WO2014196652A1 - 細胞培養器 - Google Patents
細胞培養器 Download PDFInfo
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- WO2014196652A1 WO2014196652A1 PCT/JP2014/065250 JP2014065250W WO2014196652A1 WO 2014196652 A1 WO2014196652 A1 WO 2014196652A1 JP 2014065250 W JP2014065250 W JP 2014065250W WO 2014196652 A1 WO2014196652 A1 WO 2014196652A1
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D133/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Coating compositions based on derivatives of such polymers
- C09D133/04—Homopolymers or copolymers of esters
- C09D133/14—Homopolymers or copolymers of esters of esters containing halogen, nitrogen, sulfur or oxygen atoms in addition to the carboxy oxygen
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- 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
- C08F230/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal
- C08F230/02—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing phosphorus, selenium, tellurium or a metal containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L101/00—Compositions of unspecified macromolecular compounds
- C08L101/02—Compositions of unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L43/00—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing boron, silicon, phosphorus, selenium, tellurium or a metal; Compositions of derivatives of such polymers
- C08L43/02—Homopolymers or copolymers of monomers containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D143/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and containing boron, silicon, phosphorus, selenium, tellurium, or a metal; Coating compositions based on derivatives of such polymers
- C09D143/02—Homopolymers or copolymers of monomers containing phosphorus
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D201/00—Coating compositions based on unspecified macromolecular compounds
- C09D201/02—Coating compositions based on unspecified macromolecular compounds characterised by the presence of specified groups, e.g. terminal or pendant functional groups
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
- C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
- C09D5/16—Antifouling paints; Underwater paints
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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
Definitions
- the present invention relates to a cell culture vessel, a method for producing the same, and a method for producing a cell aggregate (also referred to as a sphere) using the same.
- the present invention relates to a cell culture container and a method for producing the same, characterized in that the surface is coated with a biological material, in particular, a copolymer having a cell adhesion inhibitory ability.
- cells and / or tissues cultured by this technique are organs lost due to evaluation of efficacy and toxicity of chemical substances, pharmaceuticals, etc., mass production of useful substances such as enzymes, cell growth factors, antibodies, and diseases and defects. It is used in various fields, such as regenerative medicine that supplements tissues and cells, plant breed improvement, and creation of genetically modified crops.
- Suspension cells are cells that do not require a scaffold for growth / proliferation
- adherent cells are cells that require a scaffold for growth / proliferation, but most cells that make up living organisms are the latter adherent cells.
- Known culture methods for adherent cells include monolayer culture, dispersion culture, embedding culture, microcarrier culture, and cell aggregate (sphere) culture.
- the present inventors paid attention to a polymer having a phosphate ester group, which is expected as a coating material having an ability to suppress adhesion of various biological substances, and conducted intensive studies.
- the cell culture container whose surface is coated with a copolymer containing a specific organic group suppresses the adhesion of cells to the container surface (wall surface), and the coating adheres firmly to the container surface. Therefore, it was found that the coating was useful as a cell culture container with improved elution into the culture medium and radiation resistance, and the present invention was completed.
- T c , T d and U d each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and R c and R d each independently represent:
- T c and T d each independently represent a hydrogen atom or a methyl group
- U d represents a hydrogen atom
- R c and R d each independently represents an ethylene group or a propylene group, 4.
- U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms, and An ⁇ represents a halogen atom Coating on at least a portion of the surface of the container; and a coated container at a temperature of ⁇ 200 ° C. to about ⁇ 200 ° C., which represents an anion selected from the group consisting of fluoride ion, inorganic acid ion, hydroxide ion and isothiocyanate ion
- the repeating units containing an organic group represented by the above formulas (a) and (b) are represented by the following formulas (A) and (B):
- T a , T b , U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
- Q a and Q b each independently represent a single bond, an ester bond or an amide bond
- R a and R b each independently represent 1 to 10 carbon atoms which may be substituted with a halogen atom.
- An ⁇ represents an anion selected from the group consisting of halide ion, inorganic acid ion, hydroxide ion and isothiocyanate ion, and m represents an integer of 0 to 6
- the production method according to 6 above which is a repeating unit derived from a monomer represented by: 8).
- the 1st aspect of this invention is a copolymer containing the repeating unit containing the organic group represented by following formula (a), and the repeating unit containing the organic group represented by following formula (b):
- U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
- An ⁇ represents a halogen atom
- the cell in the present invention is the most basic unit constituting an animal or a plant, and has a cytoplasm and various organelles inside a cell membrane as its elements.
- the nucleus containing DNA may or may not be contained inside the cell.
- the animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting the living body, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells separated from the living body, and separated from the living body.
- somatic cells constituting a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, bones Cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, Glial cells, neurons, oligodendrocytes, microglia, astrocytes, heart cells, esophageal cells, muscle cells (eg, smooth or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (eg, Cord blood-derived CD34 positive cells), mononuclear cells and the like.
- the somatic cells are, for example, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, blood vessel Includes cells taken from any tissue such as tissue, blood (including umbilical cord blood), bone marrow, heart, eye, brain or nerve tissue.
- a stem cell is a cell that has the ability to replicate itself and to differentiate into cells of other multiple lineages.
- Examples thereof include, but are not limited to, embryonic stem cells (ES cells) Embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, Hair follicle stem cells are included.
- Examples of the pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells among the stem cells.
- a progenitor cell is a cell that is in the process of being differentiated from the stem cell into a specific somatic cell or germ cell. Cancer cells are cells that have been derived from somatic cells and have acquired unlimited proliferative capacity.
- Examples of cell lines include, but are not limited to, HEK293 (human embryonic kidney cells), MDCK, MDBK, BHK, C-33A, AE-1, 3D9, Ns0 / 1, NIH3T3, PC12, S2 , Sf9, Sf21, High Five (registered trademark), Vero, and the like.
- Examples of hepatocyte cell lines include, but are not limited to, HepG2, Hep3B, HepaRG®, JHH7, HLF, HLE, PLC / PRF / 5, WRL68, HB611, SK-HEP-1, Examples include HuH-4 and HuH-7.
- the plant-derived cells in the present invention include cells separated from each tissue of the plant body, and also include protoplasts obtained by artificially removing cell walls from the cells.
- the container coated with the copolymer constituting the cell culture container of the present invention may be any type of container that can be used in this technical field, for example, a petri dish generally used for cell culture. , Flask, plastic bag, Teflon (registered trademark) bag, dish, petri dish, tissue culture dish, multi-dish, microplate, microwell plate, multiplate, multiwell plate, chamber slide, cell culture flask, spinner flask, A tube, a tray, a culture bag, a roller bottle, etc. are mentioned.
- a multiwell plate having 6 to 1536 holes and a petri dish are used.
- the resin may be either a natural resin or a synthetic resin.
- the natural resin include cellulose, cellulose triacetate (CTA), cellulose having dextran sulfate immobilized thereon
- the synthetic resin includes, for example, poly Acrylonitrile (PAN), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester (PE), polypropylene (PP), polyvinylidene fluoride (PVDF), various ion exchange resins or polyethersulfone (PES).
- PAN poly Acrylonitrile
- PEPA polyester polymer alloy
- PS polystyrene
- PSF polysulfone
- PET polyethylene terephthalate
- PMMA polymethyl methacrylate
- PVA polyvinyl alcohol
- the cell culture container of the present invention is characterized in that at least a part of the surface of the container is coated with a specific copolymer.
- the copolymer according to the present invention is a copolymer containing a repeating unit containing an organic group represented by the following formula (a) and a repeating unit containing an organic group represented by the following formula (b):
- U a1 , U a2 , U b1 , U b2 and U b3 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms
- An ⁇ represents a halogen atom Represents an anion selected from the group consisting of fluoride ion, inorganic acid ion, hydroxide ion and isothiocyanate ion).
- the copolymer according to the present invention is a copolymer including a repeating unit containing an organic group represented by the above formula (a) and a repeating unit containing an organic group represented by the above formula (b).
- the copolymer is preferably obtained by radical polymerization of a monomer containing an organic group represented by the above formula (a) and a monomer containing an organic group represented by the above formula (b). Those obtained by polycondensation and polyaddition reaction can also be used.
- Examples of the copolymer include vinyl polymer polymer reacted with olefin, polyamide, polyester, polycarbonate, polyurethane and the like.
- vinyl polymer polymer or (meth) acrylate compound reacted with olefin was polymerized.
- a (meth) acrylic polymer is desirable.
- the (meth) acrylate compound means both an acrylate compound and a methacrylate compound.
- (meth) acrylic acid means acrylic acid and methacrylic acid.
- the monomers containing the organic groups represented by the above formulas (a) and (b) are represented by the following formulas (A) and (B), respectively:
- examples of the “straight or branched alkyl group having 1 to 5 carbon atoms” include, for example, methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group or 1 -An ethylpropyl group is mentioned.
- ester bond means —C ( ⁇ O) —O— or —O—C ( ⁇ O) —
- the “linear or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom” means a linear or branched alkylene group having 1 to 10 carbon atoms, or one or more halogen atoms. Means a linear or branched alkylene group having 1 to 10 carbon atoms and substituted with
- the “linear or branched alkylene group having 1 to 10 carbon atoms” is a divalent organic group in which one hydrogen atom is further removed from the alkyl group, and examples thereof include a methylene group, an ethylene group, and a propylene group.
- an ethylene group, a propylene group, an octamethylene group, and a decamethylene group are preferable, and a linear or branched alkylene group having 1 to 5 carbon atoms such as an ethylene group, a propylene group, a trimethylene group, and a tetramethylene group are more preferable.
- An ethylene group or a propylene group is particularly preferable.
- a linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms means that one or more arbitrary hydrogen atoms of such an alkylene group are replaced with halogen atoms. In particular, those in which some or all of the hydrogen atoms of the ethylene group or propylene group are replaced with halogen atoms are preferred.
- T a and T b are preferably each independently a hydrogen atom, a methyl group or an ethyl group, more preferably each independently a hydrogen atom or a methyl group. It is.
- U a1 , U a2 , U b1 , U b2 and U b3 are preferably each independently a hydrogen atom, a methyl group or An ethyl group.
- U a1 and U a2 are more preferably a hydrogen atom.
- U b1 , U b2 (and U b3 ) are more preferably a methyl group or an ethyl group, and particularly preferably a methyl group.
- R a and R b are preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, More preferably, they are each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.
- m preferably represents an integer of 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.
- formula (A) examples include vinylphosphonic acid, acid phosphooxyethyl (meth) acrylate, 3-chloro-2-acid phosphooxypropyl (meth) acrylate, acid phosphooxypropyl (meth) acrylate, and acid phospho Examples include oxymethyl (meth) acrylate, acid phosphooxypolyoxyethylene glycol mono (meth) acrylate, and acid phosphooxypolyoxypropylene glycol mono (meth) acrylate.
- These compounds may contain a (meth) acrylate compound having two functional groups as represented by the general formula (C) or (D) described later at the time of synthesis.
- formula (B) examples include dimethylaminoethyl (meth) acrylate, diethylaminoethyl (meth) acrylate, dimethylaminopropyl (meth) acrylate, 2- (t-butylamino) ethyl (meth) acrylate, and methacryloyl.
- Choline chloride and the like can be mentioned, among which dimethylaminoethyl (meth) acrylate, methacryloylcholine chloride or 2- (t-butylamino) ethyl (meth) acrylate is preferably used.
- the proportion of the repeating unit containing the organic group represented by the formula (a) in the copolymer (or the repeating unit represented by the formula (a1)) is from 20 mol% to 80 mol%, preferably 30 It is from mol% to 70 mol%, more preferably from 40 mol% to 60 mol%.
- the copolymer which concerns on this invention may contain the repeating unit (or repeating unit represented by Formula (a1)) containing the 2 or more types of organic group represented by Formula (a).
- the ratio of the repeating unit containing the organic group represented by the formula (b) in the copolymer (or the repeating unit represented by the formula (b1)) is based on the formula (a) ( Or the remainder which subtracted the ratio of Formula (a1)) may be sufficient, and the remainder which subtracted the total ratio of the said Formula (a) (or Formula (a1)) and the 3rd component described below may be sufficient.
- the copolymer which concerns on this invention may contain the repeating unit (or repeating unit represented by Formula (b1)) containing the organic group represented by 2 or more types of formula (b).
- an optional third component may be copolymerized.
- a (meth) acrylate compound having two or more functional groups may be copolymerized as the third component, and a part of the polymer may be partially three-dimensionally crosslinked.
- a third component for example, the following formula (C) or (D):
- T c and T d are preferably each independently a hydrogen atom, a methyl group or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group. It is.
- U d is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom.
- R c and R d are preferably each independently a linear or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, More preferably, they are each independently an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.
- Preferred examples of the bifunctional monomer represented by the formula (C) include ethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, and propylene glycol di (meth) acrylate.
- the bifunctional monomer represented by the formula (D) is preferably bis (methacryloyloxymethyl) phosphate, bis [2- (methacryloyloxy) ethyl phosphate, bis [2- (methacryloyloxy) propyl phosphate ] Etc. are mentioned.
- the optional third component may be a trifunctional monomer.
- a trifunctional monomer as the third component include phosphiniridine tris (oxy-2,1-ethanediyl) triacrylate.
- ethylene glycol di (meth) acrylate represented by the following formula (C-1) and bis [2- (methacryloyloxy) ethyl phosphate represented by the following formula (D-1) are particularly preferable. .
- the copolymer may contain one or more of these third components.
- the bifunctional monomer represented by the formula (D) is preferable, and the bifunctional monomer represented by the formula (D-1) is particularly preferable.
- the ratio of the crosslinked structure derived from the third component in the copolymer, for example, the bifunctional monomer represented by the formula (C) or (D) is 0 mol% to 50 mol%.
- ⁇ Method for producing copolymer> As a method for synthesizing the copolymer according to the present invention, it can be synthesized by methods such as radical polymerization, anionic polymerization, and cationic polymerization, which are general methods for synthesizing acrylic polymer or methacrylic polymer.
- the form can be various methods such as solution polymerization, suspension polymerization, emulsion polymerization and bulk polymerization.
- the reaction solvent may be water, an alcohol such as a phosphate buffer solution or ethanol, or a mixed solvent in combination of these, but it is preferable to contain water or ethanol. Furthermore, it is preferable that 10 mass% or more and 100 mass% or less of water or ethanol are included. Furthermore, it is preferable to contain 50 mass% or more and 100 mass% or less of water or ethanol. Furthermore, it is preferable to contain 80% by mass or more and 100% by mass or less of water or ethanol. Furthermore, it is preferable that 90 mass% or more and 100 mass% or less of water or ethanol are included. The total of water and ethanol is preferably 100% by mass.
- the concentration in the reaction solvent of the monomer containing the organic group represented by the formula (a) and the monomer containing the organic group represented by the formula (b) is 0.01% by mass. It is preferable to set it as 4 to 4 mass%.
- the concentration is 4% by mass or more, for example, the copolymer may gel in the reaction solvent due to the strong association property of the phosphate group represented by the formula (a).
- the concentration is 0.01% by mass or less, since the concentration of the obtained varnish is too low, it is difficult to produce a coating film forming composition for obtaining a coating film having a sufficient film thickness. More preferably, the concentration is 0.01 mass% to 3 mass%, for example 3 mass% or 2 mass%.
- an acidic phosphate monomer (half salt) described in Formula (1) may be prepared and then polymerized to prepare a copolymer.
- the phosphoric acid group-containing monomer is a monomer that easily associates, when dropped into the reaction system, it may be dropped little by little in the reaction solvent so that it can be quickly dispersed. Further, the reaction solvent may be heated (for example, 40 ° C. to 100 ° C.) to increase the solubility of the monomer and polymer.
- polymerization initiators include 2,2'-azobis (isobutyronitrile), 2,2'-azobis (2-methylbutyronitrile), 2,2'-azobis (2,4-dimethylvaleronitrile) ), 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobis (4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis (cyclohexane-1-carbonitrile) 1-[(1-cyano-1-methylethyl) azo] formamide, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2′-azobis [2 -(2-imidazolin-2-yl) propane], 2,2'-azobis (2-methylpropionamidine) dihydrochloride, 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) propion
- the addition amount of the polymerization initiator is 0.05% by mass to 10% by mass with respect to the total weight of the monomers used for the polymerization.
- the reaction conditions are as follows.
- the reaction vessel is heated to 50 ° C. to 200 ° C. with an oil bath or the like and stirred for 1 hour to 48 hours, more preferably 80 ° C. to 150 ° C. for 5 hours to 30 hours.
- a copolymer according to the invention is obtained.
- the reaction atmosphere is preferably a nitrogen atmosphere.
- all the reactants may be put in a reaction solvent at room temperature and then heated to the above temperature for polymerization, or all or a part of the mixture of reactants may be placed in a preheated solvent. You may add it little by little.
- the concentration of the compound represented by the above formula (A) or (B) in the reaction solvent may be, for example, 0.01% by mass to 10% by mass. it can. In this case, even if the concentration exceeds 4% by mass, the dropping phase and the reaction phase become a transparent and uniform solution before the reaction, and the gelation of the copolymer after the reaction in the reaction solvent can be suppressed.
- the molecular weight of the copolymer according to the present invention may be about several thousand to several million, preferably 5,000 to 5,000,000. More preferably, it is 10,000 to 2,000,000.
- any of a random copolymer, a block copolymer, and a graft copolymer may be used, and the copolymerization reaction itself for producing the copolymer is not particularly limited, and includes radical polymerization, ionic polymerization, and photopolymerization.
- a method synthesized in a known solution such as polymerization, macromer, polymerization using emulsion polymerization, or the like can be used.
- any one of the copolymers of the present invention can be used alone, or a plurality of copolymers can be mixed and the ratio thereof can be changed.
- the various copolymers thus produced may be two-dimensional polymers or three-dimensional polymers, and are in a state of being dispersed in a solution containing water. That is, it is not preferable that the varnish containing these polymers can cause non-uniform gelation or cloudiness precipitation, and is preferably a transparent varnish, a dispersed colloidal varnish, or a sol.
- a second aspect of the present invention is a copolymer comprising a repeating unit containing an organic group represented by the following formula (a) and a repeating unit containing an organic group represented by the following formula (b):
- U a1 , U a2 , U b1 , U b2 and U b3 , and An ⁇ are as defined above
- a method for producing a cell culture vessel comprising a step of drying at ⁇ 200 ° C. to 200 ° C.
- the copolymer is coated on at least a part of the surface of the container.
- the container is a round-bottom multi-well plate, only the well indentation may be coated, or the entire plate may be coated.
- the container and the copolymer are as described in the above items ⁇ container> and ⁇ copolymer>, respectively.
- the coating step is not particularly limited, and may be performed by any coating means known to those skilled in the art (for example, application, immersion, etc.) that can contact the container and the copolymer.
- it can be carried out by applying a varnish containing a copolymer to a container, or immersing the container in a varnish containing a copolymer.
- it implements by immersing a container in the varnish containing a copolymer.
- the varnish containing the copolymer may be prepared by dissolving the copolymer obtained in the above item ⁇ Method for producing copolymer> in a suitable solvent at a desired concentration, or such a production method.
- the reaction solution containing the copolymer obtained in (1) may be used as a varnish as it is or after being diluted to a desired solid content concentration.
- the solvent contained in the varnish include water, phosphate buffer (PBS), and alcohol.
- the concentration of the copolymer in the varnish is 0.01 to 4% by mass, more preferably 0.01 to 3% by mass, more preferably 0.01 to 2% by mass, and still more preferably 0.01 to 1% by mass. %.
- concentration of the copolymer is 0.01% by mass or less, a coating having a sufficient film thickness cannot be formed.
- the copolymer concentration is 4% by mass or more, the storage stability of the varnish is deteriorated, and precipitation of a dissolved substance or gel is caused. May occur.
- other substances can be added to the varnish as necessary in a range that does not impair the performance of the coating obtained.
- examples of other substances include preservatives, surfactants, primers that enhance adhesion to the substrate (container), fungicides, and sugars.
- the pH of the varnish containing the copolymer may be adjusted in advance.
- the pH adjustment may be carried out, for example, by adding a pH adjusting agent to the varnish containing the copolymer so that the varnish has a pH of 3.5 to 8.5, preferably 4.0 to 8.0.
- the kind and amount of the pH adjuster that can be used are appropriately selected according to the concentration of the copolymer in the varnish, the abundance ratio of the anion and cation of the copolymer, and the like.
- pH adjusters include ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, organic amines such as tris (hydroxymethyl) aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; Alkali metal halides such as potassium and sodium chloride; inorganic acids such as sulfuric acid, phosphoric acid, hydrochloric acid and carbonic acid or alkali metal salts thereof; quaternary ammonium cations such as choline; or a mixture thereof (for example, phosphate buffered saline) Etc.).
- alkali metal hydroxides such as potassium hydroxide and sodium hydroxide
- Alkali metal halides such as potassium and sodium chloride
- inorganic acids such as sulfuric acid, phosphoric acid, hydrochloric acid and carbonic acid or alkali metal salts thereof
- quaternary ammonium cations such as choline; or a mixture thereof (for example, phosphate buffered
- ammonia, diethanolamine, N-methyl-D-glucamine, tris (hydroxymethyl) aminomethane, sodium hydroxide and choline are preferable, and ammonia, diethanolamine, sodium hydroxide and choline are particularly preferable.
- a varnish containing such a copolymer is brought into contact with a container to form a coating on at least a part of its surface.
- the coating is desirably formed over the entire surface of the container.
- the surface of the container may be subjected to a known UV / ozone treatment and washed before the coating step.
- a cleaning step can be performed using a commercially available UV / ozone cleaner or the like.
- the coated container is dried at a temperature of -200 ° C to 200 ° C.
- the solvent in the composition for forming a coating film is removed by drying, and the formulas (a) and (b) of the copolymer according to the present invention form ionic bonds and are completely fixed to the substrate.
- the thickness of the coating of the blood filter of the present invention is preferably 10 to 1000 mm, more preferably 10 to 500 mm.
- the present inventors do not require a treatment at a high temperature, a coating having a desired property is formed on the surface of the container by a treatment at a low temperature, and several tens to It was found to be excellent in durability despite a thin film thickness of several hundred mm.
- Drying can be performed, for example, at room temperature (10 ° C. to 35 ° C., for example, 25 ° C.), but may be performed, for example, at 40 ° C. to 50 ° C. in order to form a coating film more quickly. Further, it may be carried out at an extremely low temperature to a low temperature (around ⁇ 200 ° C. to ⁇ 30 ° C.) by freeze drying. Freeze-drying is called vacuum freeze-drying, and is a method in which what is usually dried is cooled with a refrigerant and the solvent is removed by sublimation in a vacuum state.
- Common refrigerants used in freeze drying include dry ice and methanol mixed media ( ⁇ 78 ° C.), liquid nitrogen ( ⁇ 196 ° C.), and the like.
- a more preferable drying temperature is 10 ° C. to 180 ° C., and a more preferable drying temperature is 25 ° C. to 150 ° C.
- the surface of the coated container may be washed with alcohol having 1 to 5 carbon atoms such as ethanol and / or water.
- This washing step can be carried out at a temperature of 0 ° C. to 60 ° C., preferably 25 ° C. (room temperature) to 40 ° C. for 30 minutes to 48 hours, preferably 1 to 24 hours.
- the material is selected from the group consisting of water and an aqueous solution containing an electrolyte.
- the aqueous solution containing water or electrolyte may be heated in the range of 40 ° C. to 95 ° C., for example.
- the aqueous solution containing the electrolyte is preferably PBS and physiological saline (containing only sodium chloride), Dulbecco's phosphate buffered saline, Tris buffered saline, HEPES buffered saline and veronal buffered saline, and PBS is particularly preferred. preferable.
- the coating formed on the surface of the container does not elute and remains firmly adhered to the substrate (ie, the container).
- the formed coating has an ability to suppress adhesion of various biological materials including cells. Therefore, the cell culture container of the present invention is excellent in durability to solvents and radiation in addition to suppression of cell adhesion.
- a known sterilization treatment such as radiation, electron beam, ethylene oxide, or autoclave may be performed.
- the third aspect of the present invention is characterized by using the cell culture container of the present invention and the cell culture container obtained by the production method of the present invention (hereinafter, both are referred to as the “cell culture container of the present invention”). And a method for producing a cell aggregate.
- the cell culture container of the present invention is used, adhesion of cell aggregates to the surface (wall surface) of the container is suppressed and elution of the coating into the culture solution is suppressed, so that there is no irritation from the container. Cell aggregates can be cultured.
- the cell aggregate is preferably cultured using a medium containing a polysaccharide (in particular, deacylated gellan gum) having an effect of suspending cells or tissues in the cell culture container of the present invention.
- a polysaccharide in particular, deacylated gellan gum
- the specific composition and culture method of such a medium are disclosed in, for example, International Publication No. 2014/017513.
- ⁇ Measurement method of raw material composition The concentration (mass%) of each phosphorus-containing compound in the raw material containing the phosphorus-containing compound was measured by 31 P-NMR. The absolute concentration (absolute mass%) of each phosphorus-containing compound contained in the raw material was calculated using the following standard substance.
- a dropping funnel into which the mixed solution was introduced was set, and the mixed solution was dropped into a boiling liquid of pure water and ethanol in 0.5 hours.
- 506.05 g of a transparent polymerization liquid having a solid content of about 2% by mass was obtained by stirring with heating while maintaining the environment for 24 hours.
- the weight average molecular weight in GFC of the obtained transparent liquid was about 810,000.
- 0.9 g of pure water and 0.1 g of ethanol were added to 1.00 g of the copolymer-containing varnish, and the mixture was sufficiently stirred to prepare a surface treating agent L1 (solid content 1% by mass).
- Treatment 1 After the surface treatment agent (L1 or L2) prepared in Synthesis Example 1 or 2 was filtered using a filter having a mesh size of 0.22 ⁇ m, it was placed in a well of a 96-well cell culture plate (see the following test example). It added so that it might become 200 microliters (solid content 1 mass%) / well, and after leaving still at room temperature for 1 hour, the excess surface treating agent was removed.
- Treatment 2 It was dried overnight at 50 degrees using an oven (Advantech Toyo Co., Ltd., dryer FC-612).
- Treatment 3 ⁇ -ray sterilization ⁇ Method-1>: normal conditions Gas-impermeable packaging container with a chuck made of a coated 96-hole titer plate made of aluminum / PET laminate film with light-shielding, moisture-proof and oxygen barrier properties (product name; (Lamid Zip AL-22, produced by Nihon Production Co., Ltd.), sealed in an air atmosphere, left to stand for about 24 hours or more, and then sterilized by irradiation with 15 kGy of gamma rays.
- product name (Lamid Zip AL-22, produced by Nihon Production Co., Ltd.
- ⁇ Method-2> Vacuum condition Gas-impermeable packaging container with chuck made of aluminum / PET laminate film with light-shielding, moisture-proof and oxygen barrier properties on a coated 96-hole titer plate (Product name: Lamidip AL-22, production The product was placed in a Japan company), heat sealed under vacuum using a vacuum sealer, allowed to stand for about 24 hours or longer, and then sterilized by irradiation with 15 kGy of gamma rays.
- ⁇ Method-3> Antioxidant and desiccant bundled, vacuum conditions Coated 96-well titer plate, desiccant (product name; silica gel Mix, manufactured by Toyoda Chemical Co., Ltd.), and oxygen scavenger (product name; secur) AP-500 (manufactured by Nisso Fine Co., Ltd.) in a gas impermeable packaging container with a chuck made of an aluminum / PET laminate film with a light-shielding, moisture-proof, and oxygen barrier property (Product name: Lamidip AL-22, produced by Nippon Shokubai) The sample was heated and sealed under vacuum using a vacuum sealer, and allowed to stand for about 24 hours or more, and then sterilized by irradiation with 15 kGy of gamma rays. ;
- Treatment 4 After adding 200 ⁇ L of ultrapure water (Milli-Q water) that has passed through a filter having a mesh size of 0.22 ⁇ m to each well, the entire amount is removed. This treatment was performed 3 times.
- Example 1 Cell adhesion inhibitory effect 1 of cell culture plate coated with surface treatment agent> (Preparation of coating plate) A well of a flat bottom 96-well cell culture plate (BD Bioscience, # 351172) was coated with the surface treatment agent L1 or L2 by the coating method shown in Example 1. At this time, ⁇ Method-1> was used as the ⁇ -ray sterilization method. As a negative target, an uncoated plate was used. As a positive control sample, a commercially available cell low adhesion plate (Corning, # 3474) was used.
- Human embryonic kidney cell line Hek293 (DS Pharma Biomedical), human hepatoma cell line HepG2 (DS Pharma Biomedical), and mouse macrophage cell line RAW264.7 (DS Pharma Biomedical) were used as cells. .
- the culture medium used for culturing these cells was Hek293: EMEM medium containing 10% (v / v) FBS (manufactured by WAKO), HepG2: DMEM medium containing 10% (v / v) FBS (manufactured by WAKO), RAW264.7: DMEM / F-12 medium (Sigma) containing 10% (v / v) FBS was used.
- the cells were statically cultured for 2 days or more using a petri dish (10 mL of medium) having a diameter of 10 cm while maintaining a 5% carbon dioxide concentration in a 37 ° C. CO 2 incubator. Subsequently, the cells were washed with 5 ml of PBS, 1 ml of trypsin-EDTA solution (manufactured by Invitrogen) was added, the cells were detached, and suspended in 10 ml of the above medium. After centrifuging this suspension (manufactured by Kubota Corporation, model number 5900, 1500 rpm / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.
- Cell attachment experiment To the plate prepared above, 100 ⁇ L of each cell suspension was added so as to be 2 ⁇ 10 4 cells / well. Thereafter, the sample was allowed to stand in a CO 2 incubator at 37 ° C. for 4 days while maintaining a 5% carbon dioxide concentration.
- Example 2 Cell adhesion inhibitory effect 2 of cell culture plate coated with surface treatment agent> (Preparation of coating plate)
- the wells of a flat bottom 96-well cell culture plate (BD Bioscience, # 351172) were coated with the surface treatment agent L1 by the coating method shown in Example 1.
- ⁇ Method-1>, ⁇ Method-2>, and ⁇ Method-3> were used as the ⁇ -ray sterilization method.
- As a negative target an uncoated plate was used.
- a human fetal kidney cell line Hek293 (manufactured by DS Pharma Biomedical) was used.
- an EMEM medium (manufactured by WAKO) containing 10% (v / v) FBS was used.
- the cells were statically cultured for 2 days or more using a petri dish (10 mL of medium) having a diameter of 10 cm while maintaining a 5% carbon dioxide concentration in a 37 ° C. CO 2 incubator.
- the cells were washed with 5 ml of PBS, 1 ml of trypsin-EDTA solution (manufactured by Invitrogen) was added, the cells were detached, and suspended in 10 ml of the above medium. After centrifuging this suspension (manufactured by Kubota Corporation, model number 5900, 1500 rpm / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.
- trypsin-EDTA solution manufactured by Invitrogen
- Example 3 Cell adhesion inhibitory effect 3 of cell culture plate coated with surface treatment agent> (Preparation of coating plate)
- the well of a U-shaped 96-well cell culture plate (BD Bioscience, # 353227) was coated with the surface treatment agent L1 or L2 by the coating method shown in Example 1. At this time, ⁇ -ray sterilization was not performed.
- As a negative target an uncoated plate was used.
- As a positive control sample a commercially available cell low adhesion plate (manufactured by Sumitomo Bakelite Co., Ltd., # MS-9096U) was used.
- hepatoma cell line HepG2 (DS Pharma Biomedical) was used.
- DMEM medium manufactured by WAKO
- the cells were statically cultured for 2 days or more using a petri dish (10 mL of medium) having a diameter of 10 cm while maintaining a 5% carbon dioxide concentration in a 37 ° C. CO 2 incubator. Subsequently, the cells were washed with 5 ml of PBS, 1 ml of trypsin-EDTA solution (manufactured by Invitrogen) was added, the cells were detached, and suspended in 10 ml of the above medium.
- the mixture was dissolved by stirring while heating, and the aqueous solution was autoclaved at 121 ° C. for 20 minutes.
- This solution was added to a DMEM medium (manufactured by WAKO) containing 10% (v / v) FBS while stirring well so as to be a deacylated gellan gum having a final concentration of 0.015% (w / v).
- the cell culture container of the present invention is coated on at least a part of its surface with a copolymer containing a specific organic group.
- a coating suppresses adhesion of cells to the container surface (wall surface), and the coating can firmly adhere to the container surface, so that elution of the coating into the culture medium and radiation resistance are improved. Therefore, the cell culture container of the present invention is advantageous in that cell aggregates can be cultured without any stimulation from the container.
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Abstract
Description
1.下記式(a)で表される有機基を含む繰り返し単位と、下記式(b)で表される有機基を含む繰り返し単位とを含む共重合体:
2.上記式(a)及び(b)で表される有機基を含む繰り返し単位が、それぞれ、下記式(A)及び(B):
3.mが1であり、Ra及びRbが、それぞれ独立して、エチレン基又はプロピレン基を表す、上記2に記載の細胞培養容器;
4.上記共重合体が、さらに下記式(C)又は(D):
5.Tc及びTdが、それぞれ独立して、水素原子又はメチル基を表し、Udが、水素原子を表し、Rc及びRdが、それぞれ独立して、エチレン基又はプロピレン基を表す、上記4に記載の細胞培養容器;
6.下記式(a)で表される有機基を含む繰り返し単位と、下記式(b)で表される有機基を含む繰り返し単位とを含む共重合体:
コーティングされた容器を-200℃乃至200℃にて乾燥させる工程
を含む、細胞培養容器の製造方法;
8.上記式(a)及び(b)で表される有機基を含む繰り返し単位が、それぞれ、下記式(A)及び(B):
8.コーティング工程が、共重合体を含むワニスを用いて実施される、上記6又は7に記載の製造方法;
9.共重合体を含むワニスが、予めpH調整されている、上記8に記載の製造方法;
10.さらに、コーティングされた細胞培養容器を、乾燥工程前及び/又は後に、洗浄する工程を含む、上記6乃至9何れかに記載の製造方法;
11.乾燥工程後の洗浄が、水及び電解質を含む水溶液からなる群より選ばれる少なくとも1種の溶媒を用いて実施される、上記10に記載の製造方法;
12.さらに、コーティングされた細胞培養容器を、乾燥後に、放射線処理にて滅菌する工程を含む、上記6乃至11何れかに記載の製造方法;
13.上記1乃至5何れかに記載の細胞培養容器又は上記6乃至12何れかに記載の製造方法により製造された細胞培養容器を用いることを特徴とする、細胞凝集塊の製造方法;
14.細胞培養容器中の培地が、ナノファイバー形成し細胞又は組織を浮遊させる効果を有する多糖類を含むことを特徴とする、上記13に記載の細胞凝集塊の製造方法;
15.多糖類が脱アシル化ジェランガムであることを特徴とする、上記14に記載の細胞凝集塊の製造方法。
本発明における細胞とは、動物或いは植物を構成する最も基本的な単位であり、その要素として細胞膜の内部に細胞質と各種の細胞小器官をもつものである。この際、DNAを内包する核は、細胞内部に含まれても含まれなくてもよい。例えば、本発明における動物由来の細胞には、***や卵子などの生殖細胞、生体を構成する体細胞、幹細胞(多能性幹細胞等)、前駆細胞、生体から分離された癌細胞、生体から分離され不死化能を獲得して体外で安定して維持される細胞(細胞株)、生体から分離され人為的に遺伝子改変が成された細胞、生体から分離され人為的に核が交換された細胞等が含まれる。生体を構成する体細胞の例としては、以下に限定されるものではないが、線維芽細胞、骨髄細胞、Bリンパ球、Tリンパ球、好中球、赤血球、血小板、マクロファージ、単球、骨細胞、骨髄細胞、周皮細胞、樹状細胞、ケラチノサイト、脂肪細胞、間葉細胞、上皮細胞、表皮細胞、内皮細胞、血管内皮細胞、肝実質細胞、軟骨細胞、卵丘細胞、神経系細胞、グリア細胞、ニューロン、オリゴデンドロサイト、マイクログリア、星状膠細胞、心臓細胞、食道細胞、筋肉細胞(たとえば、平滑筋細胞又は骨格筋細胞)、膵臓ベータ細胞、メラニン細胞、造血前駆細胞(例えば、臍帯血由来のCD34陽性細胞)、及び単核細胞等が含まれる。当該体細胞は、例えば皮膚、腎臓、脾臓、副腎、肝臓、肺、卵巣、膵臓、子宮、胃、結腸、小腸、大腸、膀胱、前立腺、精巣、胸腺、筋肉、結合組織、骨、軟骨、血管組織、血液(臍帯血を含む)、骨髄、心臓、眼、脳又は神経組織などの任意の組織から採取される細胞が含まれる。幹細胞とは、自分自身を複製する能力と他の複数系統の細胞に分化する能力を兼ね備えた細胞であり、その例としては、以下に限定されるものではないが、胚性幹細胞(ES細胞)、胚性腫瘍細胞、胚性生殖幹細胞、人工多能性幹細胞(iPS細胞)、神経幹細胞、造血幹細胞、間葉系幹細胞、肝幹細胞、膵幹細胞、筋幹細胞、生殖幹細胞、腸幹細胞、癌幹細胞、毛包幹細胞などが含まれる。多能性幹細胞としては、前記幹細胞のうち、ES細胞、胚性生殖幹細胞、iPS細胞が挙げられる。前駆細胞とは、前記幹細胞から特定の体細胞や生殖細胞に分化する途中の段階にある細胞である。癌細胞とは、体細胞から派生して無限の増殖能を獲得した細胞である。細胞株とは、生体外での人為的な操作により無限の増殖能を獲得した細胞である。
がん細胞株の例としては、以下に限定されるものではないが、ヒト乳がん細胞株としてHBC-4、BSY-1、BSY-2、MCF-7、MCF-7/ADR RES、HS578T、MDA-MB-231、MDA-MB-435、MDA-N、BT-549、T47D、ヒト子宮頸がん細胞株としてHeLa、ヒト肺がん細胞株としてA549、EKVX、HOP-62、HOP-92、NCI-H23、NCI-H226、NCI-H322M、NCI-H460、NCI-H522、DMS273、DMS114、ヒト大腸がん細胞株としてCaco-2、COLO-205、HCC-2998、HCT-15、HCT-116、HT-29、KM-12、SW-620、WiDr、ヒト前立腺がん細胞株としてDU-145、PC-3、LNCaP、ヒト中枢神経系がん細胞株としてU251、SF-295、SF-539、SF-268、SNB-75、SNB-78、SNB-19、ヒト卵巣がん細胞株としてOVCAR-3、OVCAR-4、OVCAR-5、OVCAR-8、SK-OV-3、IGROV-1、ヒト腎がん細胞株としてRXF-631L、ACHN、UO-31、SN-12C、A498、CAKI-1、RXF-393L、786-0、TK-10、ヒト胃がん細胞株としてMKN45、MKN28、St-4、MKN-1、MKN-7、MKN-74、皮膚がん細胞株としてLOX-IMVI、LOX、MALME-3M、SK-MEL-2、SK-MEL-5、SK-MEL-28、UACC-62、UACC-257、M14、白血病細胞株としてCCRF-CRM、K562、MOLT-4、HL-60TB、RPMI8226、SR、UT7/TPO、Jurkat等が挙げられる。細胞株の例としては、以下に限定されるものではないが、HEK293(ヒト胎児腎細胞)、MDCK、MDBK、BHK、C-33A、AE-1、3D9、Ns0/1、NIH3T3、PC12、S2、Sf9、Sf21、High Five(登録商標)、Vero等が含まれる。肝細胞株の例としては、以下に限定されるものではないが、HepG2、Hep3B、HepaRG(登録商標)、JHH7、HLF、HLE、PLC/PRF/5、WRL68、HB611、SK-HEP-1、HuH-4、HuH-7等が挙げられる。
本発明の細胞培養容器を構成する、共重合体がコーティングされる容器は、当技術分野で使用され得る任意の形態の容器類であればよく、例えば、細胞の培養に一般的に用いられるシャーレ、フラスコ、プラスチックバック、テフロン(登録商標)バック、ディッシュ、ペトリデッシュ、組織培養用ディッシュ、マルチディッシュ、マイクロプレート、マイクロウエルプレート、マルチプレート、マルチウエルプレート、チャンバースライド、細胞培養フラスコ、スピナーフラスコ、チューブ、トレイ、培養バック、ローラーボトル等が挙げられる。好ましくは、6~1536穴のマルチウェルプレート及びシャーレが挙げられる。
本発明の細胞培養容器は、容器の表面の少なくとも一部が、特定の共重合体でコーティングされていることを特徴とする。本発明に係る共重合体は、下記式(a)で表される有機基を含む繰り返し単位と、下記式(b)で表される有機基を含む繰り返し単位とを含む共重合体:
本発明において、「ハロゲン化物イオン」は、ハロゲン原子のアニオンを意味し、フッ化物イオン、塩化物イオン、臭化物イオン、ヨウ化物イオンが挙げられ、好ましくは、塩化物イオンである。
本発明において、「無機酸イオン」とは、炭酸イオン、硫酸イオン、リン酸イオン、リン酸水素イオン、リン酸二水素イオン、硝酸イオン、過塩素酸イオン又はホウ酸イオンを意味する。
上記An-として好ましいのは、ハロゲン化物イオン、硫酸イオン、リン酸イオン、水酸化物イオン及びイソチオシアネートイオンであり、特に好ましいのはハロゲン化物イオンである。
上記共重合体中における第三成分、例えば、上記式(C)又は(D)で表される2官能性モノマーから誘導される架橋構造の割合は、0モル%乃至50モル%である。
本発明に係る共重合体の合成方法としては、一般的なアクリルポリマー又はメタクリルポリマー等の合成方法であるラジカル重合、アニオン重合、カチオン重合などの方法により合成することができる。その形態は溶液重合、懸濁重合、乳化重合、塊状重合など種々の方法が可能である。
本発明の第二の態様は、下記式(a)で表される有機基を含む繰り返し単位と、下記式(b)で表される有機基を含む繰り返し単位とを含む共重合体:
コーティングされた容器を-200℃乃至200℃にて乾燥させる工程
を含む、細胞培養容器の製造方法である。
本発明の細胞培養容器の製造方法のコーティング工程では、共重合体を、容器の表面の少なくとも一部にコーティングする。例えば、容器が丸底のマルチウェルプレートである場合、ウェルのくぼみの部分のみをコーティングしてもよいが、プレート全体をコーティングしてもよい。ここで、容器及び共重合体は、それぞれ前述の項目<容器>及び<共重合体>で述べた通りである。
コーティング工程後、コーティングされた容器を-200℃乃至200℃の温度にて乾燥させる。乾燥により、上記コーティング膜形成用組成物中の溶媒を取り除くと共に、本発明に係る共重合体の式(a)及び式(b)同士がイオン結合を形成して基体へ完全に固着する。本発明の血液フィルターのコーティングの膜厚は、好ましくは10~1000Åであり、さらに好ましくは10~500Åである。本発明者らは、本発明の細胞培養容器の製造方法では、高温での処理を要さず、低温での処理により、所望の特性を有するコーティングが容器の表面に形成され、しかも数十乃至数百Å程度の薄い膜厚にも係らず、耐久性に優れることを見出した。
本発明の第三の態様は、本発明の細胞培養容器及び本発明の製造方法により得られた細胞培養容器(以下、両者を合わせて「本発明の細胞培養容器」という)を用いることを特徴とする、細胞凝集塊の製造方法である。本発明の細胞培養容器を用いると、細胞凝集塊の容器の表面(壁面)への付着が抑制され、かつコーティングの培養液への溶出が抑制されることから、容器からの刺激のない状態で細胞凝集塊を培養することができる。細胞凝集塊は、好ましくは、本発明の細胞培養容器中で細胞又は組織を浮遊させる効果を有する多糖類(特に、脱アシル化ジェランガム)を含むことを特徴とする培地を用いて培養される。そのような培地の具体的な組成や、培養方法は、例えば、国際公開第2014/017513号公報に開示されている。
リン含有化合物を含む原料の、各リン含有化合物の濃度(質量%)測定は、31P-NMRにより行った。下記標準物質を用いて原料中に含まれる各リン含有化合物の絶対濃度(絶対質量%)を算出した。
・モード:逆ゲートデカップリングモード(定量モード)
・装置:varian 400 MHz
・溶媒:CD3OD(重メタノール)(30重量%)
・回転数:0 Hz
・データポイント:64000
・フリップ角:90°
・待ち時間:70 s
・積算回数:16回,n=4,
・標準物質:トリメチルリン酸+D2O (75%TMP溶液を調製)
アシッドホスホオキシエチルメタアクリレート(製品名;ホスマーM、ユニケミカル社製、乾固法100℃・1時間における不揮発分:91.8%、アシッドホスホオキシエチルメタクリレート(44.2質量%)、リン酸ビス[2-(メタクリロイルオキシ)エチル](28.6質量%)、その他の物質(27.2質量%)の混合物)6.00gに純水12.40gを加え十分に溶解し、エタノール12.40g、メタクリル酸2-(ジメチルアミノ)エチル4.12g(東京化成工業(株)社製)、2、2’-アゾ(2-メチル-N-(2-ヒドロキシエチル)プロピオンアミド)(製品名;VA-086、和光純薬社製)0.10gを20℃以下に保ちながら順に加え滴下ロートに導入した。一方で、純水471.13g、エタノール37.20gを冷却管付きの3つ口フラスコに加えて窒素フローし、撹拌しながらリフラックス温度まで昇温した。この状態を維持しつつ、混合液を導入した滴下ロートをセットし、0.5時間で混合液を純水とエタノールの沸騰液内に滴下した。滴下後、24時間環境を維持した状態で加熱撹拌することで固形分約2質量%の透明重合液506.05gを得た。得られた透明液体のGFCにおける重量平均分子量は約810,000であった。その後、共重合体含有ワニス1.00gに、純水0.9g、エタノール0.1gを加えて十分に攪拌し、表面処理剤L1(固形分1質量%)を調製した。
アシッドホスホオキシエチルメタアクリレート(製品名;ホスマーM、ユニケミカル(株)社製、乾固法100℃・1時間における不揮発分:91.8%、アシッドホスホオキシエチルメタクリレート(44.2質量%)、リン酸ビス[2-(メタクリロイルオキシ)エチル](28.6質量%)、その他の物質(27.2質量%)の混合物)10.00gに純水68.88gを加え十分に溶解し、エタノール29.52g、メタクリル酸2-(ジメチルアミノ)エチル7.63g(東京化成工業(株)社製)、2,2’-アゾビス[N-(2―カルボキシエチル)-2-メチルプロピオンアミジン](製品名;VA-057、和光純薬工業(株)社製)0.09gを20℃以下に保ちながら順に加え滴下ロートに導入した。一方で、純水373.89g、エタノール29.52gを冷却管付きの3つ口フラスコに加えて窒素フローし、撹拌しながらリフラックス温度まで昇温した。この状態を維持しつつ、混合液を導入した滴下ロートをセットし、0.5時間で混合液を純水とエタノールの沸騰液内に滴下した。滴下後、24時間環境を維持した状態で加熱撹拌することで固形分約3.5質量%の透明重合液509.60gを得た。得られた透明液体のGFCにおける重量平均分子量は約280,000であった。その後、共重合体含有ワニス1.00gに、純水1.56g、エタノール0.94gを加えて十分に攪拌し、表面処理剤L2(固形分1質量%)を調製した。
以下の各処理工程を順番に実施し、本発明における細胞培養容器を調製した。
処理1:合成例1或いは2で作成した表面処理剤(L1又はL2)を、メッシュサイズが0.22μmのフィルターを用いてろ過した後、96穴細胞培養プレート(下記試験例参照)のウェルに200μL(固形分1質量%)/ウェルとなるよう添加し、室温にて1時間静置後、余剰の表面処理剤を除去した。
<方法-1>:通常条件
コーティング済みの96穴タイタープレートを遮光・防湿・酸素バリヤー性を持つアルミ/PETラミネートフィルムからなるチャック付ガス不透過性包装容器(製品名;ラミジップAL-22、生産日本社製)に入れ、大気雰囲気下で密封し、約24時間以上放置してからガンマ線を15kGy照射し滅菌を行った。
<方法-2>:真空条件
コーティング済みの96穴タイタープレートを遮光・防湿・酸素バリヤー性を持つアルミ/PETラミネートフィルムからなるチャック付ガス不透過性包装容器(製品名;ラミジップAL-22、生産日本社製)に入れ、バキュームシーラーを用いて真空下で加熱密封し、約24時間以上放置してからガンマ線を15kGy照射し滅菌を行った。
<方法-3>:酸化防止剤・乾燥剤同梱・真空条件
コーティング済みの96穴タイタープレートと、乾燥剤(製品名;シリカゲルMix、豊田化工社製)と、脱酸素剤(製品名;セキュールAP-500、ニッソーファイン社製)を遮光・防湿・酸素バリヤー性を持つアルミ/PETラミネートフィルムからなるチャック付ガス不透過性包装容器(製品名;ラミジップAL-22、生産日本社製)に入れ、バキュームシーラーを用いて真空下で加熱密封し、約24時間以上放置してからガンマ線を15kGy照射し滅菌を行った。;
(コーティングプレートの調製)
実施例1に示したコーティング法により平底96穴細胞培養プレート(BDバイオサイエンス社製、#351172)のウェルを、表面処理剤L1又はL2にてコーティングした。この際、γ線滅菌方法は<方法-1>を用いた。陰性対象としては、コーティング処理をしていないプレートを用いた。陽性対照のサンプルとしては、市販の細胞低接着プレート(コーニング社製、#3474)を用いた。
細胞は、ヒト胎児腎細胞株Hek293(DSファーマバイオメディカル社製)、ヒト肝癌細胞株HepG2(DSファーマバイオメディカル社製)、マウスマクロファージ細胞株RAW264.7(DSファーマバイオメディカル社製)を用いた。
それら細胞の培養に用いた培地は、Hek293:10%(v/v)FBSを含むEMEM培地(WAKO社製)、HepG2:10%(v/v)FBSを含むDMEM培地(WAKO社製)、RAW264.7:10%(v/v)FBSを含むDMEM/F-12培地(シグマ社製)を用いた。細胞は、37℃CO2インキュベーター内にて5%二酸化炭素濃度を保った状態で、直径10cmのシャーレ(培地10mL)を用いて2日間以上静置培養した。引き続き、本細胞をPBS5mlで洗浄した後、トリプシン-EDTA溶液(インビトロジェン社製)1mLを添加して細胞を剥がし、上記の培地10mLにてそれぞれ懸濁した。本懸濁液を遠心分離(久保田商事株式会社製、型番5900、1500rpm/3分、室温)後、上清を除き、上記の培地を添加して細胞懸濁液を調製した。
上記にて調製したプレートに対して、それぞれの細胞懸濁液を2×104cells/wellとなるように各100μL加えた。その後、5%二酸化炭素濃度を保った状態で、37℃で4日間CO2インキュベーター内にて静置した。
培養4日間後、表面処理剤L1又はL2にてコーティングした平底96穴タイタープレート、陰性対照、陽性対照に対する細胞の付着を倒立型顕微鏡(オリンパス社製、CKX31)による観察に基づき比較した。その結果を下記表1に示す。また、細胞観察の代表例として表面処理剤L1でコーティングしたプレートにて培養したHepG2細胞の顕微鏡写真(倍率:40倍)を図1に示す。
(コーティングプレートの調製)
実施例1に示したコーティング法により平底96穴細胞培養プレート(BDバイオサイエンス社製、#351172)のウェルを、表面処理剤L1にてコーティングした。この際、γ線滅菌方法は<方法-1><方法-2><方法-3>を用いた。陰性対象としては、コーティング処理をしていないプレートを用いた。
細胞は、ヒト胎児腎細胞株Hek293(DSファーマバイオメディカル社製)を用いた。本細胞の培養には、10%(v/v)FBSを含むEMEM培地(WAKO社製)を用いた。細胞は、37℃CO2インキュベーター内にて5%二酸化炭素濃度を保った状態で、直径10cmのシャーレ(培地10mL)を用いて2日間以上静置培養した。引き続き、本細胞をPBS5mlで洗浄した後、トリプシン-EDTA溶液(インビトロジェン社製)1mLを添加して細胞を剥がし、上記の培地10mLにてそれぞれ懸濁した。本懸濁液を遠心分離(久保田商事株式会社製、型番5900、1500rpm/3分、室温)後、上清を除き、上記の培地を添加して細胞懸濁液を調製した。
上記にて調製したプレートに対して、Hek293細胞懸濁液を2×104cells/wellとなるように各100μL加えた。その後、5%二酸化炭素濃度を保った状態で、37℃で4日間CO2インキュベーター内にて静置した。
培養4日間後、表面処理剤L1にてコーティングした平底96穴タイタープレート及び陰性対照に対する細胞の付着を倒立型顕微鏡(オリンパス社製、CKX31)による観察に基づき比較した。その結果を下記表2に示す。
(コーティングプレートの調製)
実施例1に示したコーティング法によりU字底96穴細胞培養プレート(BDバイオサイエンス社製、#353227)のウェルを、表面処理剤L1又はL2にてコーティングした。この際、γ線滅菌は実施しなかった。陰性対象としては、コーティング処理をしていないプレートを用いた。陽性対照のサンプルとしては、市販の細胞低接着プレート(住友ベークライト社製、#MS-9096U)を用いた。
細胞は、ヒト肝癌細胞株HepG2(DSファーマバイオメディカル社製)を用いた。本細胞の培養には、10%(v/v)FBSを含むDMEM培地(WAKO社製)を用いた。細胞は、37℃CO2インキュベーター内にて5%二酸化炭素濃度を保った状態で、直径10cmのシャーレ(培地10mL)を用いて2日間以上静置培養した。引き続き、本細胞をPBS5mlで洗浄した後、トリプシン-EDTA溶液(インビトロジェン社製)1mLを添加して細胞を剥がし、上記の培地10mLにてそれぞれ懸濁した。本懸濁液を遠心分離(久保田商事株式会社製、型番5900、1500rpm/3分、室温)後、上清を除き、上記の培地或いは終濃度0.015%(w/v)の脱アシル化ジェランガムを添加した培地を用いて細胞懸濁液をそれぞれ調製した。ここで、0.015%(w/v)の脱アシルジェランガムを添加した培地は以下の様に調製した。すなわち、脱アシル化ジェランガム(KELCOGEL CG-LA、三晶株式会社製)を0.3%(w/v)となるように超純水(Milli-Q水)に懸濁した後、90℃にて加熱しながらの撹拌により溶解し、本水溶液を121℃で20分オートクレーブ滅菌した。本溶液を10%(v/v)FBSを含むDMEM培地(WAKO社製)に終濃度0.015%(w/v)の脱アシル化ジェランガムとなるようによく撹拌しながら添加した。
上記にて調製したプレートに対して、それぞれの細胞懸濁液を2×103cells/wellとなるように各100μL加えた。その後、5%二酸化炭素濃度を保った状態で、37℃で5日間CO2インキュベーター内にて静置した。
培養5日間後、表面処理剤L1又はL2にてコーティングしたU字底96穴タイタープレート、陰性対照、陽性対照に対する細胞の付着を倒立型顕微鏡(オリンパス社製、CKX31)による観察に基づき比較した。その結果を下記表3に示す。また、細胞観察の代表例として表面処理剤L1にてコーティングしたプレートにて培養したHepG2細胞の顕微鏡写真(倍率:40倍)を図2に示す。
Claims (14)
- 上記式(a)及び(b)で表される有機基を含む繰り返し単位が、それぞれ、下記式(A)及び(B):
Ta、Tb、Ua1、Ua2、Ub1、Ub2及びUb3は、それぞれ独立して、水素原子又は炭素原子数1乃至5の直鎖若しくは分岐アルキル基を表し、Qa及びQbは、それぞれ独立して、単結合、エステル結合又はアミド結合を表し、Ra及びRbは、それぞれ独立して、ハロゲン原子で置換されていてもよい炭素原子数1乃至10の直鎖又は分岐アルキレン基を表し、An-は、ハロゲン化物イオン、無機酸イオン、水酸化物イオン及びイソチオシアネートイオンからなる群から選ばれる陰イオンを表し、そしてmは、0乃至6の整数を表す
で表されるモノマーから誘導される繰り返し単位である、請求項1に記載の細胞培養容器。 - mが1であり、Ra及びRbが、それぞれ独立して、エチレン基又はプロピレン基を表す、請求項2に記載の細胞培養容器。
- Tc及びTdが、それぞれ独立して、水素原子又はメチル基を表し、Udが、水素原子を表し、Rc及びRdが、それぞれ独立して、エチレン基又はプロピレン基を表す、請求項4に記載の細胞培養容器。
- 上記式(a)及び(b)で表される有機基を含む繰り返し単位が、それぞれ、下記式(A)及び(B):
Ta、Tb、Ua1、Ua2、Ub1、Ub2及びUb3は、それぞれ独立して、水素原子又は炭素原子数1乃至5の直鎖若しくは分岐アルキル基を表し、Qa及びQbは、それぞれ独立して、単結合、エステル結合又はアミド結合を表し、Ra及びRbは、それぞれ独立して、ハロゲン原子で置換されていてもよい炭素原子数1乃至10の直鎖又は分岐アルキレン基を表し、An-は、ハロゲン化物イオン、無機酸イオン、水酸化物イオン及びイソチオシアネートイオンからなる群から選ばれる陰イオンを表し、そしてmは、0乃至6の整数を表す
で表されるモノマーから誘導される繰り返し単位である、請求項6に記載の製造方法。 - コーティング工程が、共重合体を含むワニスを用いて実施される、請求項6又は7に記載の製造方法。
- 共重合体を含むワニスが、予めpH調整されている、請求項8に記載の製造方法。
- さらに、コーティングされた細胞培養容器を、乾燥工程前及び/又は後に、洗浄する工程を含む、請求項6乃至9何れか1項に記載の製造方法。
- 乾燥工程後の洗浄が、水及び電解質を含む水溶液からなる群より選ばれる少なくとも1種の溶媒を用いて実施される、請求項10に記載の製造方法。
- 請求項1乃至5何れか1項に記載の細胞培養容器又は請求項6乃至11何れか1項に記載の製造方法により製造された細胞培養容器を用いることを特徴とする、細胞凝集塊の製造方法。
- 細胞培養容器中の培地が、細胞又は組織を浮遊させる効果を有する多糖類を含むことを特徴とする、請求項12に記載の細胞凝集塊の製造方法。
- 多糖類が脱アシル化ジェランガムであることを特徴とする、請求項13に記載の細胞凝集塊の製造方法。
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