WO2016159153A1 - Élément de base pour l'utilisation en culture cellulaire, son procédé de production et procédé de culture cellulaire l'utilisant - Google Patents

Élément de base pour l'utilisation en culture cellulaire, son procédé de production et procédé de culture cellulaire l'utilisant Download PDF

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WO2016159153A1
WO2016159153A1 PCT/JP2016/060513 JP2016060513W WO2016159153A1 WO 2016159153 A1 WO2016159153 A1 WO 2016159153A1 JP 2016060513 W JP2016060513 W JP 2016060513W WO 2016159153 A1 WO2016159153 A1 WO 2016159153A1
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group
block
cell culture
polymer
cells
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PCT/JP2016/060513
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Japanese (ja)
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山田悟
今富伸哉
近藤聡
前島雪絵
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東ソー株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F297/00—Macromolecular compounds obtained by successively polymerising different monomer systems using a catalyst of the ionic or coordination type without deactivating the intermediate polymer

Definitions

  • the present invention relates to a cell culture substrate that enables cell detachment in a short time, a production method thereof, and a cell culture method using the same. Furthermore, the present invention relates to a cell culture substrate that enables cells to be detached with a maximum diameter of 5 ⁇ m to 300 ⁇ m, a method for producing the same, and a cell culture method using the same.
  • biopolymers such as collagen, fibronectin and laminin.
  • many cells having adhesiveness in cell culture need to adhere to some kind of substrate when culturing.
  • surface-treated glass or polymer has been used as a substrate.
  • a substrate obtained by subjecting polystyrene to ⁇ -ray irradiation or silicone coating.
  • a support on which a biopolymer such as collagen or fibronectin is coated on the surface is also used.
  • proteolytic enzymes are responsible for decomposing proteins on the cell surface and breaking the bonds between cells and substrates and between cells.
  • proteolytic enzymes have a great influence on the survival rate of cells, and a technique of separating cells from a substrate without using proteolytic enzymes is important as a method that does not damage cells.
  • regenerative medicine there is a need to separate cells or organized cells from the base material and return them to the body without damaging cells cultured outside the body and further breaking the bonds between the cells. Therefore, there is a need for a method of separating from a substrate without using a proteolytic enzyme.
  • a cell culture substrate in which a temperature-responsive polymer is coated on the substrate surface is disclosed.
  • Patent Document 1 a cell culture substrate in which a temperature-responsive polymer is coated on the substrate surface.
  • a base material According to such a base material, it is possible to weaken the adhesive force on the surface of the base material by the sol transition of the temperature-responsive polymer due to a temperature drop in the surrounding environment, detach the cells, and collect and recover the cells without causing damage. it can.
  • cells need to be adhered and cultured at around body temperature, and after culturing, a substrate capable of peeling cells at body temperature or lower is required.
  • the temperature-responsive polymer When using the above temperature-responsive polymer as a cell culture substrate, it is necessary to lower the temperature of the cell culture substrate below the critical lysis temperature, but at the same time, the temperature of the cell is lowered. Furthermore, the cells that peel off are in the form of a sheet. After that, in order to process the cells, it was necessary to disperse the cells by pipetting while maintaining a low temperature, and the cooling time was prolonged. . Since the lowering of the cell temperature decreases the cell activity, it is necessary to shorten the cooling time.
  • An object of the present invention is to provide a cell culture substrate that enables cell detachment in a short time, a method for producing the same, and a cell culture method using the same. Furthermore, another object is to provide a cell culture substrate capable of exfoliating cells with a maximum diameter of 5 ⁇ m to 300 ⁇ m and omitting cell dispersion, a method for producing the same, and a cell culture method using the same. It is in.
  • the present inventors have conducted extensive research, and as a result, coated a block copolymer obtained by block copolymerization of a temperature-responsive polymer with a hydrophilic polymer on a substrate. It was found that forming a membrane enables cell detachment in a short time, and that cells can be detached with a maximum diameter of 5 ⁇ m to 300 ⁇ m, and the present invention has been completed.
  • a cell culture substrate whose surface is coated with a block copolymer containing the following blocks.
  • a cell culture substrate whose surface is coated with a block copolymer containing the following blocks (A) and (B).
  • B A hydrophilic polymer block having no LCST in the range of 0 ° C. to 50 ° C. and having an HLB value (Griffin method) in the range of 9-20.
  • the block (B) is a carboxylic acid group, a carboxylic acid ester group, a carboxylic acid metal salt, a sulfonic acid group, a sulfonic acid ester group, a sulfonic acid metal salt, a hydroxy group, an alkoxy group, a phenoxy group, an amide group, amino Alkyl group, carbamoyl group, sulfonamido group, sulfamoyl group, carbamate group, phosphate group, metal salt of phosphate group, oxyphosphate group, metal salt of oxyphosphate group, phosphobetaine group, sulfobetaine group, carbobetaine group,
  • the cell culture substrate according to [1] which is a polymer of a monomer having at least one hydrophilic group selected from a polyethylene glycol group and a pyrrolidone group.
  • R 1 is a hydrogen atom or a methyl group
  • R 2 and R 3 are each independently a hydrogen group, a hydrocarbon group having 1 to 6 carbon atoms, a furfuryl group or a tetrahydrofurfuryl group; 2 and R 3 may be bonded to each other to form a pyrrolidine ring, piperidine ring or morpholine ring.
  • the cell according to any one of [1] to [3], which is a block of a (co) polymer comprising at least one block unit (a) among the block units represented by Culture substrate. [5]
  • the repeating unit of the block (A) is represented by the following general formula (2)
  • R 6 represents a hydrogen atom or a methyl group
  • R 7 represents a hydrocarbon group having 1 to 6 carbon atoms.
  • the repeating unit of the block (B) is represented by the following general formula (4)
  • R 8 is a hydrogen atom or a methyl group
  • R 9 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or a (poly) oxyethylene group
  • R 10 is a group having 1 to 10 carbon atoms
  • 4 is a divalent hydrocarbon group
  • R 11 , R 12 , and R 13 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms
  • a 1 is an ester bond, an amide
  • This is a divalent bond selected from the group consisting of a bond, a urethane bond, and an ether bond.
  • the cell according to any one of [1] to [6], which is a block of a (co) polymer comprising at least one type of block unit (b) among the block units represented by Culture substrate.
  • R 14 represents a hydrogen atom or a methyl group
  • R 15 represents — (CH 2 CH 2 O) b — (CH 2 CH (CH 3 ) O) c —R 16
  • R 16 represents hydrogen An atom, an alkyl group having 1 to 30 carbon atoms
  • b is an integer of 1 to 300
  • c is an integer of 0 to 60
  • R 17 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms
  • a furfuryl group a tetrahydrofurfuryl group
  • a hydrogen atom
  • the repeating unit of the block (B) is represented by the following general formula (6)
  • R 18 is a hydrogen atom or a methyl group
  • R 19 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or a (poly) oxyethylene group
  • R 20 is a group having 1 to 4 is a divalent hydrocarbon group
  • R 21 and R 22 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms
  • a 2 is an ester bond, an amide bond, a urethane bond, And a divalent bond selected from the group consisting of ether bonds
  • X is a sulfonate anion group, a carboxylate anion group, or a phosphate anion group.
  • the cell according to any one of [1] to [6], which is a block of a (co) polymer comprising at least one type of block unit (b) among the block units represented by Culture substrate.
  • the repeating unit of the block (B) is represented by the following general formula (7)
  • R 23 is a hydrogen atom or a methyl group
  • R 24 and R 25 are each independently a hydrogen atom or a methyl group.
  • the repeating unit of the block (B) is represented by the following general formula (8)
  • R 26 is a hydrogen atom or a methyl group.
  • the repeating unit of the block (B) is represented by the following general formula (9)
  • R 27 is a hydrogen atom or a methyl group
  • R 28 is a divalent hydrocarbon group having 1 to 10 carbon atoms
  • R 29 and R 30 are each independently a hydrogen atom or a carbon number. 1 to 4 hydrocarbon groups
  • a 3 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond.
  • the cell according to any one of [1] to [6], which is a block of a (co) polymer comprising at least one type of block unit (b) among the block units represented by Culture substrate.
  • the ratio according to any one of [1] to [12], wherein the ratio of the block unit (a) to the total block unit [(a) + (b)] is 5 to 95 mol% Cell culture substrate.
  • the following cell culture substrate is provided.
  • a cell culture substrate whose surface is coated with a block copolymer containing the following blocks (A) and (B).
  • B A block that satisfies the following requirements (i) to (iii).
  • I No LCST in the range of 0 ° C to 50 ° C.
  • R 1 is a hydrogen atom or a methyl group
  • R 2 and R 3 are each independently a hydrogen group, a hydrocarbon group having 1 to 6 carbon atoms, a furfuryl group or a tetrahydrofurfuryl group; 2 and R 3 may be bonded to each other to form a pyrrolidine ring, piperidine ring or morpholine ring.
  • the repeating unit of the block (A) is represented by the following general formula (2)
  • the repeating unit of the block (A) is represented by the following general formula (3)
  • R 6 represents a hydrogen atom or a methyl group
  • R 7 represents a hydrocarbon group having 1 to 6 carbon atoms.
  • the repeating unit of the hydrophilic polymer of the block (B) is represented by the following general formula (4)
  • R 8 is a hydrogen atom or a methyl group
  • R 9 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or a (poly) oxyethylene group
  • R 10 is a group having 1 to 10 carbon atoms
  • 4 is a divalent hydrocarbon group
  • R 11 , R 12 , and R 13 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms
  • a 1 is an ester bond, an amide
  • This is a divalent bond selected from the group consisting of a bond, a urethane bond, and an ether bond.
  • R 14 represents a hydrogen atom or a methyl group
  • R 15 represents — (CH 2 CH 2 O) b — (CH 2 CH (CH 3 ) O) c —R 16
  • R 16 represents hydrogen An atom, an alkyl group having 1 to 30 carbon atoms
  • b is an integer of 1 to 300
  • c is an integer of 0 to 60
  • R 17 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms
  • a furfuryl group a tetrahydrofurfuryl group
  • a hydrogen atom
  • R 18 is a hydrogen atom or a methyl group
  • R 19 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or a (poly) oxyethylene group
  • R 20 is a group having 1 to 4 is a divalent hydrocarbon group
  • R 21 and R 22 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms
  • a 2 is an ester bond, an amide bond, a urethane bond, And a divalent bond selected from the group consisting of ether bonds
  • X is a sulfonate anion group, a carboxylate anion group, or a phosphate anion group.
  • the cell according to any one of [16] to [20], which is a block of a (co) polymer comprising at least one type of block unit (b) Culture substrate.
  • the repeating unit of the hydrophilic polymer of the block (B) is represented by the following general formula (7)
  • R 23 is a hydrogen atom or a methyl group
  • R 24 and R 25 are each independently a hydrogen atom or a methyl group.
  • the repeating unit of the hydrophilic polymer of the block (B) is represented by the following general formula (8)
  • R 26 is a hydrogen atom or a methyl group.
  • the repeating unit of the hydrophilic polymer of the block (B) is represented by the following general formula (9)
  • R 27 is a hydrogen atom or a methyl group
  • R 28 is a divalent hydrocarbon group having 1 to 10 carbon atoms
  • R 29 and R 30 are each independently a hydrogen atom or a carbon number. 1 to 4 hydrocarbon groups
  • a 3 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond.
  • the ratio of the block unit (a) to the total block unit [(a) + (b)] is 5 to 95 mol%, according to any one of [16] to [26] Cell culture substrate.
  • Mn number average molecular weight
  • the following manufacturing methods are provided.
  • a method for producing a cell culture substrate comprising dissolving a block copolymer containing the following blocks (A) and (B) in a solvent, applying the solution to a substrate, and drying the solution.
  • B A hydrophilic polymer block having no LCST in the range of 0 ° C. to 50 ° C. and having an HLB value (Griffin method) in the range of 9-20.
  • the block (B) is a carboxylic acid group, a carboxylic acid ester group, a carboxylic acid metal salt, a sulfonic acid group, a sulfonic acid ester group, a sulfonic acid metal salt, a hydroxy group, an alkoxy group, a phenoxy group, an amide group, amino Alkyl group, carbamoyl group, sulfonamido group, sulfamoyl group, carbamate group, phosphate group, metal salt of phosphate group, oxyphosphate group, metal salt of oxyphosphate group, phosphobetaine group, sulfobetaine group, carbobetaine group, [29]
  • the production method according to [29] which is a polymer of a monomer having at least one hydrophilic group selected from a polyethylene glycol group and a pyrrolidone group.
  • a method for producing a cell culture substrate comprising dissolving a block copolymer containing the following blocks (A) and (B) in a solvent, applying the solution to a substrate, and drying the solution.
  • a method for producing a cell culture substrate comprising immobilizing a block copolymer containing the following blocks (A) and (B) to the substrate by chemical bonding.
  • LCST lower critical solution temperature
  • Block (B) is a carboxylic acid group, a carboxylic acid ester group, a carboxylic acid metal salt, a sulfonic acid group, a sulfonic acid ester group, a sulfonic acid metal salt, a hydroxy group, an alkoxy group, a phenoxy group, an amide group, amino Alkyl group, carbamoyl group, sulfonamido group, sulfamoyl group, carbamate group, phosphate group, metal salt of phosphate group, oxyphosphate group, metal salt of oxyphosphate group, phosphobetaine group, sulfobetaine group, carbobetaine group,
  • the production method according to [32] which is a polymer of a monomer having at least one hydrophilic group selected from a polyethylene glycol group and a pyrrolidone group.
  • a method for producing a cell culture substrate comprising immobilizing a block copolymer containing the following blocks (A) and (B) to the substrate by chemical bonding.
  • B A block that satisfies the following requirements (i) to (iii).
  • I No LCST in the range of 0 ° C to 50 ° C.
  • the present invention also provides the following cell culture method.
  • [35] Using the cell culture substrate according to any one of [1] to [28], cells are cultured at a temperature higher than the lower critical lysis temperature (LCST), and the temperature is lower than the LCST after cell growth. And then detaching the proliferating cells from the substrate.
  • LCST lower critical lysis temperature
  • the cell culture method according to [35] wherein the cultured cells are detached with a maximum diameter of 5 ⁇ m to 300 ⁇ m.
  • the cell culture method according to [35] or [36] wherein the cultured cells are detached as single cells.
  • a cell culture method comprising culturing cells using the cells detached by the cell culture method according to any one of [35] to [37].
  • a cell culture substrate is coated with a membrane obtained from a block copolymer obtained by block copolymerization of a temperature-responsive polymer with a hydrophilic polymer, the hydrophilicity of the substrate surface due to temperature drop after cell culture. Acceleration is enabled and cell detachment in a short time becomes possible.
  • separating cells with a maximum diameter of 5 ⁇ m to 300 ⁇ m, it is possible to simplify the cell dispersal operation that was necessary when separating cells. Specifically, by omitting the cell detachment step by enzymatic digestion, it is possible to reduce the time required for the work, the reduction of the step, and the error for each operator. This also provides a cell culture substrate that can be separated and collected in a short time without damaging the cells even after cooling treatment after the cell culture.
  • the present embodiment a mode for carrying out the present invention (hereinafter simply referred to as “the present embodiment”) will be described in detail.
  • the following embodiments are exemplifications for explaining the present invention, and are 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 gist.
  • the cell culture substrate of the present invention is a substrate having a surface coated with a block copolymer containing specific (A) and specific (B) blocks.
  • One of the cell culture substrates of the present invention is the following (A) and (B) (A) A block of a temperature-responsive polymer having a lower critical solution temperature (LCST) in water in the range of 0 ° C to 50 ° C. (B) A hydrophilic polymer block having no LCST in the range of 0 ° C. to 50 ° C. and having an HLB value (Griffin method) in the range of 9-20.
  • a cell culture substrate whose surface is coated with a block copolymer containing the above block.
  • the lower critical solution temperature means that the polymer dissolves in water at a temperature lower than this temperature and becomes a transparent solution, but becomes insoluble at a temperature higher than this temperature. The temperature at which the solution becomes cloudy or precipitates and the phases are separated.
  • the block (A) in the first invention is a block of a temperature-responsive polymer having an LCST in the range of 0 ° C. to 50 ° C.
  • Block (A) for the purpose of separating and recovering cells without damaging them while imparting cell adhesion near the body temperature when the culture substrate of the present invention is used.
  • the LCST is preferably in the range of 25 ° C. to 45 ° C., more preferably in the range of 28 ° C. to 40 ° C. If the LCST is less than 0 ° C., it becomes difficult to detach without damaging the cells, and if it exceeds 50 ° C., the cells cannot be adhered near the body temperature, and cell culture becomes difficult.
  • the block (A) in the first invention is not particularly limited, but the following general formula (1)
  • a (co) polymer block comprising at least one block unit (a) can be used.
  • R 1 is a hydrogen atom or a methyl group, and a hydrogen atom is used to bring the LCST into a range of 25 ° C. to 45 ° C.
  • R 2 and R 3 are each independently a hydrogen group, a hydrocarbon group having 1 to 6 carbon atoms, a furfuryl group or a tetrahydrofurfuryl group, and R 2 and R 3 are bonded to each other to form a pyrrolidine ring, a piperidine ring or A morpholine ring may be formed.
  • hydrocarbon group having 1 to 6 carbon atoms examples include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • -Butyl group, n-hexyl group and isohexyl group can be exemplified, but n-propyl group and isopropyl group are preferably used in order to make LCST in the range of 25 ° C to 45 ° C.
  • N N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N- Isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, 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- Examples include (co) polymers of at least one monomer selected from (lopenyl) piperidine, 4-
  • the block (A) in the first invention is not particularly limited, but the following general formula (2)
  • a (co) polymer block comprising at least one type of block unit (a) can be used.
  • R 4 represents a hydrogen atom or a methyl group, to a range of 25 ° C. ⁇ 45 ° C.
  • the LCST, hydrogen atom is used.
  • R 5 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 in order to bring the LCST into a range of 25 ° C. to 45 ° C.
  • hydrocarbon group having 1 to 6 carbon atoms examples include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • -Butyl group, n-hexyl group and isohexyl group can be exemplified, but a methyl group, an ethyl group and an n-propyl group are preferably used in order to bring the LCST into a range of 25 ° C to 45 ° C.
  • A is an integer of 1 to 10, and an integer of 1 to 3 is used to make LCST in the range of 25 ° C to 45 ° C.
  • the block (A) represented by the general formula (2) in the first invention is preferably a polymer of 2- (2-ethoxy) ethoxyethyl vinyl ether so that the LCST is in the range of 25 ° C. to 45 ° C. Can be used.
  • the block (A) in the first invention is not particularly limited, but the following general formula (3)
  • a (co) polymer block comprising at least one block unit (a) can be used.
  • R 6 represents a hydrogen atom or a methyl group, and a hydrogen atom is used to make LCST in the range of 25 ° C. to 45 ° C.
  • R 7 represents a hydrocarbon group having 1 to 6 carbon atoms, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • -Butyl group, n-hexyl group and isohexyl group can be exemplified, but a methyl group and an ethyl group are preferably used in order to bring the LCST into a range of 25 ° C to 45 ° C.
  • a polymer of methyl vinyl ether can be preferably used so that the LCST is in the range of 25 ° C to 45 ° C.
  • HLB Hydrophile-Lipophile Balance
  • Griffin J. et al. Soc. Cosmetic Chemists, 1, 311 (1949).
  • Atlas method Griffin method, Davis method, and Kawakami method as the method to be determined by calculation.
  • the value calculated by the Griffin method is used, and the formula amount of the hydrophilic part in the block unit and the total of the block unit are used. It calculated
  • the block (B) in the first invention is a hydrophilic polymer block having an HLB value in a specific range and having no LCST in the range of 0 ° C. to 50 ° C.
  • 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, sulfonamido group, sulfamoyl group, carbamate group, phosphate group, metal salt of phosphate group, oxyphosphate group, metal salt of oxyphosphate group, phosphobetaine group, sulfobetaine group, carbo
  • Examples thereof include (co) polymers of monomers having at least one hydrophilic group selected from a betaine group, a polyethylene glycol group, and a pyrrolidone group.
  • the block (B) in the first invention is preferably a block having no affinity for biopolymers such as proteins, peptides, glycoproteins or the like, in order to shorten the cooling time required for cell detachment.
  • the HLB value in the first invention is in the range of 9 to 20, but is preferably in the range of 11 to 20, more preferably in the range of 13 to 20 for the purpose of shortening the cooling time required for cell detachment. It is in. When the HLB value is less than 9, the cooling time required for cell detachment becomes longer, leading to a decrease in cell activity.
  • the block (B) in the first invention is preferably a block exhibiting solubility in water in order to shorten the cooling time required for cell detachment, and more preferably 0 in 100 mL of water at 20 ° C. A block that dissolves 5 g or more, more preferably a block that dissolves 1 g or more in 100 mL of water at 20 ° C.
  • the block (B) in the first invention is not particularly limited, but the following general formula (4)
  • a (co) polymer block comprising at least one type of block unit (b) can be used.
  • R 8 represents a hydrogen atom or a methyl group, and a methyl group is used for the purpose of shortening the cooling time required for cell detachment.
  • R 9 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or (poly) oxyethylene group: — (OCH 2 CH 2 ) n — (wherein n is suitable for the block copolymer of the present invention).
  • the (poly) oxyethylene group is a group A 1 through —O—.
  • a divalent alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, propylene, butylene, pentylene, hexylene, etc., for the purpose of shortening the cooling time required for cell detachment. And more preferably ethylene.
  • R 10 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as methylene, ethylene, propylene, butylene for the purpose of shortening the cooling time required for cell detachment. More preferably, it is ethylene.
  • R 11 , R 12 , and R 13 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and for the purpose of shortening the cooling time required for cell detachment, preferably R 11 11 , R 12 and R 13 are simultaneously a hydrogen atom or a methyl group, more preferably a methyl group.
  • a 1 is a divalent bond selected from the group consisting of an ester bond, an amide bond, a urethane bond, and an ether bond, and for the purpose of shortening the cooling time required for cell detachment, preferably an ester bond, An amide bond, particularly preferably an ester bond.
  • the block (B) represented by the general formula (4) in the first invention includes 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3- (meth) acryloyloxypropyl phosphorylcholine, 4- (meth) Acryloyloxybutyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10- (meth) acryloyloxydecylphosphorylcholine, ⁇ - (meth) acryloyl (poly) oxyethylene phosphorylcholine, 2-acrylamidoethylphosphorylcholine, 3-acrylamidopropylphosphorylcholine, 4-acrylamidobutylphosphorylcholine, 6-acrylamidehexylphosphorylcholine, 10-acrylamidedecylphosphorylcholine, ⁇ - ( (Meth) acrylamide (poly) oxyethylene phosphorylcholine, 2-methacryloyloxye
  • a polymer can be exemplified, but 2-methacryloyloxyethylphosphine is preferable for the purpose of shortening the cooling time required for cell detachment. It can be used a polymer of Rirukorin.
  • the block (B) in the first invention is not particularly limited, but the following general formula (5)
  • a (co) polymer block comprising at least one type of block unit (b) can be used.
  • R 14 represents a hydrogen atom or a methyl group, and a methyl group is used for the purpose of shortening the cooling time required for cell detachment.
  • R 15 is — (CH 2 CH 2 O) b — (CH 2 CH (CH 3 ) O) c —R 16 (wherein R 16 is a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, and b is A polyoxyalkylene group represented by the formula: —CH 2 —O—R 17 (wherein R 17 is a hydrogen atom, a carbon number of 1 to 6 is a substituent represented by (6), a furfuryl group, a tetrahydrofurfuryl group, and a hydrogen atom.
  • Examples of the alkyl group having 1 to 30 carbon atoms used for R 16 include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, 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 for the purpose of shortening the cooling time required for cell detachment.
  • Examples of the alkyl group having 1 to 6 carbon atoms used for R 17 include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, 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 for the purpose of shortening the cooling time required for cell detachment.
  • the block (B) represented by the general formula (5) in the first invention includes polyethylene glycol methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, hydroxymethyl acrylate, hydroxymethyl methacrylate, 2-methoxyethyl acrylate.
  • polyethylene glycol methacrylate preferably selected from polyethylene glycol methacrylate, 2-methoxyethyl acrylate or tetrahydrofurfuryl acrylate. It can be used at least one monomer (co) polymers to be.
  • the block (B) in the first invention is not particularly limited, but the following general formula (6)
  • a (co) polymer block comprising at least one type of block unit (b) can be used.
  • R 18 represents a hydrogen atom or a methyl group, and a methyl group is used for the purpose of shortening the cooling time required for cell detachment.
  • R 19 is a divalent hydrocarbon group having 1 to 10 carbon atoms or a (poly) oxyethylene group: — (OCH 2 CH 2 ) n — (wherein n is suitable for the block copolymer of the present invention).
  • the (poly) oxyethylene group is a group A 1 through —O—.
  • a divalent alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, propylene, butylene, pentylene, hexylene, etc., for the purpose of shortening the cooling time required for cell detachment. And more preferably ethylene.
  • R 20 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as methylene, ethylene, propylene, butylene for the purpose of shortening the cooling time required for cell detachment. More preferably, it is ethylene.
  • R 21 and R 22 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms, and preferably R 21 and R 22 for the purpose of shortening the cooling time required for cell detachment. Are simultaneously a hydrogen atom or a methyl group, more preferably a methyl group.
  • a 2 is an ester bond, an amide bond, a urethane bond, and a divalent bond selected from the group consisting of ether bond, for the purpose of shortening the cooling time required for cell detachment, preferably an ester bond, An amide bond, particularly preferably an ester bond.
  • X is a sulfonate anion group, a carboxylic acid anion group, or a phosphate anion group.
  • Examples of the block (B) represented by the general formula (6) in the first invention include dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl).
  • One monomer (co) polymer can be used.
  • the block (B) in the first invention is not particularly limited, but the following general formula (7)
  • a (co) polymer block comprising at least one type of block unit (b) can be used.
  • R 23 represents a hydrogen atom or a methyl group, and a methyl group is used for the purpose of shortening the cooling time required for cell detachment.
  • R 24 and R 25 are each independently a hydrogen atom or a methyl group.
  • a (co) polymer of at least one monomer selected from acrylamide or N, N-dimethylacrylamide can be used.
  • the block (B) in the first invention is not particularly limited, but the following general formula (8)
  • a (co) polymer block comprising at least one type of block unit (b) can be used.
  • R 26 represents a hydrogen atom or a methyl group, and a methyl group is used for the purpose of shortening the cooling time required for cell detachment.
  • a (co) polymer of N-vinylpyrrolidone can be used as the block (B) represented by the general formula (8) in the first invention.
  • the block (B) in the first invention is not particularly limited, but the following general formula (9)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 27 represents a hydrogen atom or a methyl group, a methyl group is used to shorten the cooling time required for the cell detachment.
  • R 28 is a divalent hydrocarbon group having 1 to 10 carbon atoms, and preferably has a carbon number of 1 such as methylene, ethylene, propylene, butylene, pentylene, hexylene, etc. in order to shorten the cooling time required for cell detachment. To 6 divalent alkylene groups, more preferably ethylene.
  • R 29 and R 30 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms. In order to shorten the cooling time required for cell detachment, it is preferable that R 29 and R 30 are simultaneously A hydrogen atom or a methyl group, more preferably a methyl group at the same time.
  • a 3 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 in order to shorten the cooling time required for cell detachment. And particularly preferably an ester bond.
  • the block (B) represented by the general formula (9) in the first invention includes aminomethyl (meth) acrylate, N, N-dimethylaminomethyl (meth) acrylate, N, N-diethylaminomethyl (meth) acrylate Aminoethyl (meth) 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) acrylamide methylamine, dimethyl [(meth) acrylamidomethyl] amine, diethyl [(meth) acrylamidomethyl] amine, (Meth) acrylamidoethylamine, Me
  • the block copolymer used for the cell culture substrate of the first invention is preferably used for the purpose of imparting cell adhesion and shortening the cooling time required for cell detachment when coated on the substrate.
  • a block copolymer comprising a block (A) of a (co) polymer comprising the block unit (a) and a block (B) of the (co) polymer comprising the block unit (b) can be used. More preferably, a block copolymer containing a block (A) of a polymer of N-isopropylacrylamide and a block (B) of a polymer of 2-methacryloyloxyethyl phosphorylcholine can be used.
  • the ratio of (a) to all block units [(a) + (b)] is 1 to 95 mol%.
  • cell adhesion is imparted and cooling time required for cell detachment is increased.
  • the content is preferably 5 to 85 mol%. If it is less than 1 mol%, the cell adhesiveness is lowered, and if it exceeds 95 mol%, the cooling time required for cell detachment becomes longer.
  • the block copolymer used in the first invention can contain monomer units other than the block unit (a) or (b) in the block (A) or (B).
  • Monomers that generate monomer units other than the block unit (a) or (b) include aromatic vinyl compounds such as styrene, 1-vinylnaphthalene, 2-vinylnaphthalene, 9-vinylanthracene, 1-vinylpyrene and derivatives thereof, Nn-octyl (meth) acrylamide, Nn-decyl (meth) acrylamide, Nn-dodecyl (meth) acrylamide, Nn-hexadecyl (meth) acrylamide, Nn-octadecyl (meth) acrylamide, etc.
  • N-vinylamide compounds such as (meth) acrylamide compounds, N-vinyl-n-octylamide, N-vinyl-n-decylamide, N-vinyl-n-dodecylamide, N-vinyl-n-hexadecylamide, N -N-acrylates such as cyclohexylmaleimide and N-phenylmaleimide Kill maleimide compound, fumarate -tert- butyl, fumaric acid diester compounds such as fumarate -n- butyl, vinyl chloride, vinyl acetate, can be exemplified (meth) acrylonitrile, N- vinylimidazole, N- vinylcarbazole.
  • block copolymer used in the first invention can contain a polymer block other than the block (A) or (B).
  • the number average molecular weight (Mn) of the block copolymer used in the first invention is in the range of 3,000 to 1,000,000, preferably 4,000 to 500,000, more preferably 5,000. More than 100,000. In the case of less than 3,000, even if the cell culture substrate is coated, it is eluted from the substrate into the medium during cell culture. On the other hand, when it exceeds 1,000,000, the solution viscosity becomes high and it becomes difficult to coat the cell culture substrate.
  • the method for synthesizing the block copolymer used in the first invention is not particularly limited, but is published by NTS Corporation, “Radical Polymerization Handbook”, p. 161 to 225 (2010), and a copolymerization method can be used.
  • the monomer producing the block unit (a) is (co) polymerized, and after further removing the unreacted monomer, the monomer producing the block unit (b) is ( (Co) polymerization method, (co) polymerization of the monomer that produces the block unit (a), after removing the unreacted monomer, (co) polymerization of the monomer that produces the block unit (b), After removing the monomer, a method of (co) polymerizing the monomer that generates the block unit (a), (co) polymerizing the monomer that generates the block unit (b), removing the unreacted monomer, a method of (co) polymerizing a monomer that generates a monomer unit other than a) or (b), further removing an unreacted monomer, and (co) polymerizing a monomer that generates a block unit (a), (Co) polymerization of the monomer that produces a), after removing the unreacted monomer
  • Another cell culture substrate of the present invention includes the following (A) and (B) (A) A block of a temperature-responsive polymer having a lower critical solution temperature (LCST) in water in the range of 0 ° C to 50 ° C. (B) A block that satisfies the following requirements (i) to (iii). (I) No LCST in the range of 0 ° C to 50 ° C.
  • LCST lower critical solution temperature
  • the block (A) in the second invention is a block of a temperature-responsive polymer having an LCST in the range of 0 ° C. to 50 ° C.
  • the LCST of the block (A) is used to impart cell adhesion at around body temperature, detach the cells when the temperature drops, and collect and collect the cells without damage. Is preferably in the range of 25 ° C to 45 ° C, more preferably in the range of 28 ° C to 40 ° C.
  • the block (A) in the second invention is not particularly limited, but the following general formula (1)
  • a polymer block comprising at least one type of block unit (a) can be used.
  • R 1 is a hydrogen atom or a methyl group, and a hydrogen atom is used to bring the LCST into a range of 25 ° C. to 45 ° C.
  • R 2 and R 3 are each independently a hydrogen group, a hydrocarbon group having 1 to 6 carbon atoms, a furfuryl group or a tetrahydrofurfuryl group, and R 2 and R 3 are bonded to each other to form a pyrrolidine ring, a piperidine ring or A morpholine ring may be formed.
  • hydrocarbon group having 1 to 6 carbon atoms examples include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • -Butyl group, n-hexyl group and isohexyl group can be exemplified, but n-propyl group and isopropyl group are preferably used in order to make LCST in the range of 25 ° C to 45 ° C.
  • the block (A) represented by the general formula (1) in the second invention includes N, N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N- Isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylamide, 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- Examples include polymers of at least one monomer selected from (lopenyl) piperidine, 4- (1-o
  • N, N-diethyl acrylamide, Nn-propyl acrylamide, N-isopropyl acrylamide, Nn-propyl methacrylamide, N-ethoxyethyl acrylamide, N-tetrahydroflur in order to be in the range of from 0 to 45 ° C.
  • a polymer of N, N-diethylacrylamide or N-isopropylacrylamide is more preferably used in order to make the polymer of furylacrylamide and N-tetrahydrofurfurylmethacrylamide in the range of 28 to 40 ° C. LCST.
  • the block (A) in the second invention is not particularly limited, but the following general formula (2)
  • a polymer block comprising at least one type of block unit (a) can be used.
  • R 4 represents a hydrogen atom or a methyl group, and a hydrogen atom is used to make LCST in the range of 25 ° C. to 45 ° C.
  • R 5 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 in order to bring the LCST into a range of 25 ° C. to 45 ° C.
  • hydrocarbon group having 1 to 6 carbon atoms examples include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • -Butyl group, n-hexyl group and isohexyl group can be exemplified, but a methyl group, an ethyl group and an n-propyl group are preferably used in order to bring the LCST into a range of 25 ° C to 45 ° C.
  • a is an integer of 1 to 10
  • an integer of 1 to 3 is used to make LCST in the range of 25 ° C to 45 ° C.
  • the block (A) represented by the general formula (2) in the second invention is preferably a polymer of 2- (2-ethoxy) ethoxyethyl vinyl ether so that the LCST is in the range of 25 ° C. to 45 ° C. Is used.
  • the block (A) in the second invention is not particularly limited, but the following general formula (3)
  • a polymer block comprising at least one type of block unit (a) can be used.
  • R 6 represents a hydrogen atom or a methyl group
  • a hydrogen atom is used to make LCST in the range of 25 ° C. to 45 ° C.
  • R 7 represents a hydrocarbon group having 1 to 6 carbon atoms, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, isobutyl group, tert.
  • n-hexyl group and isohexyl group can be exemplified, but a methyl group and an ethyl group are preferably used in order to bring the LCST into a range of 25 ° C to 45 ° C.
  • a polymer of methyl vinyl ether is preferably used so that the LCST is in the range of 25 ° C to 45 ° C.
  • the block (B) in the second invention is the following (i) to (iii) (I) No LCST in the range of 0 ° C to 50 ° C.
  • the block (B) in the second invention is preferably a block having no affinity for biopolymers such as proteins, peptides, glycoproteins or the like, in order to shorten the cooling time required for cell detachment.
  • a polymer of a block and having at least one hydrophilic group selected from a phosphobetaine group, a sulfobetaine group, a carbobetaine group, a polyethylene glycol group, a methoxyethylene group, a furfuryl group, a dialkylaminoalkyl group, and a pyrrolidone group Is a block using a hydrophilic polymer.
  • the block (B) in the second invention is preferably a block showing solubility in water in order to shorten the cooling time necessary for cell detachment, and more preferably 0 in 100 mL of water at 20 ° C.
  • hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (4)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 8 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • R 9 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or (poly) oxyethylene group: — (OCH 2 CH 2 ) n — (wherein n is suitable for the block copolymer of the present invention).
  • the (poly) oxyethylene group is a group A 1 through —O—.
  • a divalent alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, propylene, butylene, pentylene, hexylene, etc., in order to shorten the cooling time required for cell detachment. More preferably, it is ethylene.
  • R 10 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as methylene, ethylene, propylene, butylene, etc. in order to shorten the cooling time required for cell detachment, Ethylene is preferable.
  • R 11 , R 12 and R 13 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms. In order to shorten the cooling time required for cell detachment, preferably R 11 , R 12 and R 13 are simultaneously a hydrogen atom or a methyl group, more preferably a methyl group.
  • a 1 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 in order to shorten the cooling time required for cell detachment. And particularly preferably an ester bond.
  • hydrophilic polymer represented by the general formula (4) in the second invention examples include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3- (meth) acryloyloxypropyl phosphorylcholine, 4- (meth) Acryloyloxybutyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10- (meth) acryloyloxydecylphosphorylcholine, ⁇ - (meth) acryloyl (poly) oxyethylene phosphorylcholine, 2-acrylamidoethylphosphorylcholine, 3-acrylamidopropylphosphorylcholine, 4-acrylamidobutylphosphorylcholine, 6-acrylamidehexylphosphorylcholine, 10-acrylamidedecylphosphorylcholine, ⁇ - (meta ) Acrylamide (poly) oxyethylene phosphorylcholine, 2-methacryloyl
  • hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (5)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 14 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • R 15 is — (CH 2 CH 2 O) b — (CH 2 CH (CH 3 ) O) c —R 16 (wherein R 16 is a hydrogen atom, an alkyl group having 1 to 30 carbon atoms, and b is A polyoxyalkylene group represented by the formula: —CH 2 —O—R 17 (wherein R 17 is a hydrogen atom, a carbon number of 1 to 6 is a substituent represented by (6), a furfuryl group, a tetrahydrofurfuryl group, and a hydrogen atom.
  • Examples of the alkyl group having 1 to 30 carbon atoms used for R 16 include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, 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.
  • Examples of the alkyl group having 1 to 6 carbon atoms used for R 17 include methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, 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.
  • hydrophilic polymer represented by the general formula (5) in the second invention examples include polyethylene glycol methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, hydroxymethyl acrylate, hydroxymethyl methacrylate, 2-methoxyethyl acrylate.
  • hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (6)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 18 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • R 19 is a divalent hydrocarbon group having 1 to 10 carbon atoms, or (poly) oxyethylene groups :-( OCH 2 CH 2) n - (wherein, n suitable for the block copolymer of the present invention
  • n suitable for the block copolymer of the present invention
  • the (poly) oxyethylene group is a group A 1 through —O—.
  • R 20 is a divalent hydrocarbon group having 1 to 4 carbon atoms, and is preferably an alkylene group such as methylene, ethylene, propylene, butylene, etc. in order to shorten the cooling time required for cell detachment, Ethylene is preferable.
  • R 21 and R 22 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms. In order to reduce the cooling time required for cell detachment, preferably R 21 and R 22 are simultaneously A hydrogen atom or a methyl group, more preferably a methyl group at the same time.
  • a 2 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 in order to shorten the cooling time required for cell detachment. And particularly preferably an ester bond.
  • X is a sulfonate anion group, a carboxylic acid anion group, or a phosphate anion group.
  • hydrophilic polymer represented by the general formula (6) in the second invention examples include dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl).
  • These polymers can be used.
  • hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (7)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 23 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • R 24 and R 25 are each independently a hydrogen atom or a methyl group.
  • a polymer of at least one monomer selected from acrylamide or N, N-dimethylacrylamide can be used as the hydrophilic polymer represented by the general formula (7) in the first invention.
  • hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (8)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 26 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • hydrophilic polymer represented by the general formula (8) in the second invention a polymer of N-vinylpyrrolidone can be used.
  • the hydrophilic polymer of the block (B) in the second invention is not particularly limited, but the following general formula (9)
  • a polymer block comprising at least one type of block unit (b) can be used.
  • R 27 represents a hydrogen atom or a methyl group, and a methyl group is used to shorten the cooling time required for cell detachment.
  • R 28 is a divalent hydrocarbon group having 1 to 10 carbon atoms, and preferably has a carbon number of 1 such as methylene, ethylene, propylene, butylene, pentylene, hexylene, etc. in order to shorten the cooling time required for cell detachment.
  • methylene ethylene
  • propylene propylene
  • butylene pentylene
  • hexylene etc.
  • divalent alkylene groups more preferably ethylene.
  • R 29 and R 30 are each independently a hydrogen atom or a hydrocarbon group having 1 to 4 carbon atoms. In order to shorten the cooling time required for cell detachment, it is preferable that R 29 and R 30 are simultaneously A hydrogen atom or a methyl group, more preferably a methyl group at the same time.
  • a 3 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 in order to shorten the cooling time required for cell detachment. And particularly preferably an ester bond.
  • hydrophilic polymer represented by the general formula (9) in the second invention examples include aminomethyl (meth) acrylate, N, N-dimethylaminomethyl (meth) acrylate, and N, N-diethylaminomethyl (meth) acrylate.
  • a polymer of at least one monomer selected from ethyl (meth) acrylate, dimethyl [(meth) acrylamidomethyl] amine, and dimethyl [(meth) acrylamidoethyl] amine can be used.
  • the monomer unit further contained in the hydrophilic polymer includes aromatic vinyl compounds such as styrene, 1-vinylnaphthalene, 2-vinylnaphthalene, 9-vinylanthracene, 1-vinylpyrene and derivatives thereof.
  • the ratio of (a) to the block unit [(a) + (b)] is 1 to 95 mol%.
  • the content is preferably 5 to 85 mol%. If it is less than 1 mol%, the cell adhesiveness is lowered, and if it exceeds 95 mol%, the cooling time required for cell detachment becomes longer.
  • the amount of the block unit (a) constituting the block A is 4 to 94 mol% with respect to the amount of all block units constituting the block copolymer, Is preferably 10 to 85 mol% in order to impart a low temperature and shorten the cooling time required for cell detachment. If it is less than 4 mol%, the cell adhesiveness is lowered, and if it exceeds 94 mol%, the cooling time required for cell detachment becomes longer.
  • the number average molecular weight (Mn) of the block copolymer used in the second invention is in the range of 3,000 to 1,000,000, preferably 4,000 to 500,000, more preferably 5,000. More than 100,000. In the case of less than 3,000, even if the cell culture substrate is coated, it is eluted from the substrate into the medium during cell culture. On the other hand, when it exceeds 1,000,000, the solution viscosity becomes high and it becomes difficult to coat the cell culture substrate.
  • the method for synthesizing the block copolymer used in the second invention is not particularly limited, but is published by NTS Corporation, “Radical Polymerization Handbook”, p. 161 to 225 (2010), and a copolymerization method can be used.
  • the order of monomers to be polymerized in the second invention is as follows: 1) copolymerize a monomer having a hydrophilic group and an aromatic vinyl compound, remove the unreacted monomer, and then polymerize the monomer that forms the block unit (a). 2) A method of polymerizing a monomer that forms the block unit (a), removing an unreacted monomer, and then copolymerizing a monomer having a hydrophilic group and an aromatic vinyl compound, 3) having a hydrophilic group After copolymerizing the monomer and the aromatic vinyl compound, removing the unreacted monomer, polymerizing the monomer that forms the block unit (a), and further removing the unreacted monomer, the monomer having a hydrophilic group and the aromatic 4.
  • a method of copolymerizing a monomer having a hydrophilic group and a (meth) acrylamide compound 8) producing a block unit (a)
  • the monomer having a hydrophilic group and the (meth) acrylamide compound are copolymerized, and after further removing the unreacted monomer, the monomer that forms the block unit (a) is obtained.
  • the cell culture substrate of the present invention can be produced by, for example, dissolving the block copolymer in various solvents, applying the solution to the substrate, and then drying.
  • a solvent used for coating ethanol, a mixed solvent of water and ethanol, which has little influence on cultured cells even if it remains, is preferably used.
  • the substrate is not particularly limited, but various hydrophobic polymer materials are preferably used.
  • the hydrophobic polymer material include acrylic polymers such as polymethyl methacrylate, various silicone rubbers such as polydimethylsiloxane, polystyrene, polyethylene terephthalate, and polycarbonate.
  • a metal substrate, a ceramic substrate, or a glass substrate that has been surface-treated with a silane coupling agent can also be used.
  • 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 the plate-like substrate.
  • various commonly known methods such as brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating and blade coating are used. It is possible.
  • the thickness of the block copolymer coated on the culture substrate of the present invention is 1 nm or more and 10 ⁇ m or less, preferably 10 nm or more and 5 ⁇ m or less, more preferably 30 nm or more and 500 nm or less, and further preferably 50 nm or more and 200 nm. It is as follows. When the thickness is less than 1 nm, the cooling time required for cell detachment becomes long when the cell culture substrate is coated. When the thickness exceeds 10 ⁇ m, the adhesion of cells decreases when the cell culture substrate is coated.
  • the surface of the culture substrate of the present invention may have a microphase separation structure for the purpose of promoting hydrophilicity of the substrate surface due to a temperature drop after cell culture and shortening the cooling time required for cell detachment. preferable.
  • the domain diameter and domain spacing of the microphase separation structure can be arbitrarily 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, the domain diameter and the domain interval should be larger than the cell growth factor and smaller than the cell in order to promote the hydrophilicity of the substrate surface due to the temperature drop and shorten the cooling time required for cell detachment. Is preferred.
  • the method for producing the cell culture substrate of the present invention is not particularly limited, but in addition to the method of coating / drying the block copolymer, a method of immobilizing the block copolymer with a chemical bond can be used. .
  • the method of chemically fixing the block copolymer after synthesis is not particularly limited, but a monomer unit containing a specific functional group is introduced into the block copolymer in advance, and the specific functional group on the base material is introduced.
  • a method of irradiating with ultraviolet rays after coating can be exemplified.
  • a hydroxyl group and an amino group examples include mercapto group and maleimide group, mercapto group and mercapto group, carboxyl group and amino group, carboxyl group and hydroxyl group, and hydroxyl group and hydroxyl group.
  • a surface initiated living radical polymerization technique can be used as a method of immobilizing the block copolymer simultaneously with the synthesis of the block copolymer.
  • the block copolymer chain is obtained by chemically bonding a polymerization initiator to the surface of the base material from which the block copolymer chain starts, and by performing living radical polymerization using the polymerization initiator as a starting point.
  • a method described in JP 2009-59659 A or JP 2010-218984 A can be applied.
  • the base material used in the surface-initiated living radical polymerization is not particularly limited, but iron and iron alloys such as cast iron, steel, and stainless steel, non-ferrous and non-ferrous alloys such as aluminum and copper, and silicon wafer, glass, quartz
  • a material capable of chemically bonding a polymerization initiator to its surface, which is necessary for producing the cell culture substrate of the present invention by surface-initiated living radical polymerization can be used.
  • a silicon wafer, a cleaving mineral such as a mica peeling piece, or a planar substrate having a reactive substituent such as a hydroxyl group on the surface is preferably used.
  • the polymerization initiator is not particularly limited, but a compound having a group capable of being chemically bonded to the substrate and a radical generating group is preferable.
  • the polymerization initiator disclosed in JP 2010-218984 A, , TEMPO, ATRP, RAFT, and RTCP polymerization initiators can be used.
  • TEMPO-based, RAFT-based, and RTCP-based polymerization initiators are more preferable.
  • DEPN polymerization initiators are particularly preferable.
  • Examples of groups that can be chemically bonded to the substrate include -SiCl 3 , -Si (CH 3 ) Cl 2 , -Si (CH 3 ) 2 Cl, and -Si (OR) 3 (in these formulas, R Represents methyl, ethyl, propyl or butyl).
  • the method for chemically bonding the polymerization initiator to the surface of the substrate is not particularly limited, but a polymerization initiator solution is prepared by dissolving or dispersing the polymerization initiator in a solvent.
  • the method of immersing a base material etc. are mentioned.
  • a block copolymer chain can be formed on the surface of the base material by immersing the base material chemically bonded with the polymerization initiator in a polymerization reaction solution containing a monomer and heating as necessary.
  • the polymerization reaction solution may contain various radical initiators and components necessary for the polymerization reaction such as a solvent.
  • Cell culture using the cell culture substrate of the present invention is performed at a temperature higher than the LCST of the block copolymer coated on the surface of the culture substrate, but when human-derived cells are used, high culture efficiency is obtained.
  • it is preferably carried out near body temperature, more preferably in the temperature range of 35 to 39 ° C, and further preferably in the temperature range of 36 to 38 ° C.
  • the other culture conditions are not particularly limited, and the culture may be performed under conditions normally performed in this field.
  • the medium may be a medium supplemented with serum such as fetal bovine serum or a serum-free medium.
  • the ambient temperature a temperature lower than LCST, preferably 10 ° C. or lower than LCST. It can be used in or in other medium solutions, and can be selected according to the purpose.
  • the cell culture substrate may be tapped or shaken, or the medium may be stirred using a pipette or the like.
  • cultured cells can be detached with a maximum diameter of 5 ⁇ m to 300 ⁇ m 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 adjusted by selecting the composition and molecular weight of the block copolymer, the structure of the cell culture substrate, the cell culture substrate production method, the cell culture method, and the type of cells to be cultured. . For example, by increasing the ratio of the block (B) in the block copolymer, increasing the thickness of the block copolymer by the cell culture substrate production method, and increasing the unevenness of the culture substrate surface The size of the cell aggregate can be reduced, and the cells can be detached with a single cell.
  • the cell cultured using the cell culture substrate of the present invention is not particularly limited as long as it can adhere to the surface before applying a stimulus due to a temperature drop.
  • various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human fetal kidney-derived cells HEK293 cells and human cervical cancer-derived HeLa cells, each tissue and organ in the living body is constituted.
  • Epithelial cells and endothelial cells skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiomyocytes, neuronal cells that make up the nervous system, glial cells, fibroblasts, liver parenchymal cells involved in the metabolism of the body, liver non-parenchymal cells
  • stem cells adipocytes, and cells having differentiation ability, stem cells present in various tissues, and further cells induced to differentiate from them can be used.
  • cells live cells contained in blood, lymph, spinal fluid, sputum, urine or stool, microorganisms, viruses, protozoa, etc. present in the body or environment can be exemplified.
  • the thickness of the membrane was determined by observing a cross section of the cell culture substrate with a transmission electron microscope (TEM).
  • Example 1 [Introduction of polymerization initiator to glass petri dish surface] A solution prepared by mixing 0.5 g of 3- (3- (triethoxysilyl) propylthio) propyl-2-bromo-2-methylpropanoate as an immobilization initiator, 50 g of ethanol, and 2.8 g of ammonia was made of 25 mm glass. The petri dish was immersed and allowed to stand for 24 hours. Then, the petri dish by which the polymerization initiator was introduce
  • the glass petri dish having a polymerization initiator introduced on the surface produced above was immersed in the polymerization reaction solution prepared above, heated to 65 ° C., and stirred to start the reaction.
  • the reaction time was 6 hours, and after completion of the reaction, washing with ethanol was performed to obtain a glass petri dish having the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) introduced on the surface.
  • the obtained glass petri dish into which the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) was introduced was treated with sulfuric acid to isolate the 2-methacryloyloxyethyl phosphorylcholine polymer block and dissolved in 100 mL of water at 20 ° C.
  • the formula amount of the hydrophilic part in the block unit of the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) is a total of 8 carbons, 17 hydrogens, 1 nitrogen, 6 oxygens and 1 phosphorus (254.2). Yes, the total formula amount in block units was 295.3, and the HLB value (Griffin method) was 17.
  • the glass petri dish having the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) introduced on the surface produced above is immersed in the polymerization reaction solution prepared above, heated to 65 ° C., and stirred. The reaction started. The reaction time was 6 hours, and after completion of the reaction, washing with ethanol was performed to obtain a cell culture substrate having a temperature-responsive membrane introduced on the surface. The thickness of the film was 450 nm.
  • the obtained cell culture substrate was treated with sulfuric acid, the block copolymer was isolated, and the results of evaluating the polymer composition, Mw, Mn, and Mw / Mn are shown in Table 1.
  • the detached cells were removed with an aspirator, and the number of cells was confirmed again with a 10 ⁇ 10 magnification microscope. By cooling for 15 minutes, the cells were detached 100% in the form of single cells having a maximum diameter of 20 ⁇ m.
  • Reference example 1 Cell culture evaluation Except that a glass petri dish (base material) in which only the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) was introduced on the surface synthesized in Example 1 [Synthesis of polymer block (B)] was used. Cell culture evaluation similar to Example 1 [Cell culture evaluation and peeling evaluation] was carried out for 5 days, but the cells did not adhere to the substrate, and proliferation could not be confirmed.
  • Example 2 [Synthesis of polymer block (B)] The synthesis was performed in the same manner as in Example 1 [Synthesis of polymer block (B)] except that the reaction time was 48 hours, and the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) was introduced on the surface. A glass petri dish was obtained. The obtained glass petri dish into which the 2-methacryloyloxyethyl phosphorylcholine polymer block (B) was introduced was treated with sulfuric acid to isolate the 2-methacryloyloxyethyl phosphorylcholine polymer block and dissolved in 100 mL of water at 20 ° C. As a result, 10 g could be dissolved without any insoluble part.
  • the obtained cell culture substrate was treated with sulfuric acid, the block copolymer was isolated, and the results of evaluating the polymer composition, Mw, Mn, and Mw / Mn are shown in Table 1.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and the cells were detached 100% in the form of a single cell having a maximum diameter of 19 ⁇ m in 15 minutes.
  • Example 3 Synthesis was performed in the same manner as in Example 1 [Synthesis of polymer block (B)] except that 2.0 g of 2-methoxyethyl acrylate was used instead of 2.0 g of 2-methacryloyloxyethyl phosphorylcholine.
  • a glass petri dish into which the 2-methoxyethyl acrylate polymer block (B) was introduced was obtained.
  • the glass petri dish into which the obtained 2-methoxyethyl acrylate polymer block (B) was introduced was treated with sulfuric acid, and the 2-methoxyethyl acrylate polymer block was isolated and dissolved in 100 mL of water at 20 ° C.
  • the formula amount of the hydrophilic part in the block unit of the 2-methoxyethyl acrylate polymer block (B) is the sum of 3 carbons, 4 hydrogens and 3 oxygens (88.1), and the total formula amount of the block units is It was 130.1, and the HLB value (Griffin method) was 14.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and in 15 minutes, the cells were detached by 85% in the form of a single cell having a maximum diameter of 20 ⁇ m.
  • Example 3 Example 3 except that a glass petri dish (base material) in which only the 2-methoxyethyl acrylate polymer block (B) was introduced on the surface synthesized in Example 3 [Synthesis of polymer block (B)] was used. Cell culture evaluation similar to 1 [cell culture evaluation and peeling evaluation] was performed for 5 days, but the cells did not adhere to the substrate and proliferation could not be confirmed.
  • Example 4 [Synthesis of polymer block (B)]
  • Example 1 [Synthesis of polymer block (B)] except that 2.0 g of dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium was used instead of 2.0 g of 2-methacryloyloxyethyl phosphorylcholine.
  • a glass petri dish having a dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium polymer block (B) introduced on the surface was obtained.
  • the resulting petri dish made of dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium polymer block (B) was treated with sulfuric acid to give dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium.
  • the polymer block was isolated and dissolved in 100 mL of water at 20 ° C. As a result, 10 g could be dissolved without any insoluble part.
  • the formula amount of the hydrophilic part in the block unit of 2-methoxyethyl acrylate dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium polymer block (B) is 6 carbons, 10 hydrogens, 1 nitrogen, 4 oxygen
  • the total formula amount in block units was 215.2, and the HLB value (Griffin method) was 15.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and the cells were peeled 90% in the form of a single cell having a maximum diameter of 20 ⁇ m in 15 minutes.
  • Example 5 [Synthesis of polymer block (B)]
  • Example 1 [Synthesis of polymer block (B)] except that 2.0 g of dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium was used instead of 2.0 g of 2-methacryloyloxyethyl phosphorylcholine
  • a glass petri dish having a dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer block (B) introduced on the surface was obtained.
  • the obtained glass petri dish into which the dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer block (B) was introduced was treated with sulfuric acid to obtain dimethyl (2-methacryloylaminopropyl) (3-sulfo
  • 10 g could be dissolved without any insoluble portion.
  • Formula amount of hydrophilic part in block unit of dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer block (B) is 5 carbons, 11 hydrogens, 1 nitrogen, 4 oxygens, sulfur The total was 1 (181.2), the total formula amount in block units was 278.4, and the HLB value (Griffin method) was 13.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and in 15 minutes, the cells were detached 80% in the form of a single cell having a maximum diameter of 21 ⁇ m.
  • Example 5 [Cell culture evaluation]
  • Example 5 [Synthesis of polymer block (B)] A glass petri dish (group) in which only the dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polymer block (B) was introduced on the surface synthesized in Example 5
  • Example 6 Synthesis of polymer block (B)
  • Example 1 Polymer Block (B
  • the glass petri dish having a polyethylene glycol methacrylate polymer block (B) introduced on its surface was obtained.
  • the obtained petri dish made of polyethylene glycol methacrylate polymer block (B) was treated with sulfuric acid to isolate the polyethylene glycol methacrylate polymer block and dissolved in 100 mL of water at 20 ° C. As a result, 10 g was insoluble. It was able to dissolve without.
  • Formula amount of hydrophilic part in block unit of dimethyl (2-methacryloylaminopropyl) (3-sulfonatopropyl) aminium polyethylene glycol methacrylate polymer block (B) is 18 carbons, 34 hydrogens, 10.5 oxygens
  • the total formula amount in block units was 474.6, and the HLB value (Griffin method) was 18.
  • Example 1 [Cells, except that 1.2 g of Nn-propylmethacrylamide was used instead of 1.2 g of N-isopropylacrylamide, a glass petri dish into which the polyethylene glycol methacrylate polymer block (B) was introduced. Synthesis was performed in the same manner as in [Synthesis of culture substrate] to obtain a cell culture substrate having a temperature-responsive membrane introduced on the surface. The thickness of the film was 400 nm. The obtained cell culture substrate is treated with sulfuric acid, the block copolymer is isolated, and the results of evaluating the polymer composition, Mw, Mn, and Mw / Mn are shown in Table 1.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and the cells were detached 100% in the form of a single cell having a maximum diameter of 20 ⁇ m in 15 minutes.
  • Example 6 Cell culture evaluation
  • Example 1 Example 1 [Except that a glass petri dish (base material) in which only the polyethylene glycol methacrylate polymer block (B) was introduced on the surface synthesized in Example 6 [Synthesis of polymer block (B)] was used. Cell culture evaluation and peeling evaluation] were carried out for 5 days, but the cells did not adhere to the substrate and proliferation could not be confirmed.
  • Example 7 Synthesis was performed in the same manner as in Example 1 [Synthesis of polymer block (B)] except that 2.0 g of 2-hydroxyethyl methacrylate was used instead of 2.0 g of 2-methacryloyloxyethyl phosphorylcholine.
  • a glass petri dish having 2-hydroxyethyl methacrylate polymer block (B) introduced on the surface was obtained.
  • the glass petri dish into which the obtained 2-hydroxyethyl methacrylate polymer block (B) was introduced was treated with sulfuric acid to isolate the 2-hydroxyethyl methacrylate polymer block and dissolved in 100 mL of water at 20 ° C.
  • the formula amount of the hydrophilic part in the block unit of the 2-hydroxyethyl methacrylate polymer block (B) is a total of 3 carbons, 5 hydrogens and 3 oxygens (89.1). The amount was 130.1 and the HLB value (Griffin method) was 14.
  • Example 1 Example 1 except that 1.2 g of N, N-diethylacrylamide was used instead of 1.2 g of N-isopropylacrylamide, a glass petri dish into which the 2-hydroxyethyl methacrylate polymer block (B) was introduced. Synthesis was performed in the same manner as in 1 [Synthesis of cell culture substrate] to obtain a cell culture substrate having a temperature-responsive membrane introduced on its surface. The film thickness was 250 nm. The obtained cell culture substrate is treated with sulfuric acid, the block copolymer is isolated, and the results of evaluating the polymer composition, Mw, Mn, and Mw / Mn are shown in Table 1.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and the cells were peeled 90% in the form of a single cell having a maximum diameter of 20 ⁇ m in 15 minutes.
  • Example 7 Cell culture evaluation Except that a glass petri dish (base material) having a 2-hydroxyethyl methacrylate polymer block (B) introduced on the surface synthesized in Example 7 [Synthesis of polymer block (B)] was used. Cell culture evaluation similar to Example 1 [Cell culture evaluation and peeling evaluation] was carried out for 5 days, but the cells did not adhere to the substrate, and proliferation could not be confirmed.
  • Example 8 Synthesis was performed in the same manner as in Example 1 [Synthesis of polymer block (B)] except that 2.0 g of N, N-dimethylaminoethyl methacrylate was used instead of 2.0 g of 2-methacryloyloxyethyl phosphorylcholine. A glass petri dish having an N, N-dimethylaminoethyl methacrylate polymer block (B) introduced on the surface was obtained.
  • the obtained petri dish with N, N-dimethylaminoethyl methacrylate polymer block (B) was treated with sulfuric acid to isolate the N, N-dimethylaminoethyl methacrylate polymer block, and 100 mL of water at 20 ° C. As a result, 5 g could be dissolved without any insoluble part.
  • the formula amount of the hydrophilic part in the block unit of the N, N-dimethylaminoethyl methacrylate polymer block (B) is the sum of 5 carbons, 10 hydrogens, 1 nitrogen and 2 oxygens (116.1). The total formula amount in block units was 157.2, and the HLB value (Griffin method) was 15.
  • Example 8 Cell culture evaluation Except that a glass petri dish (base material) in which the N, N-dimethylaminoethyl methacrylate polymer block (B) was introduced on the surface synthesized in Example 7 [Synthesis of polymer block (B)] was used. Cell culture evaluation similar to that in Example 1 [Cell culture evaluation and peeling evaluation] was performed for 5 days, but the cells did not adhere to the substrate, and proliferation could not be confirmed.
  • Example 9 [Except that only 2-hydroxyethyl methacrylate polymer block (B) was used instead of block copolymer on the surface synthesized in Example 9 [Synthesis of polymer block (B)]. A glass substrate was synthesized in the same manner as in [Synthesis of cell culture substrate].
  • Cell culture evaluation A cell culture evaluation similar to that in Example 8 [Cell culture evaluation and peeling evaluation] was performed for 5 days except that the glass substrate described above was used, but the cells did not adhere to the substrate and proliferation could not be confirmed. .
  • Table 1 shows the results of evaluating the polymer composition, Mw, Mn, and Mw / Mn of the obtained block copolymer.
  • Cell proliferation was confirmed by performing the same evaluation as in Example 1 [Evaluation of cell culture and exfoliation] except that a cell culture substrate having a temperature-responsive membrane introduced on the surface produced above was used. . Further, after culturing until the cultured cells covered 100% of the base material, the base material was cooled to 10 ° C., so that the cells were detached 75% in the form of a single cell having a maximum diameter of 20 ⁇ m in 15 minutes.
  • Example 10 Cell culture except that the random copolymer block (B) of N-vinylpyrrolidone and styrene synthesized in Example 10 [Synthesis of polymer block (B)] was used instead of the block copolymer. Synthesis was performed in the same manner as in [Synthesis of Substrate] to synthesize a cell culture substrate in which a random copolymer block of N-vinylpyrrolidone and styrene was introduced on the surface.
  • Cell culture evaluation Cell culture similar to that in Example 1 [Evaluation of cell culture and exfoliation], except that the cell culture substrate in which a random copolymer block of N-vinylpyrrolidone and styrene was introduced on the surface produced above was used. Evaluation was conducted for 5 days, but the cells did not adhere to the substrate, and proliferation could not be confirmed.
  • Example 11 A random copolymer block (B) of 2-methacryloyloxyethyl phosphorylcholine and styrene synthesized in Example 11 [Synthesis of polymer block (B)] was used in place of the block copolymer in Example 11 [ Synthesis was performed in the same manner as in [Synthesis of cell culture substrate] to synthesize a cell culture substrate having a random copolymer block of 2-methacryloyloxyethyl phosphorylcholine and styrene introduced on the surface.
  • Cell culture evaluation The same as in Example 1 [Evaluation of cell culture and exfoliation], except that a cell culture substrate in which a random copolymer block of 2-methacryloyloxyethyl phosphorylcholine and styrene was introduced on the surface prepared above was used. Cell culture evaluation was performed for 5 days, but the cells did not adhere to the substrate, and proliferation could not be confirmed.
  • Example 12 [Cell culture evaluation and exfoliation evaluation] Normal human skin fibroblasts (NHDF cells) (114 cells / mm 2 ) were prepared using the cell culture substrate in which a temperature-responsive membrane was introduced on the surface produced in Example 11 [Synthesis of cell culture substrate]. The cells were cultured at 37 ° C. and a CO 2 concentration of 5%. As a culture solution, a CS-C medium kit (No. CS4Z0500R) manufactured by DS Pharma Biomedical Co., Ltd. was used. When cell proliferation was confirmed and cultured for 72 hours, the number of cells was confirmed with a 10 ⁇ 10 magnification microscope, and the cells grew to 425 cells / mm 2 .
  • the detached cells were removed with an aspirator, and the number of cells was confirmed again with a 10 ⁇ 10 magnification microscope. By cooling for 15 minutes, the cells were detached 100% in the form of a single cell having a maximum diameter of 22 ⁇ m.
  • Example 13 Cell culture evaluation and exfoliation evaluation
  • Example 11 Synthesis of cell culture substrate
  • the cell culture substrate in which a temperature-responsive membrane was introduced on the surface was used and derived from Chinese hamster ovary instead of mouse connective tissue L929 cells (100 cells / mm 2 ). Except for using CHO cells (100 cells / mm 2 ), the same evaluation as in Example 1 [Cell culture evaluation and peeling evaluation] was performed, and cell proliferation was confirmed. Further, after culturing until the cultured cells covered 100% of the substrate, the substrate was cooled to 10 ° C., and in 15 minutes, the cells were detached by 70% in the form of a single cell having a maximum diameter of 18 ⁇ m.
  • Reference Example 12 Cell culture evaluation
  • a cell culture substrate in which only a random copolymer block of 2-methacryloyloxyethyl phosphorylcholine and styrene was introduced on the surface produced in Reference Example 11
  • mouse connective tissue L929 cells 100 cells
  • Chinese hamster ovary-derived CHO cells 100 / mm 2
  • Example 14 [Synthesis of cell culture substrate] 0.04 g of the block copolymer synthesized in Example 11 [Synthesis of block copolymer] was dissolved in 20 g of ethanol to prepare a 0.2 wt% ethanol solution of the block copolymer. Furthermore, 20 mL of 0.2 wt% ethanol solution and 180 mL of ethanol were mixed to prepare a 0.02 wt% ethanol solution. After adding 180 mL of the above-mentioned block copolymer 0.02 wt% ethanol solution to a 300 mL glass flask, stirring the ethanol solution with a stirrer, adding 10 g of Corning (R) Extended Microcarriers (No.
  • R Corning
  • the cell culture substrate was dispersed in a phosphate buffered saline (PBS) solution, and autoclaved. Thereafter, 0.5 ⁇ L was added to the hemocytometer, and the bead concentration was calculated.
  • a cell culture substrate was added to a cell seed HydroCell® 3.5 cm dish at 1 ⁇ 10 4 beads / mL and L929 cells were added at 100 cells / bead, and cultured at 37 ° C. After culturing for 72 hours, the culture solution was transferred to a tube, 2 mL of the culture solution was added, allowed to stand for 10 minutes, and then the supernatant was discarded to remove unattached floating cells and cell aggregates.
  • PBS phosphate buffered saline
  • Comparative Example 2 [Cell culture evaluation and exfoliation evaluation] Except for using CellCell Co., Ltd. UpCell (R) 35 mm ⁇ dish, the same evaluation as in Example 1 [Cell culture evaluation and exfoliation evaluation] was performed, and cell proliferation was confirmed.
  • cell exfoliation evaluation after cell proliferation By cooling for 3 minutes, the cells were peeled off in a sheet form by 30%. Further, by cooling for 15 minutes, the cells were peeled off by 65% in the form of a sheet having a maximum diameter of 1 cm.
  • Example 3 Cell proliferation was confirmed by the same evaluation as in Example 1 [Cell culture evaluation and exfoliation evaluation] except that a Corning cell culture surface treatment ⁇ 35 mm dish was used. In cell exfoliation evaluation after cell proliferation, The cells did not peel at all even after cooling for 15 minutes.
  • the mixture of exfoliated cells and trypsin EDTA solution was transferred into a centrifuge tube, 1 mL of 10 vol% FBS / DMEM medium was added, and the mixture was centrifuged at 1000 rpm for 3 minutes. After removing the supernatant, 10 vol% FBS / DMEM medium was newly added to recover L929 cells detached by trypsin treatment.
  • the recovered L929 cells (50 cells / mm 2 ) were cultured in the same manner as in Example 1 [Cultural evaluation of detached cells]. Cells increased to 80 cells / mm 2 after 24 hours and 480 cells / mm 2 after 72 hours, and the growth rate was slower than that of cells detached from the cell culture substrate of the present invention by cooling.
  • Example 4 [Cell culture evaluation and exfoliation evaluation] Evaluation was conducted in the same manner as in Example 12 [Evaluation of cell culture and exfoliation] except that CellCell Co., Ltd. UpCell (R) 35 mm ⁇ dish was used, and cell growth was confirmed. until mm 2 it had grown. The substrate was cooled to 10 ° C. and cooled for 15 minutes, so that the cells were peeled 90% in a sheet form having a maximum diameter of 1.2 cm. [Culture evaluation of exfoliated cells] Evaluation was performed in the same manner as in Example 12 [Culture evaluation of detached cells] except that the cells obtained by the above cell culture evaluation were dispersed by pipetting and then used. Cells increased to 210 cells / mm 2 after 24 hours and to 880 cells / mm 2 after 72 hours.
  • Example 5 Cell culture evaluation and exfoliation evaluation
  • the cells were evaluated in the same manner as in Example 12 [Evaluation of cell culture and exfoliation] except that a Corning cell culture surface treatment ⁇ 35 mm dish was used. When cell growth was confirmed and cultured for 72 hours, 266 cells / mm 2 were used. Was growing. The substrate was cooled to 10 ° C. and cooled for 60 minutes, but the cells did not detach. [Evaluation of exfoliated and exfoliated cells by trypsin] After removing the medium components from the cells obtained in the above cell culture evaluation, 1.5 mL of 0.25% trypsin EDTA solution (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the temperature was 37 ° C.
  • Example 12 Physical evaluation of exfoliated cells
  • the recovered NHDF cells 50 cells / mm 2
  • Cells increased to 150 cells / mm 2 after 24 hours and to 690 cells / mm 2 after 72 hours.
  • Example 6 Cell culture evaluation and exfoliation evaluation
  • CellCell Co., Ltd. UpCell (R) 35 mm ⁇ dish was used and Chinese hamster ovary-derived CHO cells (100 cells / mm 2 ) were used instead of mouse connective tissue L929 cells (100 cells / mm 2 ).
  • the same evaluation as in Example 1 [Cell culture evaluation and peeling evaluation] was performed, and cell growth was confirmed.
  • the cells were peeled 15% in a sheet form by cooling for 3 minutes. Further, by cooling for 15 minutes, the cells were separated by 50% in the form of a sheet having a maximum diameter of 1.2 cm.
  • the present invention it is possible to provide a cell culture substrate that enables cell detachment in a short time, a production method thereof, and a cell culture method using the same. Furthermore, it is possible to provide a cell culture substrate capable of exfoliating cells with a maximum diameter of 5 ⁇ m to 300 ⁇ m and omitting the cell dispersal operation, a production method thereof, and a cell culture method using the same.

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Abstract

Lorsque le polymère sensible à la température susmentionné est utilisé dans un élément de base pour une utilisation en culture cellulaire, il faut abaisser la température de l'élément de base pour l'utilisation en culture cellulaire à une température égale ou inférieure à la température de solution critique du polymère. Dans ce cas, cependant, la température des cellules est également abaissée simultanément. En outre, étant donné que des cellules détachées de l'élément de base ont une forme analogue à une feuille, il faut disperser les cellules par pipetage ou analogue tout en maintenant les cellules à une température basse, pour le traitement des cellules. Par conséquent, le temps de refroidissement des cellules est prolongé. L'abaissement de la température des cellules peut induire la réduction de l'activité des cellules et, par conséquent, il faut raccourcir le temps de refroidissement des cellules. Selon l'invention, le problème peut être résolu en ce que : un élément de base pour une utilisation en culture cellulaire, dont une surface est revêtue par un copolymère séquencé, le copolymère séquencé contenant un bloc polymère sensible à la température (A), qui présente une température inférieure de solution critique (LCST) par rapport à l'eau de 0 à 50°C, et un bloc polymère hydrophile (B) dont la LCST ne se situe pas dans la plage de 0 à 50°C et qui présente une valeur BHL (procédé de Griffin) de 9 à 20 ; et un élément de base pour une utilisation en culture cellulaire, dont la surface est revêtue par un copolymère séquencé, le copolymère séquencé contenant un bloc polymère sensible à la température (A) qui présente une température inférieure de solution critique (LCST) par rapport à l'eau de 0 à 50°C et un bloc (B) qui répond à une exigence (i) selon laquelle le bloc (B) ne présente pas une LCST dans la plage de 0 à 50°C, une exigence (ii) selon laquelle le bloc (B) comprend un polymère hydrophile d'un monomère présentant au moins un groupe hydrophile choisi parmi un groupe acide carboxylique, un groupe ester d'acide carboxylique, un sel métallique d'un acide carboxylique, un groupe acide sulfonique, un groupe ester d'acide sulfonique, un sel métallique d'un acide sulfonique, un groupe hydroxy, un groupe alcoxy, un groupe phénoxy, un groupe amide, un groupe carbamoyle, un groupe sulfonamide, un groupe sulfamoyle, un groupe carbamate, un groupe acide phosphorique, un groupe de sel métallique d'un acide phosphorique, un groupe acide oxyphosphorique, un groupe de sel métallique d'un acide oxyphosphorique, un groupe phosphobétaïne, un groupe sulfobétaïne, un groupe carbobétaïne, un groupe polyéthylèneglycol et un groupe pyrrolidone et une exigence (iii) selon laquelle le bloc (B) comprend le polymère mentionné dans (ii), le polymère contenant au moins un motif monomère choisi parmi un motif monomère pouvant former un composé aromatique de vinyle, un motif monomère pouvant former un composé (méth)acrylamide, un motif monomère pouvant former un composé diester de l'acide fumarique, un motif monomère pouvant former du chlorure de vinyle, un motif monomère pouvant former un acétate de vinyle, un motif monomère pouvant former du (méth)acrylonitrile, un motif monomère pouvant former du N-vinylimidazole et un motif monomère pouvant former du N-vinylcarbazole.
PCT/JP2016/060513 2015-03-31 2016-03-30 Élément de base pour l'utilisation en culture cellulaire, son procédé de production et procédé de culture cellulaire l'utilisant WO2016159153A1 (fr)

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WO2019102823A1 (fr) * 2017-11-22 2019-05-31 花王株式会社 Agent d'hydrophilisation
JP2019094434A (ja) * 2017-11-22 2019-06-20 花王株式会社 親水化処理剤組成物
JP2019094433A (ja) * 2017-11-22 2019-06-20 花王株式会社 親水化処理剤
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EP4166645A4 (fr) * 2020-06-12 2024-01-10 Nissan Chemical Corporation Film de revêtement biocompatible contenant un copolymère séquencé

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