JP2018174919A - Block copolymer and surface treating agent using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide block copolymers useful as a coupling agent of a cell culture substrate enabling cell detachment in a short time, and to provide coupling agents using the same.SOLUTION: The present invention provides a block copolymer consisting of a block of (A), (B), and (C), and having a sequence of (A)-(B)-(C). (A) is a thermoresponsive polymer block whose lower critical solution temperature (LCST) for water is within the range from 0 to 45°C, (B) is a hydrophobic polymer block having no LCST in the range from 0 to 45°C and having an HLB value (Griffin method) in the range from 0 to less than 9, and (C) is a hydrophilic polymer block having no LCST in the range from 0 to 45°C and having an HLB value (Griffin method) in the range from 9 to less than 20.SELECTED DRAWING: None

Description

  The present invention relates to a block copolymer useful as a surface treatment agent for a cell culture substrate that enables cell detachment in a short time.

  Cell culture is used to understand biochemical phenomena and produce useful substances, and in recent years, stem cell discovery and advances in culture technology have brought much attention to cell-based treatments including regenerative medicine. ing.

  Many of the cells have adhesiveness, and it is known that they adhere to biological macromolecules such as collagen, fibronectin, laminin, and proliferate / differentiate in the body. Similarly, in cell culture, many of the adherent cells need to adhere to a certain substrate during culture. Conventionally, surface-treated glass or polymer has been used as a substrate. For example, there is a substrate obtained by subjecting polystyrene to γ-ray irradiation or silicone coating. In addition, a substrate coated with a biopolymer such as collagen or fibronectin on the surface is also used.

  After culturing cells on a substrate, they generally need to be transferred to another substrate, and in many cases, proteolytic enzymes are used. Proteolytic enzymes degrade proteins on cell surfaces and play a role in breaking the bonds between cells and substrates and the bonds between cells. On the other hand, proteolytic enzymes are known to greatly affect cell viability, and a method of separating cells from a substrate without using proteolytic enzymes is important as a method that does not damage cells. Similarly in regenerative medicine, cells or organized cells are required to be separated from the substrate and returned to the body without damaging the cultured cells in vitro and further breaking the bond between the cells. There is a need for methods of separating from substrates without the use of proteolytic enzymes.

  In order to solve the above problems, Patent Document 1 discloses a cell culture substrate coated with a temperature responsive polymer on the substrate surface. According to such a substrate, the adhesion of the substrate surface can be weakened by the sol transition of the temperature responsive polymer due to the temperature drop of the surrounding environment, and the cells can be exfoliated and recovered. Usually, cells need to be adhered and cultured near body temperature, and after culture, a substrate capable of detaching cells below body temperature is required.

  In the documents 2 and 3, N-isopropylacrylamide polymer (LCST = 32 ° C), N-as a temperature responsive polymer having a sol transition temperature [lower limit critical solution temperature (LCST)] in water in the range not higher than body temperature. n-Propyl Acrylamide Polymer (LCST = 21 ° C.), N-n-Propyl Methacrylamide Polymer (LCST = 32 ° C.), N-Ethoxyethyl Acrylamide Polymer (LCST = about 35 ° C.), N-Tetrahydrofurfuryl Acrylamide Polymer (LCST = about 28 ° C.), N-tetrahydrofurfuryl methacrylamide polymer (LCST = about 35 ° C.), N, N-diethylacrylamide polymer (LCST = 32 ° C.), etc. are described (patented) Documents 2 and 3).

  When the above-mentioned thermoresponsive polymer is used as a cell culture substrate, it is necessary to lower the temperature of the cell culture substrate to the lower limit critical dissolution temperature or less, but depending on the time, the temperature of the cells may be lowered simultaneously. Since cooling the cells exerts a decrease in the activity of the cells, it is necessary to shorten the cooling time.

  Furthermore, Document 4 discloses a cell culture substrate coated on the surface with a block copolymer of a specific structure, as a cell culture substrate capable of shortening the cooling time for cell detachment. (Patent Document 4), further shortening of the cooling time has been required.

Unexamined-Japanese-Patent No. 2-211865 gazette Unexamined-Japanese-Patent No. 3-266980 JP-A-5-244938 JP, 2016-192957, A

  An object of the present invention is to provide a block copolymer useful as a surface treatment agent for a cell culture substrate that enables cell detachment in a short time and a surface treatment agent using the same.

In view of the above points, the inventors of the present invention have conducted intensive studies, and as a result, they have a block copolymer having a specific sequence, which is composed of a temperature responsive polymer, a hydrophobic polymer and a hydrophilic polymer. The inventors have found that coating on a material to form a film enables cell detachment in a short time, thereby completing the present invention.
[1] That is, according to the present invention, there is provided a block copolymer consisting of the following blocks (A), (B) and (C) and having the sequence (A)-(B)-(C): .

  (A) A temperature responsive polymer block having a lower limit critical solution temperature (LCST) to water in the range of 0 to 45 ° C.

  (B) A hydrophobic polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 9 without LCST in the range of 0 to 45 ° C.

(C) A hydrophilic polymer block having an HLB value (Griffin method) in the range of 9 or more and less than 20 without LCST in the range of 0 to 45 ° C.
[2] Further, according to the present invention, there is provided a surface treatment agent for a substrate, comprising the block copolymer according to [1].
[3] Also, according to the present invention, there is provided a film obtained by applying the surface treatment agent according to [2] to a substrate.
[4] Further, according to the present invention, there is provided a cell culture substrate coated with the membrane according to [3].
[5] Further, according to the present invention, there is provided the cell culture substrate according to [4], which is coated on the surface of the substrate at a rate of 5.0 μg / cm ² or less as the block (A).
[6] Furthermore, according to the present invention, cells are cultured at a temperature higher than LCST of block (A) using the cell culture substrate described in [5], and temperature is blocked after cell growth (A) A cell culture method is provided, characterized in that the proliferating cells are detached from the substrate by lowering the LCST of

  After coating a cell culture substrate comprising a temperature-responsive polymer, a hydrophobic polymer and a hydrophilic polymer and having a specific sequence, which is obtained from the block copolymer according to the present invention, after cell culture, Hydrophilization of the substrate surface by temperature drop is promoted, and the cooling time required for cell detachment can be shortened. As a result, even after a cell culture, even if a cooling treatment is performed, a cell culture substrate capable of recovering cells in a short time without damaging the cells can be obtained.

Hereinafter, modes for carrying out the present invention (hereinafter, simply referred to as “the present embodiment”) will be described in detail. The following embodiment is an example for describing the present invention, and is not intended to limit the present invention to the following contents. The present invention can be appropriately modified and implemented within the scope of the spirit of the present invention.
1. Block copolymer The block copolymer of the present invention comprises a specific block (A) and a specific block (B) and a specific block (C), and the sequence is (A)-(B)-(C) It is a block copolymer.

  In the present specification, the lower critical solution temperature (LCST) means that the polymer is dissolved in water to a clear solution at a temperature lower than this temperature, but it is insolubilized at a temperature higher than this temperature It is the temperature which becomes cloudy or precipitates and phase separates.

  The block (A) in the present invention is a temperature responsive polymer block having an LCST in the range of 0 ° C. to 45 ° C. When the block copolymer of the present invention is used as a substrate for cell culture, in order to impart cell adhesiveness at around body temperature and detach cells at a temperature drop and separately collect cells without giving damage. The LCST of block (A) is preferably in the range of 20 ° C. to 45 ° C., and more preferably in the range of 30 ° C. to 40 ° C. If LCST is less than 0 ° C., it becomes difficult to detach without damaging the cells, and if it exceeds 45 ° C., cells can not be attached at around body temperature, and cell culture becomes difficult.

In the present specification, the HLB value (HLB; Hydrophile-Lipophile Balance) refers to W.K. C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). The value is a value representing the degree of affinity to water and oil described in 1. The value is from 0 to 20. The closer to 0, the higher the hydrophobicity, and the closer to 20, the higher the hydrophilicity. There are Atlas method, Griffin method, Davis method and Kawakami method as a method of determining by calculation, but in the present invention, the value calculated by Griffin method is used, and the formula weight of hydrophilic part in the repeating unit and the total of repeating unit It calculated | required by the following formula based on formula weight.
HLB value = 20 × (formula weight of hydrophilic part) ÷ (total formula weight)
The block (B) in the present invention is a block of a hydrophobic polymer having a specific range of HLB value and having no LCST in the range of 0 ° C. to 45 ° C., and contributes to adhesion of the block copolymer to a substrate Do.

  The HLB value of the block (B) in the present invention is in the range of 0 to less than 9, but preferably in the range of 0 to 8 in order to obtain a stable film that is not applied to the substrate and peeled off in water. More preferably, it is in the range of 0 or more and 6 or less. When the HLB value is 9 or more, it is easily peeled off in water when applied to a substrate, and a stable film can not be obtained.

  The block (C) in the present invention is a block of a hydrophilic polymer having an HLB value in a specific range which does not have an LCST in the range of 0 ° C. to 45 ° C.

  As the block (C) in the present invention, carboxylic acid group, carboxylic acid ester group, carboxylic acid metal salt, sulfonic acid group, sulfonic acid ester group, sulfonic acid metal salt, hydroxy group, alkoxy group, phenoxy group, amide group, Aminoalkyl group, carbamoyl group, sulfonamide group, sulfamoyl group, carbamate group, metal salt of phosphate group, oxyphosphate group, metal salt of oxyphosphate group, phosphobetaine group, sulfobetaine group, carbobetaine group And polymers of monomers having at least one hydrophilic group selected from polyethylene glycol groups and pyrrolidone groups.

  The block (C) in the present invention is preferably a block having no affinity for biopolymers such as proteins, peptides and glycoproteins or a block having no affinity for cells in order to shorten the cooling time required for cell detachment. It is a polymer of a monomer having at least one hydrophilic group selected from phosphobetaine group, sulfobetaine group, carbobetaine group, polyethylene glycol group, methoxyethylene group, furfuryl group, dialkylaminoalkyl group and pyrrolidone group. .

  The HLB value of block (C) in the present invention is in the range of 9 or more and less than 20, but in order to shorten the cooling time required for cell detachment, it is preferably in the range of 11 or more and less than 20, more preferably 13 or more. It is in the range of less than 20. If the HLB value is less than 9, the cooling time required for cell detachment will be long, leading to a decrease in cell activity.

  The arrangement of block (A), block (B) and block (C) in the block copolymer is (A)-(B)-(C), and the block (A) and block (C) Both are easily released, and cell adhesion / growth and releasability due to temperature drop can be compatible. On the other hand, if the sequences are (A)-(C)-(B) and (C)-(A)-(B), the cell adhesion / proliferation and removability due to temperature drop can not be compatible.

  The block (A) constituting the block copolymer of the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (1) is used Can.

  R1 is a hydrogen atom or a methyl group, and a hydrogen atom is used to bring LCST in the range of 20 ° C to 45 ° C.

  R2 and R3 are each independently a hydrogen group, a hydrocarbon group having 1 to 6 carbon atoms, a furfuryl group or a tetrahydrofurfuryl group, and R2 and R3 are combined with each other to form a pyrrolidine ring, a piperidine ring or a morpholine ring You may.

  Examples of the hydrocarbon group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert. Although -butyl group, n-hexyl group and isohexyl group can be illustrated, in order to make LCST into the range of 20 degreeC-45 degreeC, preferably n-propyl group and isopropyl group are used.

  As a repeating unit represented by General formula (1) in this invention, N, N- diethyl acrylamide, N-ethyl acrylamide, N-n- propyl acrylamide, N-n- propyl methacrylamide, N- isopropyl acrylamide, N -Isopropyl methacrylamide, N-cyclopropyl acrylamide, N-cyclopropyl methacrylamide, N-ethoxyethyl acrylamide, N-ethoxyethyl methacrylamide, N-tetrahydrofurfuryl acrylamide, N-tetrahydrofurfuryl methacrylamide, 1- (1 -Oxo-2-propenyl) pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) pyrrolidine, 1- (1-oxo-2-propenyl) piperidine, 1- (1-oxo-2-methyl-) 2-propenyl The repeating unit formed by polymerizing piperidine, 4- (1-oxo-2-propenyl) morpholine and 4- (1-oxo-2-methyl-2-propenyl) morpholine can be exemplified, but the LCST is preferably 20 ° C. to 45 ° C. Preferably, N, N-diethyl acrylamide, N-n-propyl acrylamide, N-isopropyl acrylamide, N-n-propyl methacrylamide, N-ethoxyethyl acrylamide, N-tetrahydrofurfuryl acrylamide, in order to obtain a range of C. The repeating unit formed by polymerizing N-tetrahydrofurfuryl methacrylamide is more preferably N, N-diethyl acrylamide, N-isopropyl acrylamide, N-n-, in order to set the LCST in the range of 30 ° C. to 40 ° C. Propyl methacrylamide, N-etoki Ethyl acrylamide, using the repeating units produced by polymerizing N- tetrahydrofurfuryl methacrylamide.

  Although the block (A) in the present invention is not particularly limited, a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (2) can be used.

  R4 represents a hydrogen atom or a methyl group, and a hydrogen atom is used to bring LCST in the range of 20 ° C to 45 ° C.

  R5 is a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and an alkyl group having 1 to 3 carbon atoms is used to make the LCST in the range of 20 ° C to 45 ° C.

  Examples of the hydrocarbon group having 1 to 6 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert. Although -butyl group, n-hexyl group and isohexyl group can be exemplified, in order to set LCST in the range of 20 ° C to 45 ° C, methyl group, ethyl group and n-propyl group are preferably used.

  r is an integer of 1 to 10, and an integer of 1 to 3 is used to make LCST in the range of 20 ° C to 45 ° C.

  The repeating unit represented by the general formula (2) in the present invention is preferably a repeating unit formed by polymerizing 2- (2-ethoxy) ethoxyethyl vinyl ether in order to set LCST in the range of 20 ° C. to 45 ° C. Use a unit.

  The block (A) in the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (3) can be used.

  R6 represents a hydrogen atom or a methyl group, and a hydrogen atom is used to bring LCST in the range of 20 ° C to 45 ° C.

  R7 represents a hydrocarbon group having 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, tert. -Butyl group, n-hexyl group and isohexyl group can be exemplified, but methyl group and ethyl group are preferably used in order to set LCST in the range of 20 ° C to 45 ° C.

  As a repeating unit represented by General formula (3) in this invention, in order to make LCST into the range of 20 degreeC-45 degreeC, Preferably the repeating unit which polymerizes and produces | generates a methyl vinyl ether is used.

  The block (B) in the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (4) can be used.

  R8 represents a hydrogen atom or a methyl group, and the methyl group is used in order to obtain a stable film which does not peel off in water by applying to a substrate.

  Y can be exemplified by a hydrogen atom, a chlorine atom, an acetoxy group, a nitrile group, and an aromatic hydrocarbon group having 6 to 30 carbon atoms, and in order to obtain a stable film which does not peel off in water, preferably a hydrogen atom, chlorine An atom, an aromatic hydrocarbon having 6 to 30 carbon atoms can be used. As a C6-C30 aromatic hydrocarbon group, a phenyl group, 1-naphthalene group, 2-naphthalene group, 9-anthracene group, 1-pyrene group, and its derivative can be illustrated.

  As the repeating unit represented by the general formula (4) in the present invention, ethylene, vinyl chloride, vinyl acetate, acrylonitrile, styrene, 1-vinylnaphthalene group, 2-vinylnaphthalene group, 9-vinylanthracene group, 1-vinylpyrene And a repeating unit formed by polymerizing

  The block (B) in the present invention is not particularly limited, but a polymer containing at least one repeating unit of the repeating units represented by the general formula (5) can be used.

  R9 represents a hydrogen atom or a methyl group, and the methyl group is used in order to obtain a stable film which does not peel off in water by applying to a substrate.

  R10 is a hydrocarbon group having 1 to 30 carbon atoms, and a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, an isobutyl group, a tert. -Butyl group, n-hexyl group, isohexyl group, n-octyl group, n-decyl group, n-dodecyl group, n-hexadecyl group, n-octadecyl group can be exemplified, but a stable film not peeled off in water is obtained Preferably, n-butyl group, isobutyl group, tert. -Butyl group, n-hexyl group, isohexyl group, n-octyl group, n-decyl group, n-dodecyl group, n-hexadecyl group, n-octadecyl group are used.

  Z is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, preferably an ester bond or an amide bond in order to obtain a stable film which does not peel off in water Particularly preferred is an ester bond.

  As a repeating unit represented by General formula (5) in this invention, methyl (meth) acrylate, ethyl (meth) acrylate, n-propyl (meth) acrylate, isopropyl (meth) acrylate, n-butyl (meth) acrylate , Isobutyl (meth) acrylate, tert-butyl (meth) acrylate, n-pentyl (meth) acrylate, n-hexyl (meth) acrylate, n-octyl (meth) acrylate, n-decyl (meth) acrylate, n-undecyl (Meth) acrylates such as (meth) acrylate, n-dodecyl (meth) acrylate, n-tetradecyl (meth) acrylate, n-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, n-eicosyl (meth) acrylate Compound N-n-octyl (meth) acrylamide, N-n-decyl (meth) acrylamide, N-n-dodecyl (meth) acrylamide, N-n-hexadecyl (meth) acrylamide, N-n-octadecyl (meth) acrylamide, etc. Polymerization of N-vinylamide compounds such as (meth) acrylamide compounds, N-vinyl-n-octylamide, N-vinyl-n-decylamide, N-vinyl-n-dodecylamide, N-vinyl-n-hexadecylamide For example, ethyl (meth) acrylate, n-propyl (meth) acrylate, n-butyl (meth) acrylate, n-pentyl (meth) may be used to obtain a stable film which does not peel off in water. ) Acrylate, n-hexyl (meth) acrylate, n-heptyl (meth) acrylate Over DOO, n- octyl (meth) acrylate, n- tridecyl (meth) using a repeating unit produced by polymerizing a (meth) acrylate compound of acrylate.

  As the block (B) in the present invention, in addition to the above, N-alkylmaleimide compounds such as N-cyclohexylmaleimide, N-phenylmaleimide and the like, fumaric acids such as di-tert-butyl fumarate and di-n-butyl fumarate A polymer of at least one monomer selected from diester compounds, N-vinylimidazole, N-vinylcarbazole and the like can be used.

  The block (C) constituting the block copolymer of the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (6) may be used it can.

  R11 is a hydrogen atom or a methyl group, and preferably a methyl group is used to shorten the cooling time required for cell detachment.

  R 12 is a C 1 to C 10 bivalent hydrocarbon group, and is preferably a C 1 to C 6 bivalent hydrocarbon group, particularly alkylene, in order to shorten the cooling time required for cell detachment. . Examples of such alkylene include methylene, ethylene, propylene, butylene, pentamethylene, hexamethylene and the like, with preference given to ethylene.

  R13 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene having 1 to 4 carbon atoms, such as methylene, ethylene, propylene, butylene, etc., in order to shorten the cooling time required for cell detachment. Is exemplified, with ethylene being particularly preferred.

  R14, R15 and R16 are, independently of one another, a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms, such as a methyl group or an ethyl group, in particular R14 in order to shorten the cooling time required for cell detachment. , R15 and R16 are simultaneously preferably a hydrogen atom or a methyl group, especially a methyl group.

  A1 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, and the ester bond, the amide bond, in particular, an ester bond, in order to shorten the cooling time required for cell detachment. Is preferred.

  As a repeating unit represented by General formula (6) in this invention, 2-methacryloyl oxyethyl phosphoryl choline, 2-acryloyl oxy ethyl phosphoryl choline, 3- (meth) acryloyl oxy propyl phosphoryl choline, 4- (meth) acryloyl oxy butyl phosphoryl choline 6- (Meth) acryloyloxyhexyl phosphorylcholine, 10- (Meth) acryloyloxydecyl phosphorylcholine, ω- (Meth) acryloyl (poly) oxyethylene phosphoryl choline, 2-acrylamidoethyl phosphoryl choline, 3-acrylamido propyl phosphoryl choline, 4-acrylamido butyl Phosphoryl choline, 6-acrylamido hexyl phosphoryl choline, 10-acrylamido decyl phosphoryl choline, ω- (meth) a The repeating unit produced | generated by polymerizing chloramide (poly) oxy ethylene phosphoryl choline can be illustrated. These repeating units have structures similar to phospholipids that are constituents of cell membranes, and thus can suppress cell adhesion. In order to shorten the cooling time required for cell detachment when used as a cell culture substrate, preferably, a repeating unit formed by polymerizing 2-methacryloyloxyethyl phosphorylcholine is used.

  Although the block (C) in the present invention is not particularly limited, a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (7) can be used.

  R17 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.

  R18 is-(CH2CH2O) i- (CH2CH (CH3) O) j-R19 (wherein, R19 is a hydrogen atom or an alkyl group having 1 to 30 carbon atoms, i is an integer of 1 to 300, and j is It is an integer of 0 to 60. It is represented by a polyoxyalkylene group represented by), -CH2-O-R20 (wherein, R20 is a hydrogen atom, a hydrocarbon group having 1 to 6 carbon atoms). A substituent, a furfuryl group, a tetrahydrofurfuryl group, and a hydrogen atom are shown.

  As a C1-C30 alkyl group used for R19, a methyl group, an ethyl group, n-propyl group, an isopropyl group, a cyclopropyl group, n-butyl group, an isobutyl group, tert. -Butyl group, n-hexyl group and isohexyl group can be exemplified, but methyl group, ethyl group, n-propyl group and isopropyl group are preferably used in order to shorten the cooling time required for cell detachment.

  As a C1-C6 alkyl group used for R20, a methyl group, an ethyl group, n-propyl group, an isopropyl group, a cyclopropyl group, n-butyl group, an isobutyl group, tert. -Butyl group, n-hexyl group and isohexyl group can be exemplified, but methyl group, ethyl group, n-propyl group and isopropyl group are preferably used in order to shorten the cooling time required for cell detachment.

  As a repeating unit represented by General formula (7) in this invention, polyethyleneglycol methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, hydroxymethyl acrylate, hydroxymethyl methacrylate, 2-methoxyethyl acrylate, 2-methoxy The repeating unit formed by polymerizing ethyl methacrylate, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate or tetrahydrofurfuryl methacrylate can be exemplified, but preferably polyethylene glycol to shorten the cooling time required for cell detachment. A repeating unit formed by polymerizing methacrylate, 2-methoxyethyl acrylate or tetrahydrofurfuryl acrylate is used.

  The block (C) in the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (8) can be used.

  R21 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.

  R22 is a divalent hydrocarbon group having 1 to 10 carbon atoms, and preferably has 1 carbon atom such as methylene, ethylene, propylene, butylene, pentylene or hexylene to shorten the cooling time required for cell detachment. It is a -6 divalent alkylene group, and more preferably ethylene.

  R23 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as methylene, ethylene, propylene, butylene or the like to shorten the cooling time required for cell detachment, and more preferably Is ethylene.

  R24 and R25 each independently represent a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms. Preferably, R24 and R25 simultaneously represent a hydrogen atom or methyl to shorten the cooling time required for cell detachment. And more preferably simultaneously methyl groups.

  A2 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, and is preferably an ester bond or an amide bond to shorten the cooling time required for cell detachment. Particularly preferred is an ester bond.

  X is a sulfonate anion group, a carboxylate anion group, a phosphate anion group, or an oxyanion group.

  As the repeating unit represented by the general formula (8) in the present invention, dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (2-methacryloyloxyethyl) (3-carboxylatopropyl) aminium, dimethyl (2-acryloyloxyethyl) (3-carboxylatopropyl) aminium, Dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium, dimethyl (3-methacryloylaminopropyl) (4-sulfonatobutyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-sulfonatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-sulfonatoethyl) aminium, dimethyl (2-methacryloyloxyethyl) (3-sulfonatopropyl) aminium, dimethyl (2-acryloyloxyethyl) ) (3-Sulfonatopropyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-phosphonatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-phosphonatoethyl) aminium, dimethyl (2-methacryloyloxyethyl) (3 -A repeating unit formed by polymerizing phosphonatopropyl) aminium or dimethyl (2-acryloyloxyethyl) (3-phosphonatopropyl) aminium can be exemplified, but the cooling time required for cell detachment is shortened. Preferably, dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (3-methacryloylaminopropyl) (3-sulfonato) is preferred. A repeating unit formed by polymerizing propyl) aminium, dimethyl (3-methacryloylaminopropyl) (4-sulfonatobutyl) aminium or dimethyl (2-methacryloyloxyethyl) (2-sulfonatoethyl) aminium is used.

  The block (C) in the present invention is not particularly limited, but a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (9) can be used.

  R26 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.

  R 27 and R 28 each independently represent a hydrogen atom or a methyl group.

  As a repeating unit represented by General formula (9) in this invention, the repeating unit formed by superposing | polymerizing acrylamide or N, N- dimethyl acrylamide can be used.

  The block (C) in the present invention is not particularly limited, but a polymer containing at least one repeating unit among the repeating units represented by the general formula (10) can be used.

  R29 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.

  As a repeating unit represented by the following general formula (10) in the present invention, a repeating unit formed by polymerizing N-vinylpyrrolidone can be used.

  Although the block (C) in the present invention is not particularly limited, a polymer containing at least one kind of repeating unit among repeating units represented by the following general formula (11) can be used.

  R30 is a hydrogen atom or a methyl group, and preferably a methyl group is used to shorten the cooling time required for cell detachment.

  R 31 is a C 1 to C 10 bivalent hydrocarbon group, and is preferably a C 1 to C 6 bivalent hydrocarbon group, particularly alkylene, in order to shorten the cooling time required for cell detachment. . Examples of such alkylene include methylene, ethylene, propylene, butylene, pentamethylene, hexamethylene and the like, with preference given to ethylene.

  R32 and R33 are, independently of each other, a hydrogen atom or a hydrocarbon group having 1 to 2 carbon atoms, such as a methyl group or an ethyl group, and in order to shorten the cooling time required for cell detachment, particularly R32 and R33 At the same time, a hydrogen atom or a methyl group, particularly a methyl group, is preferred.

  A3 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, and in order to shorten the cooling time required for cell detachment, an ester bond, an amide bond, particularly an ester bond Is preferred.

  As the repeating unit represented by the general formula (11) in the present invention, aminomethyl (meth) acrylate, N, N-dimethylaminomethyl (meth) acrylate, N, N-diethylaminomethyl (meth) acrylate, aminoethyl Meta) acrylate, N, N-dimethylaminoethyl (meth) acrylate, N, N-diethylaminoethyl (meth) acrylate, 3-aminopropyl (meth) acrylate, 3- (N, N-dimethylamino) -propyl (meth) ) Acrylate, 3- (N, N-diethylamino) -propyl (meth) acrylate, (meth) acrylamidomethylamine, dimethyl [(meth) acrylamidomethyl] amine, diethyl [(meth) acrylamidomethyl] amine, (meth) acrylamide Ethylamine, Dimethy [(Meth) acrylamidoethyl] amine, diethyl [(meth) acrylamidoethyl] amine, 3- (meth) acrylamidopropylamine, dimethyl [3- (meth) acrylamidopropyl] amine, diethyl [3- (meth) acrylamidoethyl] For example, N, N-dimethylaminomethyl (meth) acrylate, N, N-dimethylaminoethyl (meth) is preferably used to shorten the cooling time required for cell detachment, although it is possible to exemplify a repeating unit formed by polymerizing an amine. A repeating unit formed by polymerizing acrylate), dimethyl [(meth) acrylamidomethyl] amine, dimethyl [(meth) acrylamidoethyl] amine is used.

  Each block in the block copolymer may be directly bonded or may be bonded via a spacer. In addition, at least one of the bonds between the blocks may be a divalent bond including at least one type of bond among the divalent bonds represented by the following general formulas (12) and (13).

R34 is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms, but is preferably a hydrogen atom for the purpose of obtaining a stable bond between the blocks. As a C1-C20 hydrocarbon group, a methyl group, an ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, n-hexyl group, n-octyl group can be illustrated.
The ratio of the amount of repeating units (a) constituting the block (A) to the amount of all repeating units constituting the block copolymer is 1 to 90 mol%, and when coated on a substrate, the cell adhesiveness is imparted In order to reduce the cooling time required for cell detachment, it is preferably 5 to 85 mol%. If the ratio of the amount of repeating units (a) constituting the block (A) to the amount of all repeating units is less than 1 mol%, the cell adhesion decreases, and if it exceeds 90 mol%, the cooling time required for cell detachment is long Become.

  The ratio of the amount of repeating units (b) constituting the block (B) to the amount of all repeating units constituting the block copolymer is 1 to 90 mol%, and in the case of coating on a substrate, adhesion to the substrate It is preferable that it is 5-85 mol%, in order to provide property and to shorten the cooling time required for cell detachment. If the ratio of the amount of repeating units (b) constituting the block (B) to the amount of all repeating units is less than 1 mol%, the adhesion to the substrate decreases, and if it exceeds 90 mol%, the cooling necessary for cell detachment The time will be longer.

  The ratio of the amount of repeating units (c) constituting the block (C) to the amount of all repeating units constituting the block copolymer is 1 to 90 mol%, and when it is coated on a substrate, the cell adhesiveness is imparted In order to reduce the cooling time required for cell detachment, it is preferably 5 to 85 mol%. If the ratio of the amount of repeating units (c) constituting the block (C) to the amount of all repeating units is less than 1 mol%, the cooling time required for cell detachment will be long, and if it exceeds 90 mol%, the cell adhesion will be reduced Do.

  The number average molecular weight (Mn) of the block copolymer of the present invention is in the range of 3,000 to 1,000,000, preferably 4,000 to 500,000, and more preferably 5,000 to 200, It is less than 000. If it is less than 3,000, even if it is coated on a cell culture substrate, it elutes from the substrate into the medium during cell culture. On the other hand, if it exceeds 1,000,000, the solution viscosity becomes high, which makes it difficult to coat the cell culture substrate.

  Although there is no limitation in particular as a synthesis method of the block copolymer of the present invention, "The radical polymerization handbook", p. The method of copolymerizing can be used using the living radical polymerization technique described in 161-225 (2010).

  As for the order of monomers to be polymerized, (co) polymerize monomers forming block (A), (co) polymerize monomers forming block (B), and further monomers (block (C)) Method of co-polymerization, (co) polymerize monomers forming block (C), (co) polymerize monomers forming block (B), and (co) monomers further forming block (A) A polymerization method can be used.

  There is no particular limitation on the synthesis method of the block copolymer of the present invention. Michael, J. Prakt. Chem. 48, 94 (1893), R.S. Huisgen, in 1,3-Dipolar Cycloadditi-on Chemistry, ed. by A. Padwa, Wiley, New York, Vol. 1, 1-176 (1984), C.I. W. Tornoe, C. Christensen, M. Meldal, J.A. Org. Chem. 67, 3057-3062, V.I. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. , Int. Ed. 41, 2596-2599 (2002), and a click reaction of a block having an azide group and a block having an alkyne group can be used.

  As a method of Click reaction, the block (B) having an alkyne group at the end is synthesized by living radical polymerization of a monomer forming the block (B), and the terminal is formed by living radical polymerization of a monomer forming a block (A). Method of reacting a block (B) having an alkyne group and a block (A) and then reacting a block (C) having an azide group at an end, living radical polymerization of a monomer forming the block (B) The block (B) having an azide group at the end is further synthesized, and the living radical polymerization of the monomer to form the block (A) further comprises a diblock consisting of the block (B) having an azide group at the end and the block (A) A method of reacting a block (C) having an alkyne group at the end after synthesis, to generate a block (B) The block (B) having an alkyne group at the end is synthesized by living radical polymerization of nomers, and the block (B) and the block (C) having an alkyne group at the end are formed by living radical polymerization of a monomer forming a block (C). Method of reacting a block (A) having an azido group at the end after synthesis of the diblock body, and synthesizing a block (B) having an azido group at the living radical polymerization of a monomer forming the block (B) And a block (A) having an alkyne group at the end after synthesizing a diblock consisting of a block (B) having an azido group at the end and a block (C) by living radical polymerization of a monomer that further generates a block (C). The method of making it react can be used.

By using the click reaction of the block having an azide group and the block having an alkyne group as a synthesis method of the block copolymer, at least one of the blocks in the block copolymer can be represented by the general formula (12) or (13) A bivalent bond is introduced which comprises a bivalent bond of
2. Surface treatment agent The surface treatment agent for a substrate of the present invention contains the above-mentioned block copolymer. Preferably, it is a surface treatment agent for cell culture substrates such as petri dishes, multiwell plates, flasks, microcarriers and the like.

  The surface treatment agent of the present invention can perform surface treatment only by applying to a substrate.

  The surface treatment agent of the present invention may contain various solvents capable of dissolving the present block copolymer, in addition to the above-mentioned block copolymer. The solvent capable of dissolving the block copolymer is not particularly limited, but a solvent which does not evaporate after application is preferable, and even if it remains, ethanol, a mixed solvent of water and ethanol having little influence on cultured cells is preferable. Particularly preferred. The surface treatment agent of the present invention is usually in the form of a solution, but may be in the form of a powder that can be dissolved in the above-mentioned solvent.

  The target substrate of the surface treatment agent of the present invention is not particularly limited. However, since the block copolymer adheres to the substrate by hydrophobic interaction, various hydrophobic polymer materials are preferably used. Examples of hydrophobic polymer materials include acrylic polymers such as polymethyl methacrylate, various silicone rubbers such as polydimethylsiloxane, polystyrene, polyethylene terephthalate, and polycarbonate. In addition, metal substrates, ceramic substrates or glass substrates that have been surface-treated with a silane coupling agent can also be used.

  Further, the shape of the substrate is not particularly limited, and examples thereof include plate-like, bead-like and fiber-like shapes, and holes, grooves and protrusions provided in a plate-like substrate.

  As a method for applying the surface treatment agent of the present invention to a substrate, for example, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, etc. are generally known. It is possible to use various methods.

3. Membrane The membrane of the present invention is a membrane obtained by applying the surface treatment agent to various substrates and then drying. By including the block (B) in the block copolymer, it has adhesiveness to the cell culture substrate. Also, by including block (A) in the block copolymer, the membrane surface becomes hydrophobic at 37 ° C. or higher, which is the cell culture temperature, to allow attachment of proteins and the like, and cell adhesion culture is possible. Become. Furthermore, after the cell culture, by lowering the temperature, the membrane surface becomes hydrophilic and cell exfoliation can be promoted. In addition, by including the block (C) in the block copolymer, hydrophilization of the film surface further proceeds when the temperature is lowered, and it becomes possible to shorten the cooling time necessary for peeling. Furthermore, by including a block (C) having a charge in the block copolymer, adhesion of cells can be promoted and improvement of cell proliferation can be expected. The arrangement of block (A), block (B) and block (C) in these block copolymers is (A)-(B)-(C), and block (A) and block (C) on the film surface Both are easily released, and cell adhesion / proliferation and peelability due to temperature drop can be compatible. On the other hand, if the sequence is (A)-(C)-(B), only the block (A) is likely to come out on the surface, and temperature response is expressed, but due to cell adhesion / growth and temperature drop Peelability can not be compatible. In addition, if the sequence is (C)-(A)-(B), only block (C) is likely to be exposed on the surface and it becomes difficult to express temperature response, and it is due to cell adhesion / growth and temperature drop. Peelability can not be compatible.

The film of the present invention is preferably coated on the surface of the substrate at a rate of 0.1 μg / cm ² or more and 5.0 μg / cm ² or less as block (A), more preferably 0.3 μg / cm ² or more It is 0.8 μg / cm ² or less. If it is less than 0.1 μg / cm ^{2, the} cooling time required for cell detachment will be long when coated on a cell culture substrate. If it exceeds 5.0 μg / cm ² , the cells do not adhere to the cell culture substrate and the cells do not grow. 0.3 [mu] g / cm ² or more 0.8 [mu] g / cm ² or less can be particularly collateral good cell adhesion and cell growth length of the cooling time reduction and required for peeling. In addition, what is necessary is just to follow the usual method of measurement of the coating amount here, for example, what is necessary is just to utilize the weighing using a balance.
4. Cell Culture Substrate The cell culture substrate of the present invention is a cell culture substrate in which the substrate surface is covered with the above-mentioned membrane. The surface of the culture substrate of the present invention preferably has a microphase separation structure in order to promote hydrophilization of the substrate surface by temperature drop after cell culture and to shorten the cooling time required for cell detachment.

  The domain diameter and domain spacing of the microphase separation structure can be optionally controlled by the ratio of each block unit, the molecular weight of the block copolymer, the coating method and the coating conditions. After cell culture, in order to promote hydrophilization of the substrate surface by temperature drop and shorten the cooling time required for cell detachment, it is preferable that the domain size and the domain spacing be larger than cell growth factor and smaller than cells. .

  Cell culture with the cell culture substrate of the present invention is performed at a temperature higher than the LCST of the block (A) coated on the surface of the culture substrate, but when using human-derived cells, high culture efficiency is obtained It is preferable to carry out in the vicinity of the body temperature for the purpose, more preferably in the temperature range of 35 to 39 ° C., and still more preferably in the temperature range of 36 to 38 ° C. Other culture conditions are not particularly limited, and the culture may be performed under conditions commonly used in the art. For example, the medium may be one to which serum such as fetal bovine serum is added, or may be a serum-free medium.

  After culture, in order to detach the proliferating cells from the cell culture substrate, it is only necessary to change the ambient temperature to a temperature lower than the LCST of block (A), preferably 10 ° C. lower than the LCST. It is possible in the culture solution which has been cultured, or in other medium solutions, and can be selected according to the purpose. At that time, in order to detach the proliferating cells effectively and easily, the cell culture substrate may be tapped, shaken, or the medium may be agitated using a pipette or the like.

  By using the culture substrate of the present invention, preferably, cultured cells can be detached at a maximum diameter of 5 μm to 300 μm only by cooling. More preferably, it can be detached in the form of a single cell only by cooling. The size and shape of exfoliated cells can be determined by selecting the composition and molecular weight of the block copolymer, the structure of the cell culture substrate, the method of producing the cell culture substrate, the cell culture method, and the type of cells to be cultured. It can be adjusted. For example, increasing the ratio of blocks (C) in the block copolymer, increasing the thickness of the block copolymer by the method for producing a cell culture substrate, and increasing the unevenness of the culture substrate surface Can reduce the size of the cell aggregate and further detach in single cells.

  The cells cultured using the cell culture substrate of the present invention are not particularly limited as long as they can adhere to the surface prior to the application of a stimulus due to temperature drop. For example, in addition to various cultured cell lines such as Chinese hamster ovary-derived CHO cells and mouse connective tissue L929 cells, human embryonic kidney-derived cells HEK293 cells and human cervical cancer-derived HeLa cells, for example, each tissue in vivo and organs are constituted. Epithelial cells and endothelial cells, contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neuronal cells that constitute the nervous system, glial cells, fibroblasts, hepatocytes involved in in vivo metabolism, non-hepatic cells Alternatively, as adipocytes and cells having differentiation ability, stem cells present in various tissues, and cells differentiated therefrom can be used. Besides these, cells (live cells) contained in blood, lymph, spinal fluid, sputum, urine or feces, microorganisms present in the body or in the environment, viruses, protozoa etc. can be exemplified.

  Examples of the present invention will be described below, but the present invention is not limited by these examples. In addition, unless otherwise indicated, the reagent used the commercial item.

<Composition of block copolymer, amount of monomers in block copolymer, amount of organic solvent>
It was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectrum analysis using a nuclear magnetic resonance measurement apparatus (manufactured by JEOL Ltd., trade name: JNM-ECZ400S / LI).

<Molecular weight of block copolymer, molecular weight distribution>
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 uses HLC-8320GPC manufactured by Tosoh Corp., the column uses two TSKgel α-Ms manufactured by Tosoh Corporation, the column temperature is set to 40 ° C., and the eluent contains 2,2,2 containing 10 mM sodium trifluoroacetate -Measured using trifluoroethanol. The measurement sample was prepared and measured at 1.0 mg / mL. The calibration curve of molecular weight used polymethyl methacrylate (Agilent Technologies) of known molecular weight.
<Thickness of block copolymer coat film>
The cross section of the cell culture substrate coated with the block copolymer was observed using a transmission electron microscope to evaluate the thickness of the coated film. The transmission electron microscope used JEM-2100F made from Nippon Denshi.

<Coating amount of block copolymer>
A surface treatment agent having a known concentration is applied to a cell culture substrate, left at room temperature for 5 minutes, and then the added surface treatment agent is recovered with a Pasteur pipette or the like, and the surface treatment agent remains on the cell culture substrate. The amount was weighed by an electronic balance. The amount of the block copolymer coated was calculated in units of μg / cm ² from the concentration of the surface treating agent, the amount of the surface treating agent, and the coating area on the cell culture substrate.

<Water contact angle of substrate surface>
The bubble contact angle (θ) (°) at 40 ° C. and 15 ° C. in water was measured, and the water contact angle (180-θ) (°) at 40 ° C. and 15 ° C. was calculated. The contact angle of 3 μl of bubbles in water was measured using a contact angle meter DM300 manufactured by Kyowa Interface Science Co., Ltd., for θ.

Example 1
[Composition of block (C)]
In a 50 mL Schlenk tube, 0.79 g (5.0 mmol) of N, N-dimethylaminoethyl methacrylate, 40.4 mg (0.1 mmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid as an RAFT agent. 2.) 3.28 mg (0.02 mmol) of azobisisobutyronitrile as an initiator was added and dissolved in 5 mL of 1,4-dioxane. After bubbling argon for 10 minutes, reaction was carried out at 65 ° C. for 38 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 98% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized. The hydrophilic part weight of the repeating unit of the N, N-dimethylaminoethyl methacrylate polymer block (C) is the total (116.1) of 5 carbons, 10 hydrogens, 1 nitrogen and 2 oxygens, and the repeating units The total formula weight was 157.2 and the HLB value (Griffin method) was 15.

[Synthesis of Diblock Copolymer]
To the reaction solution obtained above, 0.71 g (5.0 mol) of n-butyl methacrylate, 3.28 mg (0.02 mmol) of azobisisobutyronitrile, 5 mL of 1,4-dioxane are added, and argon bubbling is carried out The reaction was allowed to proceed at 65 ° C. for 70 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of n-butyl methacrylate was polymerized, and N, N-dimethylaminoethyl methacrylate polymer (block A diblock copolymer consisting of (C) and an n-butyl methacrylate polymer (block (B)) could be synthesized. The hydrophilic part weight of the repeating unit of the n-butyl methacrylate polymer block (B) is the sum of one carbon and two oxygen (44.0), and the total weight of the repeating unit is 142.2, and the HLB value (Griffin method) was 6.

[Synthesis of block copolymer]
To the reaction solution obtained above, 1.13 g (10.0 mmol) of N-isopropylacrylamide and 3.28 mg (0.02 mmol) of azobisisobutyronitrile are added, and argon bubbling is carried out for 10 minutes, and then 65 ° C. Reaction for 65 hours. After the reaction, a part of the reaction solution was collected and 1 H-NMR was measured. As a result, it was confirmed that 94% of the charged amount of N-isopropylacrylamide was polymerized.

  The reaction solution was poured into 0.5 L of distilled water, and the precipitated white solid was filtered. The obtained white solid was dissolved in 200 mL of chloroform, 3 g of anhydrous magnesium sulfate was added to the obtained solution, and the mixture was stirred at room temperature for 15 minutes. The resulting suspension was filtered to remove magnesium sulfate, and chloroform was distilled off from the filtrate under reduced pressure using an evaporator and concentrated to 20 mL. The resulting concentrated solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours to obtain N, N-dimethylaminoethyl methacrylate polymer (block (C)), n-butyl methacrylate polymer (block (B)) and N- as a white powder. 1.21 g of a block copolymer comprising isopropyl acrylamide polymer (block (A)) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1. Moreover, the monomer and the organic solvent were not detected by 1H-NMR measurement (measurement solvent: deuterated chloroform) of the block copolymer.

[Synthesis of surface treatment agent]
15 mg of the block copolymer was added to 3.0 g of ethanol, and all were dissolved in 30 seconds by stirring to synthesize 0.5 wt% of a surface treatment agent for a substrate.

[Film evaluation]
After adding 1.0 mL of the obtained surface treatment agent to a polystyrene dish (φ 6 cm) manufactured by Corning Co., and leaving at room temperature for 5 minutes, the added surface treatment agent was recovered with a Pasteur pipette. After drying the dish surface by leaving it to stand at room temperature for 5 minutes, the dish is further heated at 70 ° C. for 30 minutes and then washed with water to obtain N, N-dimethylaminoethyl methacrylate polymer (block (C) A cell culture substrate was prepared in which a membrane consisting of a block copolymer consisting of n-butyl methacrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.4 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
Consisting of N, N-dimethylaminoethyl methacrylate polymer (block (C)), n-butyl methacrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) prepared above Human adipose-derived mesenchymal stem cells (Cell Applications, Inc.) (100 cells / mm 2) at 37 ° C., 5% CO 2 concentration, using a cell culture substrate into which a membrane comprising a block copolymer has been introduced Cultured. As the medium, human preadipocyte growth medium (basic medium + additive) (manufactured by Cell Applications, Inc.) was used. When cell growth was confirmed and the cultured cells were cultured until they covered 100% of the substrate, the cell number was confirmed with a 10 × 10 × microscope. After cooling the cell culture substrate to 15 ° C., the detached cells were removed with an aspirator, and the cell number was confirmed again with a 10 × 10 × microscope. The cells were detached by 70% cooling for 45 minutes.

Example 2
[Composition of block (C)]
The synthesis was performed in the same manner as in Example 1 [Synthesis of block (C)] except that 0.39 g (2.5 mmol) of N, N-dimethylaminoethyl methacrylate was used for reaction for 22 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 87% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of diblock copolymer] except that 1.07 g (7.5 mmol) of n-butyl methacrylate was used and reacted for 69 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of n-butyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate polymer (block A diblock copolymer consisting of (C) and an n-butyl methacrylate polymer (block (B)) could be synthesized.

[Synthesis of block copolymer]
The synthesis was carried out in the same manner as in [Synthesis of block copolymer] except that the reaction was carried out for 90 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N-isopropylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to give N, N-dimethylaminoethyl methacrylate polymer (block (C)) and n- as a white powder. There were obtained 1.25 g of a block copolymer comprising a butyl methacrylate polymer (block (B)) and an N-isopropylacrylamide polymer (block (A)). The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. Cell culture substrate introduced with a film comprising a block copolymer comprising (block (C)), n-butyl methacrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) Was produced. The film thickness was 10 nm, and it was coated at a rate of 0.4 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured until they covered 100% of the substrate, the cells were exfoliated 100% by cooling at 15 ° C. for 45 minutes.

Comparative Example 1
[Composition of block (C)]
Synthesis was carried out in the same manner as in [Composition of block (C)]. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
To the reaction solution obtained above, 1.13 g (10.0 mmol) of N-isopropylacrylamide, 3.28 mg (0.02 mmol) of azobisisobutyronitrile, 5 mL of 1,4-dioxane are added, and argon bubbling is carried out The reaction was allowed to proceed at 65 ° C. for 70 hours. After the reaction, a portion of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 98% of the charged amount of N-isopropylacrylamide was polymerized, and N, N-dimethylaminoethyl methacrylate polymer (block A diblock copolymer consisting of (C) and an N-isopropylacrylamide polymer (block (A)) could be synthesized.

[Synthesis of block copolymer]
To the reaction solution obtained above, 0.71 g (5.0 mmol) of n-butyl methacrylate and 3.28 mg (0.02 mmol) of azobisisobutyronitrile are added, and argon bubbling is carried out for 10 minutes, and then 65 ° C. Reaction for 65 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the n-butyl methacrylate charge was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours to obtain, as a white powder, N, N-dimethylaminoethyl methacrylate polymer (block (C)), N-isopropylacrylamide polymer (block (A)) and n- 2.11 g of a block copolymer comprising a butyl methacrylate polymer (block (B)) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. Cell culture substrate to which a film comprising a block copolymer comprising (block (C)), N-isopropylacrylamide polymer (block (A)) and n-butyl methacrylate polymer (block (B)) has been introduced Was produced. The film thickness was 10 nm, and it was coated at a rate of 0.4 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured to cover 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 45 minutes.

Comparative example 2
[Composition of block (B)]
In a 50 mL Schlenk tube, 0.71 g (5.0 mmol) of n-butyl methacrylate, 40.4 mg (0.1 mmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid as an RAFT agent, an initiator 3.28 mg (0.02 mmol) of azobisisobutyro nitrile was added thereto as a solution and dissolved in 5 mL of 1,4-dioxane. After bubbling argon for 10 minutes, reaction was carried out at 65 ° C. for 38 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 93% of the charged amount of n-butyl methacrylate was polymerized, and a polymer of n-butyl methacrylate (block (B) Could be synthesized.

[Synthesis of Diblock Copolymer]
To the reaction solution obtained above, 0.79 g (5.0 mmol) of N, N-dimethylaminoethyl methacrylate, 3.28 mg (0.02 mmol) of azobisisobutyro nitrile, and 5 mL of 1,4-dioxane are added. After bubbling argon for 10 minutes, reaction was carried out at 65 ° C. for 70 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, n-butyl methacrylate polymer (block A diblock copolymer consisting of (B) and an N, N-dimethylaminoethyl methacrylate polymer (block (C)) could be synthesized.

[Synthesis of block copolymer]
To the reaction solution obtained above, 1.13 g (10.0 mmol) of N-isopropylacrylamide and 3.28 mg (0.02 mmol) of azobisisobutyronitrile are added, and argon bubbling is carried out for 10 minutes, and then 65 ° C. Reaction for 65 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 88% of the charged amount of N-isopropylacrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours to obtain n-butyl methacrylate polymer block (B), N, N-dimethylaminoethyl methacrylate polymer block (C) and N-isopropylacrylamide polymer as white powder. 2.05 g of a block copolymer comprising block (A) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and n-butyl methacrylate polymer block (B) A cell culture substrate was prepared, into which a membrane comprising a block copolymer consisting of N, N-dimethylaminoethyl methacrylate polymer block (C) and N-isopropylacrylamide polymer block (A) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.4 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C. was lower than the contact angle to water at 40 ° C., indicating temperature responsiveness.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Comparative example 3
[Synthesis of Polymer Block (B)]
2.4 g (17 mmol) n-butyl methacrylate in a 100 mL test tube equipped with a three-way cock, 108 mg (267 μmol) 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid as an RAFT agent, as an initiator 8.8 mg (53 μmol) of azobisisobutyronitrile was added and dissolved in 10 mL of 1,4-dioxane. After 10 minutes of argon bubbling, the reaction was carried out at 65 ° C. for 25 hours. After the reaction, a part of the reaction solution was collected and 1H-NMR was measured. As a result, it was confirmed that 93% of the charged amount of n-butyl methacrylate was polymerized, and a polymer of n-butyl methacrylate (polymer block (polymer block B) could be synthesized.

[Synthesis of Diblock Copolymer]
After adding 20 mL of 1,4-dioxane, 4.8 g (42 mmol) of N-isopropylacrylamide and 8.8 mg (53 μmol) of azobisisobutyronitrile to the reaction solution obtained above, argon bubbling is performed for 10 minutes. The reaction was carried out at 65 ° C. for 45 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of N-isopropylacrylamide was polymerized. The reaction solution was poured into 300 mL of distilled water, and the precipitated white solid was filtered. The obtained white solid was dissolved in 40 mL of chloroform, 1 g of anhydrous magnesium sulfate was added to the obtained solution, and the mixture was stirred at room temperature for 30 minutes. The resulting suspension was filtered to remove magnesium sulfate, and chloroform was distilled off from the filtrate under reduced pressure using an evaporator and concentrated to 10 mL. The obtained concentrated solution was poured into 100 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried at 80 ° C. under reduced pressure for 6 hours, and a di-hydrate comprising a n-butyl methacrylate polymer (block (B)) and an N-isopropylacrylamide polymer (block (A)) as a white powder. 3 g of block copolymer was obtained. The composition, Mn and Mw / Mn of the obtained diblock copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the diblock copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and n-butyl methacrylate polymer (block (B (B)) was used. ) And a N-isopropylacrylamide polymer (block (A)) were used to prepare a cell culture substrate into which a membrane consisting of a diblock copolymer was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C. was lower than the contact angle to water at 40 ° C., indicating temperature responsiveness.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured to cover 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 45 minutes.

Comparative example 4
[Film evaluation]
Table 1 shows the results of evaluating the water contact angles at 40 ° C. and 15 ° C. of Upcell® 35 mm dish made of Cellseed, in which N-isopropylacrylamide is electron beam polymerized on the surface of a polystyrene dish. The contact angle to water at 15 ° C. was lower than the contact angle to water at 40 ° C., indicating temperature responsiveness.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the same method as Example 1 [cell culture evaluation and exfoliation evaluation] except having used the above-mentioned substrate for cell culture. Cell proliferation was confirmed. After the cultured cells were cultured to cover 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 45 minutes.

Example 3
[Composition of block (C)]
The synthesis was performed in the same manner as in Example 1 [Synthesis of block (C)] except that 0.39 g (2.5 mmol) of N, N-dimethylaminoethyl methacrylate was used for reaction for 22 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 87% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 1.07 g (7.5 mmol) of n-butyl methacrylate, the reaction was carried out in the same manner as Example 1 [Synthesis of diblock copolymer] except that the reaction was carried out for 69 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of n-butyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate polymer (block A diblock copolymer consisting of (C) and an n-butyl methacrylate polymer (block (B)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above, 1.26 g (10.0 mmol) of N, N-diethylacrylamide, synthesis was carried out in the same manner as Example 1 [Synthesis of block copolymer] except that reaction was carried out for 90 hours Did. After the reaction, a part of the reaction solution was collected and 1 H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of N, N-diethylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to give N, N-dimethylaminoethyl methacrylate polymer (block (C)) and n- as a white powder. 1.21 g of a block copolymer comprising a butyl methacrylate polymer (block (B)) and an N, N-diethylacrylamide polymer (block (A)) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. For cell culture to which a film comprising a block copolymer comprising (block (C)), n-butyl methacrylate polymer (block (B)) and N, N-diethylacrylamide polymer (block (A)) has been introduced A substrate was made. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured until they covered 100% of the substrate, the cells were exfoliated 100% by cooling at 15 ° C. for 45 minutes.

Comparative example 5
[Composition of block (C)]
Synthesis was carried out in the same manner as in Example 3 [Synthesis of block (C)]. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 1.26 g (10.0 mmol) of N, N-diethylacrylamide for 90 hours, except that the reaction is carried out for 90 hours, in the same manner as in [Synthesis of diblock copolymer]. The synthesis was done. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of N, N-diethylacrylamide was polymerized, N, N-dimethylaminoethyl methacrylate polymer A diblock copolymer consisting of (block (C)) and an N, N-diethylacrylamide polymer (block (A)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.07 g (7.5 mmol) of n-butyl methacrylate, synthesis was carried out in the same manner as in Comparative Example 1 [Synthesis of block copolymer] except that the reaction was carried out for 69 hours. The After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 97% of the charged amount of n-butyl methacrylate was polymerized.

  The reaction solution obtained above is treated in the same manner as in Comparative Example 1 [Synthesis of block copolymer] to obtain N, N-dimethylaminoethyl methacrylate polymer (block (C)) and N, as a white powder. There were obtained 1.25 g of a block copolymer consisting of an N-diethylacrylamide polymer (block (A)) and an n-butyl methacrylate polymer (block (B)). The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. For cell culture in which a film comprising a block copolymer comprising (block (C)), N, N-diethylacrylamide polymer (block (A)) and n-butyl methacrylate polymer (block (B)) is introduced A substrate was made. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured to cover 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 45 minutes.

Comparative example 6
[Composition of block (B)]
The synthesis was performed in the same manner as in Comparative Example 2 [Synthesis of block (B)] except that 1.07 g (7.5 mmol) of n-butyl methacrylate was used for reaction for 69 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of n-butyl methacrylate was polymerized, and a polymer of n-butyl methacrylate (block (B) Could be synthesized.

[Synthesis of Diblock Copolymer]
Same as Comparative Example 2 [Synthesis of Diblock Copolymer] except that the reaction solution obtained above and 0.39 g (2.5 mmol) of N, N-dimethylaminoethyl methacrylate were reacted for 48 hours. The synthesis was done by the method. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 95% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, n-butyl methacrylate polymer (block A diblock copolymer consisting of (B) and an N, N-dimethylaminoethyl methacrylate polymer (block (C)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.26 g (10.0 mmol) of N, N-diethylacrylamide, synthesis was carried out in the same manner as in Comparative Example 2 [Synthesis of block copolymer] except that the reaction was carried out for 90 hours. Did. After the reaction, a part of the reaction solution was collected and 1 H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of N, N-diethylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Comparative Example 2 [Synthesis of block copolymer] to obtain n-butyl methacrylate polymer (block (B)) and N, N-dimethylamino as white powder. There were obtained 1.25 g of a block copolymer consisting of an ethyl methacrylate polymer (block (C)) and an N, N-diethylacrylamide polymer (block (A)). The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 1.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and n-butyl methacrylate polymer (block (B (B)) was used. )) For cell culture in which a film comprising a block copolymer comprising N, N-dimethylaminoethyl methacrylate polymer (block (C)) and N, N-diethylacrylamide polymer (block (A)) is introduced A substrate was made. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C. was lower than the contact angle to water at 40 ° C., indicating temperature responsiveness.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Example 4
[Composition of block (C)]
The synthesis was performed in the same manner as in Example 1 [Synthesis of block (C)] except that 0.39 g (2.5 mmol) of N, N-dimethylaminoethyl methacrylate was used for reaction for 22 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 87% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
The synthesis was carried out in the same manner as Example 1 [Synthesis of diblock copolymer] except that the reaction solution obtained above and 0.78 g (7.5 mmol) of styrene were reacted for 70 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of styrene was polymerized, N, N-dimethylaminoethyl methacrylate polymer (block (C) ) And a styrene polymer (block (B)) could be synthesized. The hydrophilic part weight of the repeating unit of the styrene polymer block (B) was 0, the total weight of the repeating unit was 104.1, and the HLB value (Griffin method) was 0.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.13 g (10.0 mmol) of N-isopropylacrylamide, the synthesis was carried out in the same manner as in [Synthesis of block copolymer] except that the reaction was carried out for 90 hours. The After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N-isopropylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to obtain, as a white powder, N, N-dimethylaminoethyl methacrylate polymer (block (C)) and styrene weight. 1.21 g of a block copolymer comprising the united (block (B)) and the N-isopropylacrylamide polymer (block (A)) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. A cell culture substrate was prepared in which a film comprising a block copolymer consisting of (block (C)), styrene polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured until they covered 100% of the substrate, the cells were exfoliated 100% by cooling at 15 ° C. for 45 minutes.

Comparative example 7
[Composition of block (C)]
Synthesis was carried out in the same manner as in Example 4 [Synthesis of block (C)]. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 87% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, N, N-dimethylaminoethyl methacrylate The following polymers (block (C)) could be synthesized.

[Synthesis of Diblock Copolymer]
Synthesis was carried out in the same manner as in Comparative Example 1 [Synthesis of Diblock Copolymer] except that the reaction solution obtained above and 1.13 g (10.0 mmol) of N-isopropylacrylamide were reacted for 90 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 95% of the charged amount of N-isopropylacrylamide was polymerized. A diblock copolymer consisting of N, N-dimethylaminoethyl methacrylate polymer (block (C)) and N-isopropylacrylamide polymer (block (A)) could be synthesized.

[Synthesis of block copolymer]
Synthesis was carried out in the same manner as in Comparative Example 1 [Synthesis of block copolymer] except that the reaction solution obtained above and 0.78 g (7.5 mmol) of styrene were used for reaction for 70 hours. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 97% of the amount of styrene charged was polymerized.

  The reaction solution obtained above is treated in the same manner as in Comparative Example 1 [Synthesis of block copolymer] to obtain N, N-dimethylaminoethyl methacrylate polymer (block (C)) and N- as a white powder. There were obtained 1.25 g of a block copolymer consisting of an isopropylacrylamide polymer (block (A)) and a styrene polymer (block (B)). The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as that described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and an N, N-dimethylaminoethyl methacrylate polymer was prepared. A cell culture substrate was prepared in which a film comprising a block copolymer consisting of (block (C)), N-isopropylacrylamide polymer (block (A)) and styrene polymer (block (B)) was introduced. . The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured to cover 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 45 minutes.

Comparative Example 8
[Composition of block (B)]
The synthesis was performed in the same manner as in Comparative Example 2 [Synthesis of block (B)] except that 0.78 g (7.5 mmol) of styrene was used for reaction for 70 hours. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 97% of the charged amount of styrene was polymerized, and a styrene polymer (block (B)) could be synthesized.

[Synthesis of Diblock Copolymer]
Same as Comparative Example 2 [Synthesis of Diblock Copolymer] except that the reaction solution obtained above and 0.39 g (2.5 mmol) of N, N-dimethylaminoethyl methacrylate were reacted for 22 hours. The synthesis was done by the method. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 87% of the charged amount of N, N-dimethylaminoethyl methacrylate was polymerized, and a styrene polymer (block (B) ) And a N, N-dimethylaminoethyl methacrylate polymer (block (C)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.13 g (10.0 mmol) of N-isopropylacrylamide, the synthesis was carried out in the same manner as in Comparative Example 2 [Synthesis of block copolymer] except that the reaction was carried out for 90 hours. The After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N-isopropylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Comparative Example 2 [Synthesis of block copolymer] to form a styrene powder (block (B)) and N, N-dimethylaminoethyl methacrylate as a white powder. There were obtained 1.25 g of a block copolymer comprising the united (block (C)) and the N-isopropylacrylamide polymer (block (A)). The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and a styrene polymer (block (B)) was used. A cell culture substrate was prepared in which a film comprising a block copolymer consisting of N, N-dimethylaminoethyl methacrylate polymer (block (C)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The contact angle to water at 15 ° C. was lower than the contact angle to water at 40 ° C., indicating temperature responsiveness.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 1 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Example 5
[Composition of block (C)]
The synthesis was carried out in the same manner as Example 1 [Synthesis of block (C)] except that reaction was carried out for 48 hours using 0.74 g (2.5 mmol) of 2-methacryloyloxyethyl phosphorylcholine. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of 2-methacryloyloxyethyl phosphorylcholine was polymerized, and a polymer of 2-methacryloyloxyethyl phosphorylcholine ( The block (C) was able to be synthesized. The hydrophilic part weight of the repeating unit of 2-methacryloyloxyethyl phosphorylcholine polymer (block (C)) is a total of 25 carbons, 17 hydrogens, 1 nitrogen, 6 oxygens and 1 phosphorus (254.2). The total repeating unit weight was 295.3, and the HLB value (Griffin method) was 17.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above and 0.79 g (7.5 mmol) of styrene, the synthesis was carried out in the same manner as Example 1 [Synthesis of diblock copolymer] except that the reaction was carried out for 48 hours. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of styrene was polymerized, and 2-methacryloyloxyethyl phosphorylcholine polymer (block (C)) A diblock copolymer consisting of a styrene polymer (block (B)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above, 1.26 g (10.0 mmol) of N, N-diethylacrylamide, synthesis was carried out in the same manner as Example 1 [Synthesis of block copolymer] except that reaction was carried out for 90 hours Did. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N, N-diethylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to give 2-methacryloyloxyethyl phosphorylcholine polymer (block (C)) and styrene polymer (white powder) as a white powder. 1.15 g of a block copolymer comprising the block (B) and the N, N-diethylacrylamide polymer (block (A)) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and 2-methacryloyloxyethyl phosphorylcholine polymer (block A cell culture substrate was prepared in which a film comprising a block copolymer consisting of (C), a styrene polymer (block (B)) and an N, N-diethylacrylamide polymer (block (A)) was introduced. . The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
Block copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine polymer (block (C)), styrene polymer (block (B)) and N, N-diethylacrylamide polymer (block (A)) prepared above Human bone marrow-derived mesenchymal stem cells (Lonza, PT-2501) (100 cells / mm 2) were cultured at 37 ° C. and a CO 2 concentration of 5% using a cell culture substrate into which a combined membrane was introduced. The medium and the addition factor used Lonza PT-3001 kit. When cell growth was confirmed and the cultured cells were cultured until they covered 100% of the substrate, the cell number was confirmed with a 10 × 10 × microscope. After cooling the cell culture substrate to 15 ° C., the detached cells were removed with an aspirator, and the cell number was confirmed again with a 10 × 10 × microscope. Cooling for 15 minutes resulted in 100% detachment of cells in the form of single cells.

Comparative Example 9
[Composition of block (C)]
Example 5 [Synthesis of block (C)] Synthesis was carried out in the same manner as described above. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of 2-methacryloyloxyethyl phosphorylcholine was polymerized, and a polymer of 2-methacryloyloxyethyl phosphorylcholine ( The block (C) was able to be synthesized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 1.26 g (10.0 mmol) of N, N-diethylacrylamide for 70 hours, except that the reaction is carried out for 70 hours, in the same manner as in [Synthesis of diblock copolymer]. The synthesis was done. After the reaction, a portion of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the charged amount of N, N-diethylacrylamide was polymerized, and 2-methacryloyloxyethyl phosphorylcholine polymer (block A diblock copolymer consisting of (C) and N, N-diethylacrylamide (block (A)) could be synthesized.

[Synthesis of block copolymer]
The synthesis was performed in the same manner as in Comparative Example 1 [Synthesis of block copolymer] except that the reaction solution obtained above and 0.79 g (7.5 mmol) of styrene were used for reaction for 48 hours. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 92% of the charged amount of styrene was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and 2-methacryloyloxyethyl phosphorylcholine polymer (block (C)), N, N-diethylacrylamide (block (A)) and styrene polymer (block) as white powder. 2.11 g of a block copolymer consisting of (B) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and 2-methacryloyloxyethyl phosphorylcholine polymer (block A cell culture substrate was prepared in which a membrane comprising a block copolymer of (C), N, N-diethylacrylamide (block (A)) and a styrene polymer (block (B)) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Comparative example 10
[Composition of block (B)]
The synthesis was performed in the same manner as in Comparative Example 2 [Synthesis of block (B)] except that 0.79 g (7.5 mmol) of styrene was used for reaction for 48 hours. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 90% of the charged amount of styrene was polymerized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 0.74 g (2.5 mmol) of 2-methacryloyloxyethyl phosphorylcholine and following the same procedure as in [Synthesis of diblock copolymer] in Comparative Example 2 except that reaction is carried out for 48 hours The synthesis was done. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 93% of the charged amount of 2-methacryloyloxyethyl phosphorylcholine was polymerized, and a styrene polymer (block (B)) A diblock copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine (block (C)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.26 g (10.0 mmol) of N, N-diethylacrylamide, synthesis was carried out in the same manner as in Comparative Example 2 [Synthesis of block copolymer] except that the reaction was carried out for 75 hours. Did. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N, N-diethylacrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and a styrene polymer (block (B)), 2-methacryloyloxyethyl phosphorylcholine (block (C)) and an N, N-diethylacrylamide polymer (block) are obtained as a white powder. 2.31 g of a block copolymer consisting of (A) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and a styrene polymer (block (B)) was used. A cell culture substrate was prepared in which a membrane consisting of a block copolymer consisting of 2-methacryloyloxyethyl phosphorylcholine (block (C)) and an N, N-diethylacrylamide polymer (block (A)) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 60 minutes.

Comparative example 11
[Composition of block (B)]
In a 50 mL Schlenk tube, 0.74 g (2.5 mmol) of 2-methacryloyloxyethyl phosphorylcholine, 0.79 g (7.5 mmol) of styrene, 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic as a RAFT agent 40.4 mg (0.1 mmol) of acid and 6.56 mg (0.04 mmol) of azobisisobutyronitrile as an initiator were added and dissolved in 10 mL of 1,4-dioxane. After bubbling argon for 10 minutes, the reaction was carried out at 65 ° C. for 48 hours. After the reaction, a portion of the reaction solution was collected and 1H-NMR was measured. As a result, it was confirmed that 98% of the charged amount of 2-methacryloyloxyethyl phosphorylcholine and styrene were polymerized, and 2-methacryloyloxyethyl phosphorylcholine And a random copolymer of styrene and styrene (block (B)) could be synthesized. The HLB value of the random copolymer of 2-methacryloyloxyethyl phosphorylcholine and styrene (block (B)) was 3.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above and 1.26 g (10.0 mmol) of N, N-diethylacrylamide, synthesis was carried out in the same manner as in Comparative Example 2 [Synthesis of block copolymer] except that the reaction was carried out for 75 hours. Did. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N, N-diethylacrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried for 2 hours under reduced pressure, and a random copolymer of 2-methacryloyloxyethyl phosphorylcholine and styrene (block (B)) and an N, N-diethylacrylamide polymer (block (A) as white powder. 2.21 g of a diblock copolymer consisting of The composition, Mn, and Mw / Mn of the obtained diblock copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the diblock copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and a random of 2-methacryloyloxyethyl phosphorylcholine and styrene was used. A cell culture substrate was prepared in which a membrane consisting of a diblock copolymer consisting of a copolymer (block (B)) and an N, N-diethylacrylamide polymer (block (A)) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured until they covered 100% of the substrate, the cells were exfoliated 30% in the form of single cells by cooling at 15 ° C. for 60 minutes.

Example 6
[Composition of block (C)]
The same method as in Example 1 [Synthesis of block (C)] except that 0.69 g (2.5 mmol) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium was used and reacted for 48 hours The synthesis was done in After the reaction, a portion of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 90% of the charged amount of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium was polymerized, A polymer (block (C)) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium could be synthesized. The hydrophilic part weight of the repeating unit of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (block (C)) is 5 carbons, 11 hydrogens, 1 nitrogen, 4 oxygens, sulfur The total of one repeating unit (181.2), the total amount of repeating units was 278.4, and the HLB value (Griffin method) was 13.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above and 0.79 g (7.5 mmol) of styrene, the synthesis was carried out in the same manner as Example 1 [Synthesis of diblock copolymer] except that the reaction was carried out for 48 hours. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 95% of the styrene charge was polymerized, and dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) A diblock copolymer consisting of an aminium polymer (block (C)) and a styrene polymer (block (B)) could be synthesized.

[Synthesis of block copolymer]
Synthesis was carried out in the same manner as Example 1 [Synthesis of block copolymer] except that the reaction solution obtained above and 1.26 g (10.0 mmol) of N-ethoxyethyl acrylamide were reacted for 90 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N-ethoxyethyl acrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to give dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (white powder). 1.22 g of a block copolymer comprising block (C), a styrene polymer (block (B)) and an N-ethoxyethyl acrylamide polymer (block (A)) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and dimethyl (3-methacryloylaminopropyl) (3 -A film comprising a block copolymer comprising a sulfonatopropyl) aminium polymer (block (C)), a styrene polymer (block (B)) and an N-ethoxyethyl acrylamide polymer (block (A)) is introduced A cell culture substrate was prepared. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Comparative Example 12
[Composition of block (C)]
Synthesis was carried out in the same manner as in Example 4 [Synthesis of block (C)]. After the reaction, a portion of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 90% of the charged amount of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium was polymerized, A polymer (block (C)) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium could be synthesized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above and 1.26 g (10.0 mmol) of N-ethoxyethyl acrylamide, synthesis was carried out in the same manner as in Comparative Example 1 [Synthesis of Diblock Copolymer] except that reaction was carried out for 70 hours Did. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 95% of the charged amount of N-ethoxyethyl acrylamide was polymerized, and dimethyl (3-methacryloylaminopropyl) (3- A diblock copolymer consisting of sulfonatopropyl) aminium polymer (block (C)) and N-ethoxyethyl acrylamide (block (A)) could be synthesized.

[Synthesis of block copolymer]
The synthesis was performed in the same manner as in Comparative Example 1 [Synthesis of block copolymer] except that the reaction solution obtained above and 0.79 g (7.5 mmol) of styrene were used for reaction for 48 hours. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 92% of the charged amount of styrene was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (block (C)) and N-ethoxyethyl acrylamide (block (block (C)) are obtained as a white powder. 2.41 g of a block copolymer comprising A) and a styrene polymer (block (B)) was obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and dimethyl (3-methacryloylaminopropyl) (3 -A cell comprising a block copolymer comprising a sulfonatopropyl) aminium polymer (block (C)), N-ethoxyethyl acrylamide (block (A)) and a styrene polymer (block (B)) A culture substrate was prepared. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 1. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Comparative Example 13
[Composition of block (B)]
The synthesis was performed in the same manner as in Comparative Example 2 [Synthesis of block (B)] except that 0.79 g (7.5 mmol) of styrene was used for reaction for 48 hours. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 90% of the charged amount of styrene was polymerized.

[Synthesis of Diblock Copolymer]
Comparative Example 2 [Diblock, except that the reaction solution obtained above, was reacted with 0.69 g (2.5 mmol) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium for 48 hours Synthesis was carried out in the same manner as in [Synthesis of copolymer]. After the reaction, a portion of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 90% of the charged amount of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium was polymerized, A diblock copolymer consisting of a styrene polymer (block (B)) and a dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (block (C)) could be synthesized.

[Synthesis of block copolymer]
Synthesis was carried out in the same manner as Comparative Example 2 [Synthesis of block copolymer] except that the reaction solution obtained above and 1.26 g (10.0 mmol) of N-ethoxyethyl acrylamide were reacted for 75 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 92% of the charged amount of N-ethoxyethyl acrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and a styrene polymer (block (B)) and a dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (block (C) as a white powder are obtained. ) And 2.21 g of a block copolymer comprising N-ethoxyethyl acrylamide polymer (block (A)) were obtained. The composition, Mn, and Mw / Mn of the obtained block copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and a styrene polymer (block (B)) was used. A film comprising a block copolymer consisting of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer (block (C)) and N-ethoxyethyl acrylamide polymer (block (A)) is introduced A cell culture substrate was prepared. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 60 minutes.

Comparative Example 14
[Composition of block (B)]
In a 50 mL Schlenk tube, 0.69 g (2.5 mmol) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium, 0.79 g (7.5 mmol) of styrene, 4-cyano-4- [4 as a RAFT agent 40.4 mg (0.1 mmol) of (dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid and 6.56 mg (0.04 mmol) of azobisisobutyronitrile as an initiator were added and dissolved in 10 mL of 1,4-dioxane . After bubbling argon for 10 minutes, the reaction was carried out at 65 ° C. for 48 hours. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was found that dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium charged amount and 95% of styrene charged amount were polymerized. Were confirmed, and a random copolymer (block (B)) of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium and styrene could be synthesized. The HLB value of a random copolymer of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium and styrene (block (B)) was 3.

[Synthesis of Diblock Copolymer]
Synthesis was carried out in the same manner as Comparative Example 2 [Synthesis of block copolymer] except that the reaction solution obtained above and 1.26 g (10.0 mmol) of N-ethoxyethyl acrylamide were reacted for 75 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 90% of the charged amount of N-ethoxyethyl acrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and a random copolymer of dimethyl (3-methacryloylaminopropyl) (3-sulfonatopropyl) aminium and styrene (block (B)) and N-ethoxy as a white powder. 2.42 g of a diblock copolymer consisting of an ethyl acrylamide polymer (block (A)) was obtained. The composition, Mn, and Mw / Mn of the obtained diblock copolymer are shown in Table 2.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the diblock copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and dimethyl (3-methacryloylaminopropyl) (3 For cell culture in which a membrane consisting of a diblock copolymer consisting of a random copolymer of (sulfonatopropyl) aminium and styrene (block (B)) and an N-ethoxyethyl acrylamide polymer (block (A)) has been introduced A substrate was made. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 2. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 3 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After the cultured cells were cultured until they covered 100% of the substrate, the cells were exfoliated 30% in the form of single cells by cooling at 15 ° C. for 60 minutes.

Example 7
[Composition of block (C)]
Example 1 [Block (C), except that 0.33 g (2.5 mmol) of 2-methoxyethyl acrylate and 31.8 mg (0.1 mmol) of cyanomethyldodecyltrithiocarbonate as a RAFT agent were used and reacted for 24 hours Synthesis was carried out in the same manner as in the synthesis of After the reaction, a part of the reaction solution was collected and 1H-NMR was measured. As a result, it was confirmed that 96% of the charged amount of 2-methoxyethyl acrylate was polymerized, and a polymer (block (block C) could be synthesized. The hydrophilic part weight of the repeating unit of the 2-methoxyethyl acrylate polymer (block (C)) is the total (88.1) of 3 carbons, 4 hydrogens and 3 oxygens, and the total amount of repeating units is 130 .1, and the HLB value (Griffin method) was 14.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 0.96 g (7.5 mmol) of n-butyl acrylate, synthesis was carried out in the same manner as Example 1 [Synthesis of diblock copolymer] except that reaction was carried out for 24 hours. went. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 96% of the charged amount of n-butyl acrylate was polymerized, 2-methoxyethyl acrylate polymer (block (C) ) And a n-butyl acrylate polymer (block (B)) were able to synthesize a diblock copolymer. The hydrophilic part weight of the repeating unit of the n-butyl acrylate polymer (block (B)) is the sum (88.1) of one carbon and two oxygen, and the total weight of the repeating unit is 128.2 The HLB value (Griffin method) was 7.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.13 g (10.0 mmol) of N-isopropylacrylamide, the synthesis was carried out in the same manner as in [Synthesis of block copolymer] except that the reaction was carried out for 48 hours. The After the reaction, a part of the reaction solution was collected and 1 H-NMR was measured. As a result, it was confirmed that 94% of the charged amount of N-isopropylacrylamide was polymerized.

  The reaction solution obtained above is treated in the same manner as in Example 1 [Synthesis of block copolymer] to give 2-methoxyethyl acrylate polymer (block (C)) and n-butyl acrylate as a white powder. 1.52 g of a block copolymer composed of the combined (block (B)) and the N-isopropylacrylamide polymer (block (A)) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 3.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane comprising a block copolymer consisting of n-butyl acrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 10 nm and was coated at a rate of 0.6 μg / cm ² as block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Example 8
[Synthesis of surface treatment agent]
24 mg of the block copolymer obtained in Example 7 was added to 3.0 g of ethanol, and all were dissolved in 120 seconds by stirring to synthesize 0.8 wt% of a surface treatment agent for a substrate.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane comprising a block copolymer consisting of n-butyl acrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 13 nm and was coated at a rate of 0.8 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Example 9
[Synthesis of surface treatment agent]
48 mg of the block copolymer obtained in Example 7 was added to 3.0 g of 2-methoxyethanol, and all was dissolved in 600 seconds by stirring to synthesize a 1.6 wt% surface treatment agent for a substrate.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane comprising a block copolymer consisting of n-butyl acrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 26 nm and was coated at a rate of 1.6 μg / cm ² as block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Although the cell adhesion shape had low spread, cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Example 10
[Synthesis of surface treatment agent]
93 mg of the block copolymer obtained in Example 7 was added to 3.0 g of 2-methoxyethanol, and all were dissolved in 600 seconds by stirring, whereby a 3.0 wt% surface treatment agent for a substrate was synthesized.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane comprising a block copolymer consisting of n-butyl acrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 50 nm and was coated at a rate of 3.0 μg / cm ² as block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Although the cell adhesion shape had low spread, cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Example 11
[Synthesis of surface treatment agent]
158 mg of the block copolymer obtained in Example 7 was added to 3.0 g of 2-methoxyethanol, and all were dissolved in 600 seconds by stirring to synthesize a 5.0 wt% surface treatment agent for a substrate.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane comprising a block copolymer consisting of n-butyl acrylate polymer (block (B)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 80 nm and was coated at a rate of 4.8 μg / cm ² as block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Although the cell adhesion shape had low spread, cell proliferation was confirmed. After cultured until the cultured cells covered 100% of the substrate, the cells were exfoliated in the form of single cells by cooling at 15 ° C. for 15 minutes.

Comparative example 15
[Composition of block (C)]
Example 5 [Synthesis of block (C)] Synthesis was carried out in the same manner as described above. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 97% of the 2-methoxyethyl acrylate charge had been polymerized, and a polymer (block (block C) could be synthesized.

[Synthesis of Diblock Copolymer]
Using the reaction solution obtained above, 1.13 g (10.0 mmol) of N-isopropylacrylamide, the reaction was carried out in the same manner as in Comparative Example 1 [Synthesis of diblock copolymer] except that reaction was carried out for 48 hours. went. After the reaction, a part of the reaction solution was collected, and 1H-NMR was measured. As a result, it was confirmed that 92% of the charged amount of N-isopropylacrylamide was polymerized, and 2-methoxyethyl acrylate polymer (block (C) ) And N-isopropylacrylamide (block (A)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 0.96 g (7.5 mmol) of n-butyl acrylate, synthesis was carried out in the same manner as in Comparative Example 1 [Synthesis of block copolymer] except that the reaction was carried out for 24 hours. The After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 96% of the n-butyl acrylate charge was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours to obtain 2-methoxyethyl acrylate polymer (block (C)), N-isopropyl acrylate (block (A)) and n-butyl acrylate polymer (block) as white powder. 1.38 g of a block copolymer consisting of (B) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 3.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 [membrane evaluation] except that the surface treatment agent obtained above was used, and a 2-methoxyethyl acrylate polymer (block ( C) A cell culture substrate was prepared in which a membrane consisting of a block copolymer comprising N-isopropyl acrylate (block (A)) and n-butyl acrylate polymer (block (B)) was introduced. The film thickness was 10 nm, and it was coated at a rate of 0.5 μg / cm ² as the block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The contact angle to water at 15 ° C was almost the same as the contact angle to water at 40 ° C and showed no temperature response.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. The cells did not adhere and could not grow.

Comparative example 16
[Composition of block (B)]
Comparative Example 2 [Block (B) except that 0.96 g (7.5 mmol) of n-butyl acrylate and 31.8 mg (0.1 mmol) of cyanomethyldodecyltrithiocarbonate as a RAFT agent were used and reacted for 24 hours Synthesis was carried out in the same manner as in the synthesis]. After the reaction, as a result of collecting a part of the reaction solution and measuring 1 H-NMR, it could be confirmed that 93% of the charged amount of styrene was polymerized.

[Synthesis of Diblock Copolymer]
By the same method as Comparative Example 2 [Synthesis of diblock copolymer] except that the reaction solution obtained above and 0.33 g (2.5 mmol) of 2-methoxyethyl acrylate were reacted for 24 hours. The synthesis was done. After the reaction, a part of the reaction solution was collected and measured by 1 H-NMR. As a result, it was confirmed that 95% of the 2-methoxyethyl acrylate charge had been polymerized, n-butyl acrylate polymer (block (B) ) And a 2-methoxyethyl acrylate polymer (block (C)) could be synthesized.

[Synthesis of block copolymer]
Using the reaction solution obtained above and 1.13 g (10.0 mmol) of N-isopropylacrylamide, the synthesis was carried out in the same manner as in Comparative Example 2 [Synthesis of block copolymer] except that the reaction was carried out for 48 hours. The After the reaction, a part of the reaction solution was collected and 1 H-NMR was measured. As a result, it was confirmed that 94% of the charged amount of N-isopropylacrylamide was polymerized.

  The resulting reaction solution was poured into 250 mL of hexane, and the precipitated white solid was filtered. The obtained white solid is dried under reduced pressure for 2 hours, and n-butyl acrylate polymer (block (B)), 2-methoxyethyl acrylate polymer (block (C)) and N-isopropylacrylamide polymer as white powder 1.37 g of a block copolymer consisting of (block (A)) was obtained. The composition, Mn and Mw / Mn of the obtained block copolymer are shown in Table 3.

[Synthesis of surface treatment agent]
The synthesis was carried out in the same manner as in Example 1 [Synthesis of surface treatment agent] except that the block copolymer obtained above was used, to synthesize a surface treatment agent.

[Film evaluation]
A cell culture substrate was prepared by the same method as described in Example 1 (membrane evaluation) except that the surface treatment agent obtained above was used, and n-butyl acrylate polymer (block (B (B)) was used. A cell culture substrate was prepared in which a membrane consisting of a block copolymer consisting of)), 2-methoxyethyl acrylate polymer (block (C)) and N-isopropylacrylamide polymer (block (A)) was introduced. . The film thickness was 10 nm and was coated at a rate of 0.6 μg / cm ² as block (A). The results of evaluating the water contact angle at 40 ° C. and 15 ° C. are shown in Table 3. The water contact angle at 15 ° C showed a temperature response lower than the water contact angle at 40 ° C.

[Cell culture evaluation and exfoliation evaluation]
It evaluated by the method similar to Example 5 [cell culture evaluation and exfoliation evaluation] except using the base material for cell cultures produced above. Cell proliferation was confirmed. After culturing until the cultured cells covered 100% of the substrate, the cells were not detached at all even when cooled at 15 ° C. for 60 minutes.

Claims (6)

The block copolymer which consists of a block of the following (A), (B), (C), and whose arrangement | sequence is (A)-(B)-(C).
(A) A temperature responsive polymer block having a lower limit critical solution temperature (LCST) to water in the range of 0 to 45 ° C.
(B) A hydrophobic polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 9 without LCST in the range of 0 to 45 ° C.
(C) A hydrophilic polymer block having an HLB value (Griffin method) in the range of 9 or more and less than 20 without LCST in the range of 0 to 45 ° C.   A surface treating agent for a substrate comprising the block copolymer according to claim 1.   A film obtained by applying the surface treatment agent according to claim 2 to a substrate. The film according to claim 3, wherein the surface of the substrate is coated at a rate of 5.0 μg / cm ² or less as the block (A).   A cell culture substrate coated with the membrane according to claim 3 or 4.   A cell culture substrate according to claim 5 is used to culture the cells at a temperature higher than the LCST of block (A), and after cell growth, the temperature is made lower than the LCST of block (A) to grow proliferating cells from the substrate A cell culture method characterized by exfoliating.