Temperature-responsive polymer surface treatment agent

Abstract

Provided are: a temperature-responsive polymer surface treatment agent containing a temperature-responsive polymer and a cell-adhesive polymer including a monomer in which the HLB value (Griffin's method) with a monomer having a functional group exhibiting acidity is in the range of 0-5.0; and a temperature-responsive cell culture substrate in which cell adhesion and cell recovery by cooling treatment are achieved by coating a substrate with the temperature-responsive polymer surface treatment agent.

Description

Technical Field

[0001] The present invention relates to a temperature-responsive polymer surface treatment agent.

Background Art

[0002] Biopharmaceuticals using cells as raw materials are expected to spread due to their high drug efficacy, and attention has been focused on technologies for efficiently culturing raw material cells. Many useful cells adhere to and grow on some substrate when cultured, but after growth, it is necessary to perform an operation of detaching and subculturing the cultured cells. Generally, enzymatic treatment using a proteolytic enzyme such as trypsin is performed, but the enzymatic treatment involves complicated operations other than the enzyme, so the efficiency is insufficient.

[0003]

[0004] In order to solve the above problems, Patent Document 1 describes a cell recovery method by cooling treatment. Further, Patent Documents 2 and 3 describe cell culture substrates modified with temperature-responsive polymers. Cells cultured using a cell culture substrate coated with a temperature-responsive polymer can be detached / collected without complicated operations by cooling to below the sol-gel transition temperature of the temperature-responsive polymer, which weakens the adhesive force on the substrate surface. On the other hand, the cell adhesiveness of the temperature-responsive polymer itself is weak, and sufficient cell adhesiveness may not be obtained depending on the coating amount and the type of cell culture substrate. Therefore, a temperature-responsive cell culture substrate that achieves both cell recovery by cooling treatment and cell adhesiveness has been demanded.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a temperature-responsive polymer surface treatment agent.

Means for Solving the Problems

[0007] In view of the above points, the present inventors have conducted intensive research. As a result, a surface treatment agent containing a temperature-responsive polymer, a monomer having a functional group exhibiting acidity, and a cell-adhesive polymer containing a monomer having an HLB value (Griffin method) in the range of 0 to 5.0 can be coated on a substrate to obtain a temperature-responsive cell culture substrate that achieves both cell adhesiveness and cell recovery by cooling treatment. Thus, the present invention has been completed. That is, the present invention includes the following aspects. <1> A surface treatment agent containing a temperature-responsive polymer and a cell-adhesive polymer composed of the following (A) and (B); (A) A monomer having a functional group exhibiting acidity, (B) A monomer having an HLB value (Griffin method) in the range of 0 to 5.0. <2> The surface treatment agent according to <1>, wherein the introduction amount of the cell-adhesive polymer is 1 to 50 wt% with respect to the introduction amount of the temperature-responsive polymer. <3> The surface treatment agent according to <1> or <2>, wherein the temperature-responsive polymer exhibits a lower critical solution temperature for water in the range of 0°C to 50°C. <4> The surface treatment agent according to any one of <1> to <3>, wherein the temperature-responsive polymer is a block copolymer composed of three or more repeating units. <5> The surface treatment agent according to any one of <1> to <4>, wherein the functional group exhibiting acidity of the constituent component (A) is selected from a hydroxy group, a carboxy group, a sulfo group, and a phosphate group. <6> The surface treatment agent according to any one of <1> to <5>, wherein the acid dissociation constant pKa of the functional group exhibiting acidity of the constituent component (A) is -5.0 to 6.0. <7> The surface treatment agent according to any one of <1> to <6>, wherein the concentration of the temperature-responsive polymer in the surface treatment agent is 0.1 to 5.0 wt%. <8> A film formed by applying the surface treatment agent according to any one of <1> to <7> to a substrate. <9> The film according to <8>, having a film thickness of 1 to 1000 nm. <10> A cell culture substrate having the film according to <8> or <9> coated on its surface. <11> The cell culture substrate according to <10>, which is in the form of a film. <12> A method for producing the cell culture substrate according to <10> or <11>, comprising a step of applying the surface treatment agent to the substrate.

Advantages of the Invention

[0008] A surface treatment agent containing a temperature-responsive polymer, a monomer having a functional group exhibiting acidity, and a cell-adhesive polymer composed of a monomer having an HLB value (Griffin method) in the range of 0 to 5.0, when coated on a substrate, can provide a temperature-responsive cell culture substrate that combines cell adhesiveness and cell recovery by cooling treatment.

Modes for Carrying Out the Invention

[0009] 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 exemplification for explaining the present invention and is not intended to limit the present invention to the following content. The present invention can be appropriately modified and implemented within the scope of its gist. In the present invention, the "surface treatment agent" may also be referred to as a "polymer solution".

[0010] The surface treatment agent of the present invention is a surface treatment agent containing a temperature-responsive polymer, a monomer having a functional group exhibiting acidity, and a cell-adhesive polymer composed of a monomer having an HLB value (Griffin method) in the range of 0 to 5.0.

[0011] The temperature-responsive polymer in this specification is not particularly limited, but preferably exhibits a lower critical solution temperature in the range of 0°C to 50°C. More preferably, 22°C to 37°C is preferred. The repeating unit of the temperature-responsive component having a lower critical solution temperature (LCST) with respect to water and its LCST with respect to water are, as an example, N-cyclopropylacrylamide (LCST = 46°C), N-isopropylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N,N-diethylacrylamide (LCST = 32°C), N-isopropylmethacrylamide (LCST = 44°C), N-n-propylmethacrylamide (LCST = 28°C), N-tetrahydrofurfurylmethacrylamide (LCST = 35°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), N-methyl-N-n-propylacrylamide (LCST = 20°C), or N,N-dimethylaminoethyl methacrylate (LCST = 47°C), etc. can be exemplified.

[0012] Among these, it is preferable to exhibit an LCST between 37°C, which is the normal cell culture temperature, and 22°C, which is a general room temperature, and N-isopropylacrylamide is particularly preferred. Also, if it has an LCST, in addition to the above-mentioned temperature-responsive repeating unit, different repeating units may be included. The LCST in the case of including different repeating units may, for example, be expressed in the range of 0°C to 100°C when the temperature-responsive repeating unit is N-isopropylacrylamide.

[0013] The structure of the temperature-responsive polymer in this specification is not particularly limited. However, when it consists of two or more components, it is preferably a block copolymer in order to exhibit a high degree of temperature responsiveness. If one or more temperature-responsive components are included, it may also contain components having other properties. Examples of such block copolymers include those containing styrene and its derivatives, 2-methoxyethyl acrylate, n-propyl acrylate, n-propyl methacrylate, n-butyl acrylate, n-butyl methacrylate, and the like. A block copolymer containing three components of a block composed of repeating units of 2-methoxyethyl acrylate, repeating units of n-butyl acrylate, and repeating units of N-isopropylacrylamide is particularly preferred because it can provide a high degree of temperature responsiveness and an improved effect of coating the substrate.

[0014] The composition ratio of the repeating units of the temperature-responsive component in the temperature-responsive polymer in this specification is not particularly limited. As an example, it is 1 to 100 mol%. Also, the number average molecular weight Mn of the temperature-responsive polymer is not particularly limited. As an example, it is 1,000 to 1,000,000.

[0015] The functional group showing the acidity of the component (A) in this specification is a functional group that ionizes in water to show anionic properties. It is not particularly limited, but as an example, it is selected from a hydroxy group, a carboxy group, a sulfo group, and a phosphate group. Two or more of these functional groups may be contained in the monomer, and it may also contain functional groups other than these, such as an aldehyde group, a carbonyl group, a nitro group, an amino group, an ether group, an ester group, and an amide group.

[0016] The acid dissociation constant in this specification refers to the acid dissociation constant in water. The pKa is determined from the side chain structure of the polymer structure. It is necessary to promote the electrostatic bond between cells and the substrate to impart cell adhesiveness. The acid dissociation constant pKa of the functional group showing acidity of the component (A) of the present invention is not particularly limited, but is -5.0 to 6.0, preferably -3.0 to 5.0. If the acid dissociation constant pKa is less than -5.0, copolymerization with the component (B) becomes difficult, and if the acid dissociation constant pKa exceeds 6.0, the promoting effect of the electrostatic bond weakens. As an example of the acid dissociation constant, since the side chain structure of acrylic acid is -COOH, it is 4.76, since the side chain structure of p-carboxystyrene is -C6H4COOH, it is 4.20, and since the side chain structure of p-styrenesulfonic acid is -C6H4SO3H, it is -2.80.

[0017] The component (A) in this specification is not particularly limited, and examples thereof include acrylic acid, vinylphenol, carboxystyrene, styrenesulfonate, and derivatives thereof.

[0018] The HLB value (HLB; Hydrophile-Lipophile Balance) in this specification is a value representing the degree of affinity for water and oil, as described in W.C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It takes values from 0 to 20, and the closer to 0, the higher the hydrophobicity, and the closer to 20, the higher the hydrophilicity. As methods for determining by calculation, there are the Atlas method, the Griffin method, the Davis method, and the Kawakami method. In the present invention, the value calculated by the Griffin method is used, and it is obtained by the following calculation formula based on the formula weight of the hydrophilic part in the repeating unit and the total formula weight of the repeating unit. HLB value = 20 × (formula weight of hydrophilic part) ÷ (total formula weight) As the definition of the hydrophilic part in the repeating unit of each block described above, examples include a sulfo group (-SO3-), a phospho group (-PO3-), a carboxy group (-COOH), an ester part (-COO-), an amide part (-CONH-), an imide part (-CON-), an aldehyde group (-CHO), a carbonyl group (-CO-), a hydroxy group (-OH), an amino group (-NH2), an acetyl group (-COCH3), an ethyleneamine part (-CH2CH2N-), an ethyleneoxy part (-CH2CH2O-), an alkali metal ion, an alkaline earth metal ion, an ammonium ion, a halide ion, and an acetate ion.

[0019] In calculating the hydrophilic part in the repeating unit, atoms constituting the hydrophilic part shall not overlap as atoms constituting other hydrophilic parts. An example of calculating the HLB value in the repeating unit is described below. For example, in the case of styrene (molecular weight: 104.15), there is no hydrophilic part, and since the molecular weight of the hydrophilic part is 0, the HLB value is 0.0. In the case of p-carboxystyrene (molecular weight: 148.15), the hydrophilic part is one carboxy group, and since the molecular weight of the hydrophilic part is 45.02, the HLB value is 6.1.

[0020] It is necessary to promote the hydrophobic bond between the cell and the substrate to impart cell adhesiveness, and the component (B) in the present specification is a monomer having an HLB value in the range of 0 to 5.0. When the HLB value exceeds 5.0, the promoting effect of the hydrophobic bond weakens.

[0021] The component (B) in the present specification is not particularly limited, and examples include styrene derivatives containing styrene, vinylnaphthalene, vinylanthracene, olefins, acrylates, and methacrylates.

[0022] The ratio of the component (A) in the cell-adhesive polymer in the present specification is not particularly limited, but is 1 to 99 mol%, preferably 10 to 50 mol%. If it is less than 10 mol%, the solubility in applicable solvents decreases, and if it exceeds 50 mol%, the balance between the electrostatic bond and the hydrophobic bond between the cell and the substrate is disrupted, making it difficult to exhibit sufficient cell adhesiveness.

[0023] The cell-adhesive polymer of the present specification may contain components other than component (A) and component (B). As an example, alkyl (meth)acrylate may be copolymerized to obtain solubility in a solvent. Further, it may be a random copolymer in which the components are randomly arranged, or it may be a block copolymer in which polymers composed of the respective components are linked.

[0024] The number average molecular weight Mn of the cell-adhesive polymer of the present specification is not particularly limited. As an example, it is 5,000 to 1,000,000.

[0025] The temperature-responsive polymer and the cell-adhesive polymer of the present specification become the surface treatment agent of the present invention by dissolving in a solvent. The solvent of the surface treatment agent is not particularly limited, but it is preferable to select a solvent in which the temperature-responsive polymer and the cell-adhesive polymer are soluble and the base material to be coated does not dissolve. As an example, 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, 3-methoxy-1-butanol, and a mixed solution selected from these can be used.

[0026] The concentration of the temperature-responsive polymer in the surface treatment agent of the present invention is not particularly limited, and any concentration can be selected according to the film-forming method and the target film thickness. As an example, it is 0.1 to 5.0 wt%, preferably 0.2 to 3.0 wt%. Further, a compound other than the temperature-responsive polymer and the cell-adhesive polymer of the present invention may be contained.

[0027] The introduction amount of the cell-adhesive polymer in the surface treatment agent of the present invention is not particularly limited. As an example, it is 1 to 50 wt%, preferably 1 to 30 wt%, more preferably 1 to 10 wt% with respect to the introduction amount of the temperature-responsive polymer. If it is less than 1 wt%, cell adhesiveness is not sufficiently expressed, and if it exceeds 50 wt%, the cell recoverability by the cooling treatment decreases.

[0028] By applying the surface treatment agent of the present invention to a substrate and drying it, a film made of the polymer compound of the present invention can be formed on the surface of the substrate. There is no particular limitation on the type of the substrate. As an example, polyethylene, polypropylene, polyolefin, acrylic polymer, methacrylic acid polymer, silicone rubber, polystyrene, polyethylene terephthalate, polycarbonate, metal, ceramics, and glass can be mentioned. These materials may be mixtures or copolymers, and may contain additives such as plasticizers. The film may be single-layer or have a multilayer structure. Substrates made of materials such as polyolefin, polystyrene, and polyethylene terephthalate are easy to mold. Also, the thickness of the film-like substrate may be a thickness having flexibility that enables molding and culture operations. For example, it is 10 to 500 μm, preferably 20 to 300 μm. The thickness of the film-like cell culture substrate is preferably 10 to 500 μm.

[0029] Also, the shape of the substrate is not particularly limited. As an example, in addition to plate-like, film-like, bead-like, and fibrous shapes, holes, grooves, protrusions, etc. provided on the plate-like substrate can also be mentioned. There is no particular limitation on the film forming method. As an example, 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. The application of the polymer solution may be performed on only one side or on all surfaces of the substrate. Also, the drying method may be natural drying or heat drying. The film thickness is not particularly limited. As an example, 1 nm to 100 μm, 10 nm to 1 μm, etc. can be mentioned, preferably 20 nm to 1 μm or 40 nm to 1 μm, and more preferably 20 nm to 50 nm.

[0030] The film-like cell culture substrate of the present invention can be used by laying it on a petri dish or a plate, or by welding the film and using it in the form of a cell culture bag.

[0031] The cell culture substrate obtained by applying and drying the surface treatment agent of the present invention on a film-like substrate is suitable for large-scale cell culture. Since a large amount of cells are required for biopharmaceuticals using cells as raw materials, there is a demand for increasing the size and reducing the cost of temperature-responsive cell culture substrates. A general temperature-responsive cell culture substrate is in the shape of a plastic petri dish and has a small surface area, so it is not suitable for culturing a large amount of cells. In addition, there has been a need for low production efficiency and unsuitability for mass production. Therefore, there has been a demand for a large-sized and highly productive temperature-responsive culture substrate. Since the film-like substrate is easy to increase in size and has excellent production efficiency, it meets the above needs.

[0032] The substrate coated with the film of the present invention on the surface can be used as a cell culture substrate. The cells applicable to the cell culture substrate of the present invention are not particularly limited. As an example, mesenchymal stem cells, Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human fetal kidney-derived cells HEK293 cells, human cervical cancer-derived HeLa cells, and further epithelial cells and endothelial cells constituting each tissue and organ in the living body, skeletal muscle cells, smooth muscle cells, cardiomyocytes, neuron cells and glial cells constituting the nervous system, fibroblasts, hepatocytes and non-parenchymal liver cells involved in the metabolism of the living body, adipocytes, stem cells existing in various tissues as cells having differentiation ability, and further cells differentiated therefrom can be used. In addition to these, cells contained in blood, lymph, cerebrospinal fluid, sputum, urine or feces, and microorganisms, viruses, protozoa, etc. existing in the body or the environment can be exemplified.

[0033] After culturing, to detach the proliferated cells from the cell culture substrate, it is only necessary to change the surrounding temperature to a temperature lower than the LCST of the temperature-responsive polymer, preferably 10 °C or lower than the LCST. Examples include medium exchange with a cooled medium and storage in a cold place. The medium exchange with a cooled medium is not particularly limited, and an example is a method of withdrawing the warm medium using a pipette and then pouring the cooled medium. It is also possible in the culture solution in which the cells were cultured and in other medium solutions, and can be selected according to the purpose.

[0034] Although the cooling time of the present invention is not particularly limited, it is preferably within 60 minutes in order to reduce damage to cells due to cooling.

Examples

[0035] Examples of the present invention will be described below, but the present invention is not limited by these examples. Unless otherwise specified, commercially available reagents were used. <Composition of Polymer> Proton nuclear magnetic resonance spectroscopy ( 1 1H-NMR) spectrum analysis was performed using a nuclear magnetic resonance measuring apparatus (manufactured by JEOL Ltd., trade name JNM-ECZ400S / L1). <Molecular Weight and Molecular Weight Distribution of Polymer> 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 apparatus used was HLC-8320GPC manufactured by Tosoh Corporation, and two columns of TSKgel Super AWM-H manufactured by Tosoh Corporation were used. The column temperature was set at 40°C, and the eluent was N,N-dimethylformamide containing 10 mM phosphoric acid and 10 mM lithium bromide for measurement. The measurement sample was prepared at 1.0 mg / mL for measurement. A calibration curve for molecular weight was prepared using polymethyl methacrylate (manufactured by Polymer Laboratories) with a known molecular weight.

[0036] <Measurement of Film Thickness> The film thickness was measured using a microscopic spectroscopic film thickness meter (manufactured by Otsuka Electronics Co., Ltd., trade name OPTM-A1). For Examples 1 to 7 and Comparative Examples 1 to 4 in the prepared culture substrate, the film thicknesses at 51 points were measured in the range of Φ48 mm, and for Examples 8 to 15 and Comparative Examples 5 to 7, the film thicknesses at 81 points were measured in the range of 40 mm × 40 mm, and the average values were determined. <Measurement of Cell Number> 10 μL was added from the cell suspension to a slide for cell counting (manufactured by Thermo Fisher Scientific, Inc., product name Countess Cell Counting Chamber Slid), and the cell count was measured using an automatic cell counter (manufactured by Thermo Fisher Scientific, Inc., product name CountessR II).

[0037] Example 1 A triblock copolymer composed of 2-methoxyethyl acrylate (MEA), n-butyl acrylate (BA), and N-isopropylacrylamide (IPAAm), with a composition ratio (molar ratio) of MEA / BA / IPAAm = 5 / 30 / 65, a number average molecular weight Mn of 128,000, and a molecular weight distribution Mw / Mn of 1.5, was prepared as a temperature-responsive polymer A. The lower critical solution temperature of the temperature-responsive polymer A was 32°C. Also, a random copolymer composed of carboxystyrene (CSt, pKa = 4.20) as component (A) and styrene (St, HLB value = 0) as component (B), with a composition ratio (molar ratio) of CSt / St = 33 / 67, a number average molecular weight Mn of 106,000, and a molecular weight distribution Mw / Mn of 1.8, was prepared as a cell-adhesive polymer a. 0.12 g of the temperature-responsive polymer A, 0.0012 g of the cell-adhesive polymer a, and 9.8788 g of the solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, the mixture was allowed to stand overnight to dissolve each polymer, thereby preparing a surface treatment agent 1.

[0038] 100 μL of the surface treatment agent 1 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 the surface treatment agent 1. The film thickness was 46 nm. Human bone marrow-derived mesenchymal stem cells (manufactured by Lonza Japan Co., Ltd., Product Code: PT-2501) were seeded on this cell culture substrate at a density of 1.0×10 5Cells were seeded and cultured at 37°C with a CO2 concentration of 5%. Dulbecco's Modified Eagle's Minimum Essential Medium (10 vol% FBS / DMEM) containing 10 vol% fetal bovine serum (produced in Columbia) was used as the culture medium. After culturing for 6 days, the culture medium was removed, and freshly cooled culture medium at 4°C was added and cooled at room temperature for 20 minutes. After 20 minutes, the cells detached with a pipette were collected and the cell count was measured. Furthermore, the cells remaining on the culture substrate were collected using trypsin and the total cell count was measured. The total cell count was 2.9×10 5 cells, and the cell recovery rate by the cooling treatment was 99%.

[0039] Example 2 0.12 g of temperature-responsive polymer A, 0.006 g of cell-adhesive polymer a, and 9.874 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, the mixture was allowed to stand overnight to dissolve each polymer, thereby preparing surface treatment agent 2.

[0040] 100 μL of surface treatment agent 2 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 surface treatment agent 2. The film thickness was 48 nm. 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. The total cell count was 2.8×10 5 cells, and the cell recovery rate by the cooling treatment was 95%.

[0041] Example 3 0.12 g of temperature-responsive polymer A, 0.012 g of cell-adhesive polymer a, and 9.868 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, the mixture was allowed to stand overnight to dissolve each polymer, thereby preparing surface treatment agent 3.

[0042] 100 μL of the surface treatment agent 3 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 the surface treatment agent 3. The film thickness was 51 nm. 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. The total cell number was 2.8×10 5 cells, and the cell recovery rate by the cooling treatment was 91%.

[0043] Example 4 0.3 g of the temperature-responsive polymer A, 0.03 g of the cell-adhesive polymer a, and 9.67 g of the solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, left standing overnight to dissolve each polymer, thereby preparing the surface treatment agent 4.

[0044] 100 μL of the surface treatment agent 4 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 the surface treatment agent 4. The film thickness was 167 nm. 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. The total cell number was 2.4×10 5 cells, and the cell recovery rate by the cooling treatment was 63%.

[0045] Example 5 A triblock copolymer composed of n-butyl acrylate (BA) and N-isopropylacrylamide (IPAAm) with a composition ratio (molar ratio) of BA / IPAAm = 35 / 65, a number average molecular weight Mn of 103,000, and a molecular weight distribution Mw / Mn of 1.5 was prepared. The lower critical solution temperature of the temperature-responsive polymer B was 32°C. 0.12 g of the temperature-responsive polymer B, 0.0012 g of the cell-adhesive polymer a, and 9.8788 g of the solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, left standing overnight to dissolve each polymer, thereby preparing the surface treatment agent 5.

[0046] 100 μL of the surface treatment agent 5 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 the surface treatment agent 5. The film thickness was 48 nm. 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. The total number of cells was 2.6×10 5 cells, and the cell recovery rate by the cooling treatment was 50%.

[0047] Example 6 0.12 g of the temperature-responsive polymer B, 0.006 g of the cell-adhesive polymer a, and 9.874 g of the solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, left standing overnight to dissolve each polymer, thereby preparing the surface treatment agent 6.

[0048] 100 μL of the surface treatment agent 6 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 the surface treatment agent 6. The film thickness was 50 nm. 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. The total number of cells was 2.6×10 5 cells, and the cell recovery rate by the cooling treatment was 47%.

[0049] Example 7 0.3 g of the temperature-responsive polymer B, 0.03 g of the cell-adhesive polymer a, and 9.67 g of the solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, left standing overnight to dissolve each polymer, thereby preparing the surface treatment agent 7.

[0050] 100 μL of the surface treatment agent 7 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 the surface treatment agent 7. The film thickness was 158 nm. 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. The total number of cells was 2.6×10 5 cells, and the cell recovery rate by the cooling treatment was 38%.

[0051] Comparative Example 1 0.10 g of temperature-responsive polymer A and 9.90 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, each polymer was dissolved by allowing it to stand overnight to prepare surface treatment agent 8.

[0052] 100 μL of surface treatment agent 8 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 surface treatment agent 8. The film thickness was 37 nm. 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. The total cell number was 0.03×10 5 cells, and the cell recovery rate was 99%.

[0053] Comparative Example 2 0.10 g of cell-adhesive polymer a and 9.90 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, each polymer was dissolved by allowing it to stand overnight to prepare surface treatment agent 9.

[0054] 100 μL of surface treatment agent 9 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 surface treatment agent 9. The film thickness was 87 nm. 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. The total cell number was 2.8×10 5 cells, and the cell recovery rate was 3%.

[0055] Comparative Example 3 A random copolymer consisting of carboxystyrene (CSt, pKa = 4.20) as component (A) and n-butyl acrylate (BA, HLB value = 6.9) as an alternative component of component (B), with a composition ratio (molar ratio) of CSt / BA = 39 / 61, a number average molecular weight Mn of 70,000, and a molecular weight distribution Mw / Mn of 1.6, was prepared as cell-adhesive polymer b. 0.10 g of temperature-responsive polymer A, 0.010 g of cell-adhesive polymer b, and 9.890 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, they were left standing overnight to dissolve each polymer, thereby preparing surface treatment agent 10.

[0056] 100 μL of surface treatment agent 10 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 surface treatment agent 10. The film thickness was 46 nm. 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. The total number of cells was 1.3×10 5 cells, and the cell recovery rate by the cooling treatment was 98%.

[0057] Comparative Example 4 0.10 g of temperature-responsive polymer B and 9.90 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, they were left standing overnight to dissolve each polymer, thereby preparing surface treatment agent 11.

[0058] 100 μL of surface treatment agent 11 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 surface treatment agent 11. The film thickness was 38 nm. 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. The total number of cells was 0.05×10 5 cells, and the cell recovery rate was 99%.

[0059]

Table 1

[0060] Example 8 Polymer solution 1 (surface treatment agent 1) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.) and air-dried to prepare the cell culture substrate 12. The film thickness was 45 nm. The circularly cut cell culture substrate 12 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells (manufactured by Lonza Japan, Product Code: PT-2501) were seeded at 1.0×10 5 cells and cultured at 37°C with a CO2 concentration of 5%. Dulbecco's modified Eagle's minimum essential medium containing 10 vol% fetal bovine serum (produced in Columbia) (10 vol% FBS / DMEM) was used as the culture medium. After culturing for 7 days, the culture medium was removed, and freshly cooled culture medium at 4°C was added and cooled at room temperature for 10 minutes. After 10 minutes, the cells detached with a pipette were collected and the cell count was measured. Furthermore, the cells remaining on the culture substrate were collected using trypsin and the total cell count was measured. The total cell count was 3.7×10 5 cells, and the cell recovery rate by the 10-minute cooling treatment was 100%.

[0061] Example 9 Polymer solution 2 (surface treatment agent 2) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.) and air-dried to prepare the cell culture substrate 13. The film thickness was 48 nm. The circularly cut cell culture substrate 13 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total cell count was 4.2×10 5 cells, and the cell recovery rate by the 30-minute cooling treatment was 100%.

[0062] Example 10 A polymer solution 3 (surface treatment agent 3) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (manufactured by OSG System Products Co., Ltd., OSP-05), and air-dried to prepare the cell culture substrate 14. The film thickness was 47 nm. The circularly cut cell culture substrate 14 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 4.3×10 5 cells, and the cell recovery rate by the 60-minute cooling treatment was 94%.

[0063] Example 11 0.12 g of temperature-responsive polymer A, 0.036 g of cell-adhesive polymer a, and 9.844 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, left standing overnight to dissolve each polymer, thereby preparing the surface treatment agent 12. The polymer solution 12 was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (manufactured by OSG System Products Co., Ltd., OSP-05), and air-dried to prepare the cell culture substrate 15. The film thickness was 58 nm. The circularly cut cell culture substrate 15 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 3.8×10 5 cells, and the cell recovery rate by the 60-minute cooling treatment was 71%.

[0064] Example 12 0.12 g of temperature-responsive polymer A, 0.060 g of cell-adhesive polymer a, and 9.820 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, the mixture was allowed to stand overnight to dissolve each polymer, thereby preparing surface treatment agent 13. Polymer solution 13 was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (manufactured by OSG System Products Co., Ltd., OSP-05), and air-dried to prepare cell culture substrate 16. The film thickness was 86 nm. The circularly cut cell culture substrate 16 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 3.6×10 5 cells, and the cell recovery rate by the 60-minute cooling treatment was 69%.

[0065] Example 13 0.30 g of temperature-responsive polymer A, 0.030 g of cell-adhesive polymer a, and 9.670 g of solvent 1-methoxy-2-propanol were added to a glass container, and after stirring, the mixture was allowed to stand overnight to dissolve each polymer, thereby preparing surface treatment agent 14. Polymer solution 14 was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (manufactured by OSG System Products Co., Ltd., OSP-05), and air-dried to prepare cell culture substrate 17. The film thickness was 145 nm. The circularly cut cell culture substrate 17 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 2.7×10 5 cells, and the cell recovery rate by the 30-minute cooling treatment was 94%.

[0066] Example 14 The polymer solution 2 (surface treatment agent 2) was applied to a 25-μm-thick polyethylene terephthalate film (Lumirror T60, manufactured by Toray Industries, Inc.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.) and air-dried to prepare the cell culture substrate 18. The film thickness was 57 nm. The circularly cut cell culture substrate 18 was placed in an untreated dish for suspension cells (AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total cell number was 4.0×10 5 cells, and the cell recovery rate by the 20-minute cooling treatment was 97%.

[0067] Example 15 The polymer solution 5 (surface treatment agent 5) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by Toho Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.) and air-dried to prepare the cell culture substrate 19. The film thickness was 45 nm. The circularly cut cell culture substrate 19 was placed in an untreated dish for suspension cells (AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total cell number was 3.7×10 5 cells, and the cell recovery rate by the 20-minute cooling treatment was 55%.

[0068] Comparative Example 5 The polymer solution 8 (surface treatment agent 8) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by Toho Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.) and air-dried to prepare the cell culture substrate 20. The film thickness was 46 nm. The circularly cut cell culture substrate 20 was placed in an untreated dish for suspension cells (AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total cell number was 0.13×10 5 cells, and the cell recovery rate by the 10-minute cooling treatment was 100%.

[0069] Comparative Example 6 The polymer solution 9 (surface treatment agent 9) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.), and air-dried to prepare the cell culture substrate 21. The film thickness was 49 nm. The circularly cut cell culture substrate 21 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 3.4×10 5 cells, and the cell recovery rate by the 60-minute cooling treatment was 18%.

[0070] Comparative Example 7 The polymer solution 11 (surface treatment agent 11) was applied to a 100-μm-thick linear low-density polyethylene film (LLDPE, manufactured by ToShin Chemical Industry Co., Ltd.) using a bar coater (OSP-05, manufactured by OSG System Products Co., Ltd.), and air-dried to prepare the cell culture substrate 22. The film thickness was 47 nm. The circularly cut cell culture substrate 22 was placed in an untreated dish for suspension cells (manufactured by AGC Techno Glass, 1010-060), and human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 8. The total number of cells was 0.05×10 5 cells, and the cell recovery rate by the 10-minute cooling treatment was 100%.

[0071]

Table 2

Claims

1. A surface treatment agent comprising a temperature-responsive polymer and a cell-adhesive polymer composed of the following (A) and (B); (A) A monomer having a functional group exhibiting acidity, (B) A monomer having an HLB value (Griffin method) in the range of 0 to 5.

0.

2. The surface treatment agent according to Claim 1, wherein the introduction amount of the cell-adhesive polymer is 1 to 50 wt% with respect to the introduction amount of the temperature-responsive polymer.

3. The surface treatment agent according to Claim 1 or 2, wherein the temperature-responsive polymer exhibits a lower critical solution temperature for water in the range of 0°C to 50°C.

4. The surface treatment agent according to any one of Claims 1 to 3, wherein the temperature-responsive polymer is a block copolymer composed of three or more repeating units.

5. The surface treatment agent according to any one of Claims 1 to 4, wherein the functional group exhibiting acidity of the constituent component (A) is selected from a hydroxy group, a carboxy group, a sulfo group, and a phosphate group.

6. The surface treatment agent according to any one of Claims 1 to 5, wherein the acid dissociation constant pKa of the functional group exhibiting acidity of the constituent component (A) is -5.0 to 6.

0.

7. The surface treatment agent according to any one of Claims 1 to 6, wherein the concentration of the temperature-responsive polymer in the surface treatment agent is 0.1 to 5.0 wt%.

8. A film formed by applying the surface treatment agent according to any one of Claims 1 to 7 to a substrate.

9. The film according to Claim 8, having a film thickness of 1 to 1000 nm.

10. A cell culture substrate having the film according to Claim 8 or 9 coated on its surface.

11. The cell culture substrate according to Claim 10, which is in the form of a film.

12. A method for producing the cell culture substrate according to Claim 10 or 11, comprising a step of applying the surface treatment agent to the substrate.

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