture was 3.44 × 10⁶. 5 They were cells.

[0051] (Example 9) In a 50 mL three-necked flask, 1.00 g (4.5 mmol) of DMA, 1.50 g (11.7 mmol) of BA, 42 mg (300 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a light brown solid polymer compound 7poly(DMA-co-BA). The composition of polymer compound 7 was DMA / BA = 30 / 70 [mol%], and the weight ratio of catechol groups was 21 wt%.

[0052] 0.05 g of polymer compound 7 and 9.95 g of 2-methoxyethanol, from which oxygen had been removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer and prepare polymer solution 7. 100 μL of polymer solution 9 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and the cell culture substrate 9 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0053] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 9 was used. The number of cells after 3 days of culture was 3.34 × 10⁶. 5 They were cells.

[0054] (Comparative Example 3) 0.10 g (0.5 mmol) of DMA, 2.40 g (18.7 mmol) of BA, 42 mg (300 μmol) of azobisisobutyronitrile, and 10 mL of N,N-dimethylformamide were added to a 50 mL three-necked flask. After purging with nitrogen gas, the mixture was heated and stirred at 60 °C for 19 hours. The reaction mixture was reprecipitated and purified with diethyl ether, and dried under reduced pressure to obtain a light brown solid polymer compound 8poly(DMA-co-BA). The composition of polymer compound 8 was DMA / BA = 3 / 97 [mol%], and the weight ratio of catechol groups was 2 wt%.

[0055] 0.05 g of polymer compound 8 and 9.95 g of 2-methoxyethanol, from which oxygen was removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer, and polymer solution 8 was prepared. 100 μL of polymer solution 9 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 10 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0056] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 9 was used. The number of cells after 3 days of culture was 2.55 × 10⁶. 5 They were cells.

[0057] [Table 3]

[0058] (Example 10) 0.40 g of polymer compound 5 and 9.60 g of 2-methoxyethanol, from which oxygen was removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer, and polymer solution 9 was prepared. 100 μL of polymer solution 9 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 7 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0059] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 11 was used. The number of cells after 3 days of culture was 3.89 × 10⁶. 5 They were cells.

[0060] (Example 11) 0.05 g of polymer compound 5 and 9.95 g of 2-butanol, from which oxygen was removed by nitrogen gas bubbling, were added to a glass container. After stirring, the mixture was allowed to stand overnight to dissolve the polymer, and polymer solution 10 was prepared. 100 μL of polymer solution 10 was dropped onto an untreated dish for suspension cells (AGC Techno Glass Co., Ltd., 1010-060), and cell culture substrate 12 was prepared by spin-coating at 3000 rpm for 60 seconds.

[0061] Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that cell culture substrate 12 was used. The number of cells after 3 days of culture was 3.78 × 10⁶. 5 They were cells.

[0062] [Table 4]

Claims

1. A surface treatment agent comprising a catechol group-containing polymer, The polymer is a surface treatment agent that is a block copolymer with the following component (A): A surface treatment agent having a polymer catechol group content of 5 to 60 wt%. (A) Monomers with HLB values ​​(Griffin method) in the range of 0 to 15.0

2. A surface treatment agent wherein the structure of the catechol group-containing polymer according to claim 1 is (meth)acrylate or (meth)acrylamide.

3. A surface treatment agent according to claim 1, wherein the polymer concentration of the surface treatment agent is 0.1 to 10 wt%.

4. The solubility parameter of the solvent for the surface treatment agent according to claim 1 is 9.0 to 14.0 (cal / cm³). ３ ) ０．５ A surface treatment agent.

5. A surface treatment agent according to claim 4, wherein the solvent of the surface treatment agent is alcohol-based.

6. A film coated with the surface treatment agent described in claim 4 or 5.

7. A cell culture substrate having the membrane described in claim 6 coated on its surface.

8. A method for producing a surface treatment agent according to any one of claims 1 to 5, characterized by comprising the step of dissolving the catechol group-containing polymer in a solvent from which oxygen has been removed.

9. A cell culture method using the cell culture substrate described in claim 7.

10. The cell culture method according to claim 9, characterized in that the cell culture method is a serum-free culture method.

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