JP6954047B2 - Block copolymer coated beads and their manufacturing method - Google Patents

Description

The present invention relates to beads coated with a block copolymer containing a temperature-responsive polymer block and a method for producing the same.

Since pluripotent stem cells have pluripotency that allows them to differentiate into cells of all strains, research is underway toward the establishment of regenerative medicine and cell medicine. On the other hand, cost reduction of consumables such as media and growth factors has become an issue, and an inexpensive and stable cell supply system is required. In general, many animal cells, including human cells, are scaffold-dependent and require a scaffold to proliferate. In order to reduce the cost, it was necessary to design a scaffold that increases cell proliferation per unit medium amount. To solve this problem, microcarriers composed of beads made from natural polymers such as synthetic polymers and polysaccharides have been developed. By putting the microcarriers into a container containing a cell suspension and culturing the cells, the cells adhere to and proliferate not only on the surface of the container but also on the surface of the microcarriers, and the cell proliferation per unit medium amount can be enhanced.

On the other hand, the proliferated cells need to be detached from the scaffold and collected. The cells are attached to the scaffold by the adhesive protein produced by the cells, and generally, a proteolytic enzyme such as trypsin is used to exfoliate and recover the cells attached to the scaffold. However, proteolytic enzymes such as trypsin decompose adhesive proteins and at the same time, cell-specific surface proteins, which causes damage to cells.

In order to solve these problems, for example, in Patent Document 1, cells are cultured using microcarriers in which a temperature-responsive polymer whose hydration power changes at 32 ° C is immobilized on chloromethylated polystyrene beads. Cell proliferation is observed at 37 ° C., and a method of exfoliating and recovering cells from microcarriers by cooling to 20 ° C. without using a proteolytic enzyme is disclosed.

However, although the microcarrier described in Patent Document 1 uses chloromethylated polystyrene beads, there is a concern that the cells may be adversely affected due to the presence of unreacted chloromethyl groups. In addition, in the cooling exfoliation of cells using microcarriers on which a temperature-responsive polymer is fixed, it is difficult to exchange the medium with a pre-cooled medium because the microcarriers are suspended in the medium, and it is necessary to cool the cells in a cold place. However, due to the high heat capacity of the medium, it took more than 20 minutes for the medium to cool sufficiently, and minimally invasive cell recovery was not always possible.

Japanese Unexamined Patent Publication No. 2013-59312

An object of the present invention is to provide beads coated with a block copolymer containing a temperature-responsive polymer block and a method for producing the same.

In view of the above points, the present inventors have conducted intensive studies and found that the HLB value (Griffin method) is in the range of 7 or more and 20 or less, and the HLB value (Griffin method) is 0 or more and less than 7. Large amounts by using block copolymer coated beads containing polymer blocks in the range and block copolymer blocks containing temperature responsive polymer blocks with a lower critical dissolution temperature (LCST) in the range 0 ° C to 50 ° C. The present invention has been completed by finding that the cells can be cultured and that the cells can be recovered in a minimally invasive manner by changing the temperature. That is, the present invention includes the following aspects.
<1> Block copolymer-coated beads, wherein the block copolymer containing the following blocks (A), (B) and (C) is coated on the beads.
(A) A polymer block having an HLB value (Griffin method) in the range of 7.0 or more and 20.0 or less.
(B) A polymer block having an HLB value (Griffin method) in the range of 0.0 or more and less than 7.0.
(C) A temperature-responsive polymer block having a lower limit critical dissolution temperature (LCST) in water in the range of 0 ° C. to 50 ° C.
<2> The block copolymer-coated beads according to <1>, wherein the beads have a diameter of 20 μm to 1000 μm.
<3> The block copolymer-coated beads according to <1> or <2>, wherein the material of the beads is a synthetic polymer material.
<4> The method for producing block copolymer coated beads according to any one of <1> to <3>, which comprises the following steps (1) and (2).
(1) A step of producing a surface treatment agent containing a block copolymer containing the blocks (A), (B) and (C) according to <1>.
(2) A step of contacting the beads with the surface treatment agent produced in the step (1) to coat the beads with a block copolymer.
<5> The production method according to <4>, wherein the beads are coated with a block copolymer while being heated to 40 ° C. or higher.
<6> The production method according to <4>, wherein the beads are coated with a block copolymer under reduced pressure.
<7> Using the block copolymer-coated beads according to any one of <1> to <3>, the cells are subjected to the block copolymer at a temperature higher than the LCST of the temperature-responsive polymer block according to <1>. A cell culturing method characterized by culturing on coated beads, lowering the temperature below LCST after cell proliferation, and exfoliating the proliferating cells from the block copolymer coated beads.

Block copolymers containing two types of polymer blocks (A) and (B) having HLB values in a specific range and a temperature-responsive polymer block (C) can be made into beads regardless of the material and shape of the beads. It is possible to coat. The beads coated with the block copolymer containing the above-mentioned blocks (A), (B) and (C) are placed on the beads coated with the block copolymer by lowering the temperature below LCST after cell proliferation. The cells proliferated in the beads can be rapidly detached from the beads.

Hereinafter, the present invention will be described in detail.

The block copolymer used for the block copolymer coated beads of the present invention is a block copolymer containing the following blocks (A), (B) and (C).
(A) A polymer block having an HLB value (Griffin method) in the range of 7.0 or more and 20.0 or less.
(B) A polymer block having an HLB value (Griffin method) in the range of 0.0 or more and less than 7.0.
(C) A temperature-responsive polymer block having a lower limit critical dissolution temperature (LCST) in water in the range of 0 ° C. to 50 ° C.

The details of the blocks (A), blocks (B) and blocks (C) contained in the block copolymer used for the block copolymer coated beads of the present invention will be described below. The "polymer" includes a "copolymer" and a "homomopolymer". That is, the repeating units constituting each of the blocks (A), (B), and (C) may be one type or two or more types.

In the present specification, the HLB value (Hydrophile-Lipophile Balance: HLB) is referred to as W. C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It is a value indicating the degree of affinity for water and oil described in the above, and takes a value from 0 to 20, and the closer it is to 0, the higher the hydrophobicity, and the closer it is to 20, the higher the hydrophilicity. There are Atlas method, Griffin method, Davis method, and Kawakami method as the calculation method of the HLB value by the calculation formula, but in this specification, the value calculated by the Griffin method is used to construct the block copolymer of the present invention. It was calculated by the following formula based on the formula amount of the hydrophilic part in the repeating unit of each block and the total formula amount of the repeating unit.

HLB value = 20 × (formula amount of hydrophilic part in repeating unit) ÷ (total formula amount of repeating unit)
As the definition of the hydrophilic part in the repeating unit of each block described above, the sulfone part (-SO _3- ), the phosphono base part (-PO _3- ), the carboxyl base part (-COOH), the ester part (-COO-), and the amide Part (-CONH-), imide part (-CON-), aldehyde base (-CHO), carbonyl base (-CO-), hydroxyl base (-OH), amino base (-NH ₂ ), acetyl base (-COCH) _3), ethyleneamine unit (-CH2CH2N-), ethyleneoxy unit _{(-CH 2} CH 2 O-), an alkali metal ion, can be exemplified alkaline earth metal ions, ammonium ions, halide ions, acetate ions ..

In the calculation of the hydrophilic part in the repeating unit, the atoms constituting the hydrophilic part must not overlap as the 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 2-dimethylaminoethyl methacrylate (molecular weight: 157.11), the hydrophilic part has one ester part and one ethyleneamine part, and the hydrophilic part has a molecular weight of 86.07. The value is 11.0. In the case of n-butyl methacrylate (molecular weight: 142.20), the hydrophilic portion has one ester portion and the hydrophilic portion has a molecular weight of 44.01, so that the HLB value is 6.2.

Further, when each block constituting the block copolymer of the present invention is a copolymer composed of different monomers (monomer 1, monomer 2 ...), it is a repeating unit generated by polymerizing each monomer. The ratio (mol%) in the copolymer can be analyzed and calculated by the following formula.

HLB value = HLB value 1 x ratio 1 + HLB value 2 x ratio 2 + ...
Here, the HLB value 1 is the HLB value of the polymer produced by the polymerization of the monomer 1, and the composition 1 is the ratio (mol%) of the repeating unit produced by the polymerization of the monomer 1 in the polymer. The HLB value 2 is the HLB value of the polymer produced by the polymerization of the monomer 2, and the composition 2 is the ratio (mol%) of the repeating unit produced by the polymerization of the monomer 2 in the polymer.

The handling of the HLB value determines whether it is 7 or more or less than the number up to the first decimal place displayed by rounding off the second decimal place.

The block (A) contained in the block copolymer used for the block copolymer coated beads of the present invention is a block having an HLB value (Griffin method) in the range of 7.0 or more and 20.0 or less, and is a block of the present invention. It is preferably 8.0 or more and 20.0 or less in terms of shortening the cooling time required for exfoliating the cells grown on the block copolymer coated beads. The repeating unit constituting the block (A) is not particularly limited as long as the HLB value is 7.0 or more and 20.0 or less, and is 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 2-methoxyethyl acrylate, and tetrahydrofurfuryl. Aacrylate, dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium , Dimethyl (3-methacryloylaminopropyl) (4-sulfonatobutyl) aminium or dimethyl (2-methacryloyloxyethyl) (2-sulfonatoethyl) aminium, 2-dimethylaminomethyl (meth) acrylate, 2-dimethylaminoethyl (meth) ) A repeating unit produced by polymerizing acrylate, dimethyl [(meth) acrylamide methyl] amine, dimethyl [(meth) acrylamide ethyl] amine as a monomer, 2-dimethylaminoethyl (meth) acrylate, 3-dimethylaminopropyl ( A repeating unit produced by polymerizing meth) acrylate, dimethyl [(meth) acrylamide ethyl] amine, and dimethyl [3- (meth) acrylamide propyl] amine as monomers, and alkyl halides having 1 to 4 carbon atoms, ethylene oxide, and propylene. Examples of repeating units produced by reacting oxide, 1,2-butylene oxide, and 2-chloroethylmethyl ether can be exemplified. Among these repeating units, 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 2-methoxyethyl acrylate, and tetrahydrofurfuryl acrylate are used in that cell adhesion can be controlled and the cooling time required for cell detachment is shortened. , 2-Dimethylaminomethyl (meth) acrylate, 2-dimethylaminoethyl (meth) acrylate, dimethyl [(meth) acrylamide methyl] amine, dimethyl [(meth) acrylamide ethyl] amine as a monomer, a repeating unit produced by polymerization. Is preferable. Further, the block (A) may be composed of one type of repeating unit or may be composed of two or more types of repeating units as long as the HLB value is 7.0 or more and 20.0 or less.

The block (B) contained in the block copolymer used for the block copolymer coated beads of the present invention is a block having an HLB value (Griffin method) in the range of 0.0 or more and less than 7.0, and is coated on the beads. It is preferably 0 or more and 6 or less in terms of obtaining a stable film when the polymer is used. The repeating unit constituting the block (B) is not particularly limited as long as the HLB value is 0.0 or more and less than 7.0, and is styrene, 1-vinylnaphthalene, 2-vinylnaphthalene, 9-vinylanthracene, and 1-vinylpyrene. Ethyl (meth) acrylate, n-propyl (meth) acrylate, n-butyl (meth) acrylate, n-pentyl (meth) acrylate, n-hexyl (meth) acrylate, A repeating unit produced by polymerizing a (meth) acrylate compound such as n-heptyl (meth) acrylate, n-octyl (meth) acrylate, or n-tridecyl (meth) acrylate as a monomer can be exemplified. Among these repeating units, in terms of obtaining a stable film when coated on beads, a repeating unit produced by polymerizing styrene as a monomer, n-propyl (meth) acrylate, and n-butyl (meth) acrylate. , N-Pentyl (meth) acrylate, n-hexyl (meth) acrylate, n-heptyl (meth) acrylate, n-octyl (meth) acrylate, etc. It is preferable to have. Further, the block (B) may be composed of one kind of repeating unit or two or more kinds of repeating units as long as the HLB value is 0.0 or more and less than 7.0. In the present specification, the lower limit critical dissolution temperature (LCST; Lower Critical Separation Temperature) means that the polymer dissolves in water at a temperature lower than this temperature, but the polymer becomes insoluble or sparingly soluble at a temperature higher than this temperature. It is the temperature at which phase separation occurs.
The block (C) contained in the block copolymer used for the block copolymer coated beads of the present invention is a temperature-responsive polymer block having a lower limit critical dissolution temperature (LCST) in water in the range of 0 ° C to 50 ° C. be. When cells are cultured using the block copolymer-coated beads of the present invention, cell adhesion is imparted at around 37 ° C., which is the body temperature, and the cells are peeled off and recovered as the temperature drops, so that the LCST of the block (C) Is preferably in the range of 10 ° C to 40 ° C, more preferably in the range of 20 ° C to 35 ° C. If the LCST is less than 0 ° C or 50 ° C or higher, the cells are damaged, and minimally invasive cell detachment becomes difficult.
The repeating unit constituting the block (C) is not particularly limited as long as the LCST is in the range of 0 ° C to 50 ° C, and is N-isopropylmethacrylicamide (LCST = about 44 ° C) and N-ethoxyethylacrylamide (LCST = about). 35 ° C.), N-tetrahydrofurfurylmethacrylamide (LCST = about 35 ° C.), N-isopropylacrylamide (LCST = about 32 ° C.), N, N-diethylacrylamide (LCST = about 32 ° C.), N-n-propyl Methacrylamide (LCST = about 28 ° C), N-tetrahydrofurfurylacrylamide (LCST = about 28 ° C), N-methyl-N-isopropylacrylamide (LCST = about 22 ° C), N-n-propylacrylamide (LCST = about 28 ° C) 22 ° C.), N-methyl-Nn-propylacrylamide (LCST = about 20 ° C.) can be exemplified as a repeating unit produced by polymerizing as a monomer. Among these repeating units, N-ethoxyethylacrylamide, N-tetrahydrofurfurylmethacrylamide, N-isopropylacrylamide, N, N-diethylacrylamide, and Nn are good in cell exfoliation due to temperature drop. It is preferably a repeating unit produced by polymerizing −propylmethacrylamide and N-tetrahydrofurylacrylamide as a monomer. Further, the block (A) may be composed of one kind of repeating unit or two or more kinds of repeating units as long as the LCST is in the range of 0 ° C. to 50 ° C.

The composition ratios of the blocks (A), blocks (B) and blocks (C) constituting the block copolymer used for the block copolymer coated beads of the present invention are not particularly limited as long as they are 1 mol% to 90 mol%, respectively. However, the ratio of the block (C) is preferably 30 mol% to 90 mol% in that the cell detachability due to the temperature drop is good. When the ratio of the block (B) is less than 10 mol%, the adhesiveness of the block copolymer to the beads is lowered, and the block copolymer elutes into the medium when the temperature drops.

The number average molecular weight (Mn) of the block copolymer used for the block copolymer coated beads of the present invention is not particularly limited, but is preferably 3,000 to 1,000,000. If Mn is less than 3,000, it will be taken up by cells when eluted from the beads, and there is a concern that it will adversely affect the cells. If Mn exceeds 1,000,000, the viscosity of the solution becomes high and it becomes difficult to coat the beads.

The arrangement order of blocks (A), blocks (B), and blocks (C) constituting the block copolymer used for the block copolymer coated beads of the present invention is not particularly limited, and (A)-(B)-( Examples can be made of sequences consisting of and / or containing C), (A)-(C)-(B), (B)-(A)-(C). Further, the block copolymer may contain each block (A), (B) and (C) twice or more, and each block may be randomly arranged. For example, (A)-(B)-(A)-(C), (A)-(B)-(C)-(A) and the like are also allowed. In the sequence order of these block copolymers, (A)-(B)-(C) is preferable in terms of shortening the cooling time required for cell detachment.

Each block constituting the block copolymer used for the block copolymer coated beads of the present invention can perform block copolymerization with different types of monomers, and thus living such as living cationic polymerization, living anionic polymerization, and living radical polymerization. It is preferably produced by polymerization. Among these living polymerizations, it is preferable to use living radical polymerization because the polymerization reaction can be easily controlled. For example, "Radical Polymerization Handbook" published by NTS Co., Ltd., p. More preferably, it is produced by using the living radical polymerization technique described in 161 to 225 (2010). Examples of living radical polymerization techniques include atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated polymerization (NMP). It is preferably produced by RAFT polymerization because it is highly versatile and does not require the use of a metal exhibiting cytotoxicity.

The shape of the beads used in the present invention is not particularly limited, but is preferably spherical in terms of excellent mechanical strength and coatability of the block copolymer. The diameter of the beads is preferably larger than that of general cells because the cells are cultured on the surface of the beads. On the other hand, if the diameter of the beads is too large, the beads will settle during cell culture with stirring. Therefore, the diameter of the beads is preferably 20 to 1000 μm, more preferably 20 μm to 400 μm.

The material of the beads used in the present invention is not particularly limited, but preferred materials include, for example, polyethylene, polypropylene, polystyrene, polyalkyl (meth) acrylate, polyalkyl (meth) acrylamide, polyester, polyurethane, polyvinyl chloride, and polycarbonate. , Or synthetic polymers such as resins of their copolymers, plant-derived polymers such as dextran and cellulose, or wood chips and ceramics. Synthesis of polystyrene, polyalkyl (meth) acrylate, polyalkyl (meth) acrylamide, polyester, polyurethane, etc. in that the specific gravity of beads can be designed to be close to the specific gravity of the culture solution and the sedimentation of beads can be suppressed during cell culture with stirring. Polymer beads are preferred, and polystyrene beads are more preferred. The material of the beads may be one kind or may include two or more kinds of materials.

The block copolymer coated beads of the present invention can be produced by a method including the following steps (1) to (3).
(1) A step of producing a surface treatment agent containing a block copolymer containing the blocks of (A), (B) and (C) in the present invention.
(2) A step of contacting the beads with the surface treatment agent produced in the step (1) to coat the beads with a block copolymer.

The surface treatment agent in the present invention is not particularly limited as long as it contains a block copolymer containing the blocks (A), (B) and (C) in the present invention, and for example, it dissolves the block copolymer. It may contain a solvent that can be used. The solvent capable of dissolving the block copolymer is not particularly limited as long as it is a solvent that does not dissolve or deform the beads coating the block copolymer. When the beads are made of polystyrene, methanol, ethanol, 1 -Propanol, 2-propanol and the like can be exemplified. The concentration of the block copolymer in the surface treatment agent is not particularly limited, but is preferably 0.01 wt% to 10 wt%, more preferably 0.01 wt% to 1 wt, because the occurrence of coating unevenness of the surface treatment agent can be reduced. %.

The method of coating the beads with the surface treatment agent containing the block copolymer in the present invention is not particularly limited, and examples thereof include an impregnation method in which the beads can be coated by impregnating the surface treatment agent with the surface treatment agent and then drying the beads. Furthermore, block copolymers containing temperature-responsive polymer blocks are known to have a unique structure by self-assembly, and the structure may change depending on the temperature at the time of coating, which may affect the development of temperature responsiveness. Therefore, the temperature at the time of coating needs to be 40 ° C. or higher in order to exclude the influence of the change in room temperature and to exhibit stable temperature responsiveness.

In the coating method, the amount of the block copolymer in the present invention can be easily adjusted. Therefore, it is preferable to coat the entire amount of the block copolymer in the surface treatment agent. For example, a method of coating beads with a block copolymer can be exemplified by adding an appropriate amount of beads and a surface treatment agent to a flask, setting the flask on a rotary evaporator, and removing the solvent while reducing the pressure. The amount of block copolymer coated per unit area of beads can be calculated from the following formula.

Block copolymer coating amount per unit area (μg / cm ² ) = (amount of block copolymer charged (μg) -weight increase of flask (μg)) / (surface area of beads (cm ² / g)) × Weight of beads (g))
Coating amount of the beads of the block copolymer in the present invention is not particularly limited, for example, can be exemplified by ^{0.5μg / cm 2 ~1000μg / cm 2} . In terms of suppressing the variation in the coating amount, preferably from ^{0.5μg / cm 2 ~100μg / cm 2} .

In cell culture using the block copolymer coated beads of the present invention, cells are cultured on the beads at a temperature higher than the LCST of block (C), and after cell proliferation, the temperature is lowered to lower than the LCST of block (C) for proliferation. It is characterized by detaching cells from the beads. The method for culturing cells using the block copolymer beads of the present invention is not particularly limited. For example, the block copolymer coated beads of the present invention are added to a cell culture container containing a medium, and after seeding the cells, the cells are seeded. Cells can be cultured by standing, stirring continuously, or stirring or shaking for a certain period of time. The cooling method for exfoliating the cells from the block copolymer coated beads of the present invention by cooling is not particularly limited, and the cells may be cooled in a cold place or the medium may be replaced with a cooled medium. Further, in order to efficiently exfoliate the cells, the culture substrate may be tapped, shaken, or pipetting may be used in combination.

The cells cultured using the block copolymer-coated beads of the present invention are not particularly limited as long as they are cells that adhere to the surface of the block copolymer-coated beads before being cooled to exfoliate the cells and proliferate. , Human bone marrow-derived mesenchymal stem cells, human adipose tissue-derived mesenchymal stem cells, human lung-derived fibroblasts, human skin fibroblasts, Chinese hamster ovary-derived CHO cells, mouse binding tissue L929 cells, human fetal kidney-derived cells HEK293 Various cultured cell lines such as cells and HeLa cells derived from human cervical cancer, epithelial cells and endothelial cells constituting each tissue and organ in the living body, contractile skeletal muscle cells, smooth muscle cells, myocardial cells, nerves Neuronal cells, glial cells, fibroblasts, hepatic parenchymal cells involved in biological metabolism, non-hepatic parenchymal cells and fat cells, stem cells existing in various tissues as cells capable of differentiation, and further, they Examples of cells induced to differentiate from the above can be illustrated.

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.

<Analysis of monomer conversion rate and block copolymer composition>
It was obtained by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectral analysis using a nuclear magnetic resonance measuring device (manufactured by JEOL Ltd., trade name: JNM-ECZ400S / L1).

<Molecular weight and molecular weight distribution analysis of block copolymers>
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 device uses HLC-8320GPC manufactured by Tosoh Corporation, the column uses two TSKgel Super AWM-H manufactured by Tosoh Co., Ltd., the column temperature is set to 40 ° C., and the eluent contains 1,1 of 10 mM sodium trifluoroacetate. , 1,3,3,3-hexafluoro-2-propanol or N, N-dimethylformamide containing 10 mM lithium bromide. The measurement sample was prepared at 1.0 mg / mL and measured. As the calibration curve of the molecular weight, polymethyl methacrylate (manufactured by Polymer Laboratories) having a known molecular weight was used.

Example 1
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.57 g (10 mmol) of 2-dimethylaminoethyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyro 3.7 mg (22 μmol) of nitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.04 g (35 mmol) of n-butyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, a 2-dimethylaminoethyl methacrylate polymer (HLB value = 10.9) as a block (A) and an n-butyl methacrylate polymer (HLB value = 6.) as a block (B). 2), A block copolymer composed of an N-isopropylacrylamide polymer (LCST = about 32 ° C.) was prepared as the block (C).
[Preparation of surface treatment agent]
20 mg of the block copolymer described above was dissolved in 19.998 g of ethanol to prepare a 0.1 wt% surface treatment agent for the block copolymer.
[Coating of surface treatment agent on beads]
To the eggplant-shaped flask, 10 g of polystyrene beads having a diameter of 200 μm and 20 g of the above-mentioned surface treatment agent were added. The solvent was completely removed by reducing the pressure with stirring at 40 ° C. using a rotary evaporator, and the surface treatment agent was coated on the beads. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
5 mg of the above-mentioned block copolymer coated beads were added to a φ35 mm IWAKI untreated dish made of AGC technoglass, and 10 ^ 5 human bone marrow-derived mesenchymal stem cells (Lonza, PT-2501) were seeded at 37 ° C., CO. _The cells were cultured at 2 concentrations of 5%. As the medium, 3 mL of Lonza PT-3001 kit was used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. After 6 days of culturing, the beads and medium were transferred to a new IWAKI untreated dish and cooled at 15 ° C. for 10 minutes, and the cells detached from the beads. The number of cells exfoliated by cooling was measured with a hemocytometer. Further, the beads and the medium were transferred to a 15 cc centrifuge tube, treated with a centrifuge having a centrifugal acceleration of 100 G for 3 minutes, and then the medium was removed with an aspirator and washed with a phosphate buffer solution. 1 cc of trypsin EDTA solution was added, and the mixture was _{allowed to stand at 37 ° C. and a CO 2} concentration of 5% for 5 minutes, and the remaining cells were detached from the beads. The number of detached cells was measured with a hemocytometer. Among the cells adhering to the beads, 90% of the cells were exfoliated by cooling.

Example 2
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.56 g (10 mmol) of 2-dimethylaminopropylmethacrylamide, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobuty 3.7 mg (22 μmol) of ronitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.04 g (35 mmol) of n-butyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, a 2-dimethylaminopropyl methacrylamide polymer (HLB value = 10.0) was used as the block (A), and an n-butyl methacrylate polymer (HLB value = 6) was used as the block (B). .2) As a block (C), a block copolymer composed of an N-isopropylacrylamide polymer (LCST = about 32 ° C.) was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in [Cell culture evaluation] of Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. In the evaluation after 6 days of culturing, 85% of the cells adhering to the beads were exfoliated by cooling.

Example 3
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.57 g (10 mmol) of 2-dimethylaminoethyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyro 3.7 mg (22 μmol) of nitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.96 g (35 mmol) of n-hexyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, 2-dimethylaminoethyl methacrylate polymer (HLB value = 10.9) as block (A) and n-hexyl methacrylate polymer (HLB value = 5. B) as block (B). 2), A block copolymer composed of an N-isopropylacrylamide polymer (LCST = about 32 ° C.) was prepared as the block (C).
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in [Cell culture evaluation] of Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. In the evaluation after 6 days of culturing, 76% of the cells adhering to the beads were exfoliated by cooling.

Example 4
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.57 g (10 mmol) of 2-dimethylaminoethyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyro 3.7 mg (22 μmol) of nitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.04 g (35 mmol) of n-butyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.72 g (45 mmol) of N, N-diethylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N, N-diethylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, a 2-dimethylaminoethyl methacrylate polymer (HLB value = 10.9) as a block (A) and an n-butyl methacrylate polymer (HLB value = 6.) as a block (B). 2) As a block (C), a block copolymer composed of an N, N-diethylacrylamide polymer (LCST = about 32 ° C.) was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in [Cell culture evaluation] of Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. In the evaluation after 6 days of culturing, 73% of the cells adhering to the beads were exfoliated by cooling.

Example 5
[Preparation of block copolymer]
In a 100 mL round bottom flask, 0.94 g (6 mmol) of 2-dimethylaminoethyl methacrylate, 0.57 g (4 mmol) of n-butyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91. 7 mg (223 μmol) and 3.7 mg (22 μmol) of azobisisobutyronitrile were added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 0.63 g (4 mmol) of 2-dimethylaminoethyl methacrylate, 4.41 g (31 mmol) of n-butyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, 1, 10 mL of 4-dioxane was added. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, 2-dimethylaminoethyl methacrylate-n-butyl methacrylate copolymer (molar ratio 6/4, HLB value = 9.0) was used as the block (A), and the block (B) was used. A block composed of a 2-dimethylaminoethyl methacrylate-n-butyl methacrylate copolymer (molar ratio 4/31, HLB value = 6.7) and an N-isopropylacrylamide polymer (LCST = about 32 ° C.) as a block (C). A copolymer was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in [Cell culture evaluation] of Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. In the evaluation after 6 days of culturing, 73% of the cells adhering to the beads were exfoliated by cooling.

Example 6
[Preparation of block copolymer]
The same block copolymer as in Example 1 was prepared.
[Preparation of surface treatment agent]
The same surface treatment agent as in Example 1 was prepared.
[Coating of surface treatment agent on beads]
To the eggplant-shaped flask, 10 g of polystyrene beads having a diameter of 350 μm and 20 g of the above-mentioned surface treatment agent were added. The surface treatment agent was coated on the beads by reducing the pressure with stirring at 40 ° C. using a rotary evaporator. From the weight measurement, the coating amount was 9 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. Further, as a result of the examination of cooling exfoliation, 84% of the cells adhered to the beads were exfoliated by cooling.

Example 7
[Preparation of temperature-responsive block copolymer]
The same block copolymer as in Example 1 was prepared.
[Preparation of surface treatment agent]
The same surface treatment agent as in Example 1 was prepared.
[Coating of surface treatment agent on beads]
To the eggplant-shaped flask, 10 g of polystyrene beads having a diameter of 100 μm and 20 g of the above-mentioned surface treatment agent were added. The surface treatment agent was coated on the beads by reducing the pressure with stirring at 40 ° C. using a rotary evaporator. From the weight measurement, the coating amount was 2 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. Further, as a result of the examination of cooling exfoliation, 71% of the cells adhered to the beads were exfoliated by cooling.

Example 8
[Preparation of block copolymer]
The same block copolymer as in Example 1 was prepared.
[Preparation of surface treatment agent]
The same surface treatment agent as in Example 1 was prepared.
[Coating of surface treatment agent on beads]
To the eggplant-shaped flask, 10 g of polymethylmethacrylate beads having a diameter of 200 μm and 20 g of the above-mentioned surface treatment agent were added. The surface treatment agent was coated on the beads by reducing the pressure with stirring at 40 ° C. using a rotary evaporator. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. Further, as a result of the examination of cooling exfoliation, 80% of the cells adhered to the beads were exfoliated by cooling.

Comparative Example 1
[Preparation of block copolymer]
5.04 g (35 mmol) of n-butyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyronitrile 3 in a 100 mL round bottom flask. .7 mg (22 μmol) was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. The recovered white precipitate is dried under reduced pressure from an n-butyl methacrylate polymer (HLB value = 6.2) as a block (B) and from an N-isopropylacrylamide polymer (LCST = about 32 ° C.) as a block (C). A block copolymer containing no block (A) was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. Further, as a result of the examination of cooling exfoliation, 10% of the cells adhered to the beads were exfoliated by cooling.

Comparative Example 2
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.57 g (10 mmol) of 2-dimethylaminoethyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyro 3.7 mg (22 μmol) of nitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.14 g (45 mmol) of N-isopropylacrylamide, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 72 hours. The monomer conversion rate of N-isopropylacrylamide after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of water at 50 ° C. to obtain a pale yellow precipitate. The recovered pale yellow precipitate was dissolved in 100 mL of chloroform, anhydrous magnesium sulfate was added, and the mixture was stirred for 30 minutes. Anhydrous magnesium sulfate was removed by filtration, and the chloroform solution was concentrated under reduced pressure on a rotary evaporator to a solution of 20 mL. The concentrated chloroform solution was added dropwise to 300 mL of hexane to give a white precipitate. By drying the recovered white precipitate under reduced pressure, a 2-dimethylaminoethyl methacrylate polymer (HLB value = 10.9) as a block (A) and an N-isopropylacrylamide polymer (LCST = about 32 ° C.) as a block (C). ), A block copolymer containing no block (B) was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm2.
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, no cell adhesion was observed on the bead surface.

Comparative Example 3
[Preparation of block copolymer]
In a 100 mL round bottom flask, 1.57 g (10 mmol) of 2-dimethylaminoethyl methacrylate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid 91.7 mg (223 μmol), azobisisobutyro 3.7 mg (22 μmol) of nitrile was added and dissolved in 10 mL of 1,4-dioxane. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 24 hours. The monomer conversion rate of 2-dimethylaminoethyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, 5.04 g (35 mmol) of n-butyl methacrylate, 3.7 mg (22 μmol) of azobisisobutyronitrile, and 10 mL of 1,4-dioxane were further added to the flask. After bubbling with argon gas for 20 minutes, the flask was stoppered and reacted at 65 ° C. for 48 hours. The monomer conversion rate of n-butyl methacrylate after the reaction was 99%. After cooling the reaction solution to room temperature, the reaction solution was added dropwise to 500 mL of hexane to obtain a white precipitate. By drying the recovered white precipitate under reduced pressure, a 2-dimethylaminoethyl methacrylate polymer (HLB value = 10.9) as a block (A) and an n-butyl methacrylate polymer (HLB value = 6.) as a block (B). A block copolymer consisting of 2) was prepared.
[Preparation of surface treatment agent]
A surface treatment agent was prepared in the same manner as in [Preparation of surface treatment agent] of Example 1 except that the block copolymer prepared above was used.
[Coating of surface treatment agent on beads]
The surface treatment agent was coated on the beads in the same manner as in [Coating the surface treatment agent on the beads] of Example 1 except that the surface treatment agent prepared above was used. From the weight measurement, the coating amount was 5 μg / cm ² .
[Cell culture evaluation]
The procedure was the same as in Example 1 except that the block copolymer coated beads described above were used. After 1 day of culturing, cell adhesion to the bead surface was confirmed. Further, as a result of the examination of cooling exfoliation, the ratio of the cells adhered to the beads by cooling was 1%.

Claims (7)

A block copolymer-coated bead, wherein a block copolymer containing the following blocks (A), (B) and (C) is coated on the beads.
(A) A polymer block having an HLB value (Griffin method) in the range of 7.0 or more and 20.0 or less.
(B) A polymer block having an HLB value (Griffin method) in the range of 0.0 or more and less than 7.0.
(C) A temperature-responsive polymer block having a lower limit critical dissolution temperature (LCST) in water in the range of 0 ° C. to 50 ° C. The block copolymer-coated beads according to claim 1, wherein the beads have a diameter of 20 μm to 1000 μm. The block copolymer-coated beads according to claim 1 or 2, wherein the material of the beads is a synthetic polymer material. The method for producing block copolymer coated beads according to any one of claims 1 to 3, which comprises the following steps (1) and (2).
(1) A step of producing a surface treatment agent containing a block copolymer containing the blocks of (A), (B) and (C) according to claim 1.
(2) A step of contacting the beads with the surface treatment agent produced in the step (1) to coat the beads with a block copolymer. The production method according to claim 4, wherein the beads are coated with a block copolymer while being heated to 40 ° C. or higher. The production method according to claim 4, wherein the beads are coated with a block copolymer under reduced pressure. Using the block copolymer-coated beads according to any one of claims 1 to 3, cells are placed on the block copolymer-coated beads at a temperature higher than the LCST of the temperature-responsive polymer block according to claim 1. A cell culturing method, which comprises culturing the cells in the block copolymer, and then exfoliating the proliferating cells from the block copolymer coated beads by lowering the temperature below the LCST after the cell proliferating.