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WO2024203650A1 - Cell culturing container - Google Patents

Cell culturing container Download PDF

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Publication number
WO2024203650A1
WO2024203650A1 PCT/JP2024/010834 JP2024010834W WO2024203650A1 WO 2024203650 A1 WO2024203650 A1 WO 2024203650A1 JP 2024010834 W JP2024010834 W JP 2024010834W WO 2024203650 A1 WO2024203650 A1 WO 2024203650A1
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WO
WIPO (PCT)
Prior art keywords
culture surface
cell culture
culture vessel
band
ratio
Prior art date
Application number
PCT/JP2024/010834
Other languages
French (fr)
Japanese (ja)
Inventor
慧 足達
千裕 鍋屋
Original Assignee
日本ゼオン株式会社
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Filing date
Publication date
Application filed by 日本ゼオン株式会社 filed Critical 日本ゼオン株式会社
Publication of WO2024203650A1 publication Critical patent/WO2024203650A1/en

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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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus

Definitions

  • the present invention relates to a cell culture vessel.
  • culture medium a liquid for culturing cells that contains nutrients necessary for the cells, such as serum
  • cultured cells are broadly divided into adherent cells and suspension cells.
  • Adherent cells grow while attached to the surface of the cell culture vessel via binding between integrins present on the cell membrane surface and extracellular matrix proteins such as fibronectin attached to the surface of the cell culture vessel.
  • Patent Document 1 discloses a polymer support having a contact surface and an interior, the contact surface having a roughness quantified by a predetermined parameter, and according to Patent Document 1, the contact surface of the polymer support has improved cell adhesion, cell proliferation, and cell recovery rate.
  • the present invention aims to provide a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.
  • the present inventors have conducted extensive research to solve the above problems, and have newly discovered that the denaturation of proteins attached to the culture surface can be effectively suppressed by using a cell culture vessel that is provided with a culture surface made of a resin composition, and in which, when the amide I region (1600 cm -1 to 1700 cm -1 ) of the infrared absorption spectrum of a recombinant human fibronectin fragment attached to the culture surface is band-decomposed, the ratio of the area of the band having a peak at 1680 cm -1 to the total area of all bands is equal to or greater than a predetermined value, thereby completing the present invention.
  • the present invention aims to advantageously solve the above problems, and provides the following cell culture vessels (1) to (4).
  • the "culture surface” refers to the surface of a cell culture vessel with which cells are in contact during culture.
  • the term "recombinant human fibronectin fragment” refers to a recombinant protein (recombinant human fibronectin CH-296) containing three functional domains, namely, the cell adhesion domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 site of human fibronectin.
  • "Recombinant human fibronectin CH-296” is commercially available as Rectronectin (registered trademark, manufactured by Takara Bio Inc.), and its amino acid sequence is disclosed as SEQ ID NO: 18 in WO 2018/021543.
  • the "infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface” can be measured by the method described in the Examples of this specification. Furthermore, in the present invention, the "area ratio of the 1680 cm -1 band” can be calculated according to the method described in the Examples of this specification.
  • the X-ray photoelectron spectroscopy spectrum (hereinafter sometimes abbreviated as "XPS spectrum") of the culture surface does not have a peak derived from a ⁇ - ⁇ * bond and the ratio of the total amount of C ⁇ O groups and O—C ⁇ O groups to the amount of C—O groups on the culture surface (hereinafter sometimes abbreviated as “abundance ratio of oxygen-containing functional groups”) is equal to or less than the above value, the denaturation of proteins attached to the culture surface can be further suppressed.
  • the "XPS spectrum of the culture surface” can be measured by the method described in the Examples of this specification.
  • the "peak derived from a ⁇ - ⁇ * bond” means a ( ⁇ - ⁇ * ) satellite peak observed in the vicinity of 291 eV in the XPS spectrum.
  • the "abundance ratio of oxygen-containing functional groups” can be calculated according to the method described in the Examples of this specification.
  • a cell culture vessel according to (1) or (2) above in which the ratio of the amount of oxygen element to the amount of carbon element on the culture surface, as obtained based on an X-ray photoelectron spectroscopy spectrum, is 2.0% or more.
  • a cell culture vessel having a ratio of the amount of oxygen element to the amount of carbon element on the culture surface equal to or greater than the above value can enhance the adsorptivity of proteins to the culture surface.
  • the "ratio of the amount of oxygen element to the amount of carbon element on the culture surface" can be measured according to the method described in the Examples of this specification.
  • the present invention provides a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.
  • the cell culture vessel of the present invention is not particularly limited as long as it is intended to culture cells therein.
  • Specific examples of the cell culture vessel of the present invention include dishes, plates, microchannel chips, bags, tubes, scaffolds, cups, jar fermenters, etc.
  • the cells to be contained in the cell culture vessel are not particularly limited and can be selected arbitrarily depending on the purpose.
  • Specific examples of the cells include adherent cells such as CHO cells, VERO cells, NIH3T3 cells, HEK293 cells, nerve cells differentiated from human iPS cells, cardiomyocytes, and liver cells.
  • adherent cells such as CHO cells, VERO cells, NIH3T3 cells, HEK293 cells, nerve cells differentiated from human iPS cells, cardiomyocytes, and liver cells.
  • the cells may be used alone or in combination of two or more types in any ratio.
  • Cell culture vessel At least the culture surface of the cell culture vessel of the present invention is formed using a resin composition containing a resin.
  • a resin composition containing a resin For example, when the cell culture vessel of the present invention is a multi-well plate, the part of the well inner wall surface that contacts the cells being cultured may be formed using the resin composition.
  • the cell culture vessel of the present invention is a bag having a laminated structure made of multiple films, the innermost film (the inner surface of the bag) may be formed using the resin composition. Alternatively, the entire cell culture vessel may be formed using the resin composition.
  • the cell culture vessel of the present invention is characterized in that the area ratio of the 1680 cm -1 band in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is 10% or more.
  • the area ratio of the 1680 cm band of the recombinant human fibronectin fragment attached to the culture surface is 10% or more, so that denaturation of the protein attached to the culture surface is suppressed.
  • the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is band-resolved in the frequency region of 1600 cm -1 to 1700 cm -1 , the ratio of the area of the band having a peak at a frequency of 1680 cm -1 to the total area of all bands (area ratio of the 1680 cm -1 band) is 10% or more.
  • the region with a frequency of 1600 to 1700 cm ⁇ 1 is called the amide I region, and is known to reflect the secondary structure of the protein.
  • the present inventors have newly discovered that in the infrared absorption spectrum of a recombinant human fibronectin fragment, a band having a peak at 1680 cm ⁇ 1 is assigned to a ⁇ -sheet structure. Therefore, the “area ratio of the 1680 cm ⁇ 1 band” can be regarded as a value related to the content (%) of ⁇ -sheet in the recombinant human fibronectin fragment molecule. In addition, since the area ratio of the 1680 cm ⁇ 1 band of the cell culture vessel of the present invention is 10% or more, it can be said that the recombinant human fibronectin fragment attached to the culture surface maintains a structure rich in ⁇ -sheet structure, that is, a structure close to the native state.
  • the area ratio of the 1680 cm ⁇ 1 band of the recombinant human fibronectin fragment attached to the culture surface of the cell culture vessel of the present invention is 10% or more, it is considered that the denaturation of the protein attached to the culture surface is sufficiently suppressed. Therefore, by using the cell culture vessel of the present invention, it is possible to suppress the denaturation of proteins attached to the culture surface.
  • the resin composition used to form the culture surface of the cell culture vessel of the present invention contains a resin, and optionally further contains components other than the resin (other components).
  • the resin contained in the resin composition is not particularly limited, and examples of resins that can be used include cycloolefin polymers, polyethylene terephthalate (PET), polystyrene (PS), acrylic resins, polycarbonates, hydrogenated styrene-conjugated diene block copolymers, etc. These resins may be used alone or in combination of two or more kinds in any ratio.
  • a cycloolefin polymer from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, it is preferable to use a cycloolefin polymer, and it is more preferable to use both a cycloolefin polymer and a hydrogenated styrene-conjugated diene block copolymer.
  • the cycloolefin polymer is a resin having an alicyclic structure in the main chain and/or side chain.
  • the alicyclic structure of the cycloolefin polymer include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure.
  • Specific examples of the cycloolefin polymer include norbornene-based polymers, monocyclic olefin-based polymers, cyclic conjugated diene-based polymers, vinyl alicyclic hydrocarbon-based polymers, and hydrogenated versions of these. Among these, norbornene-based polymers and hydrogenated versions of these are preferred from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface.
  • norbornene-based polymers examples include a ring-opening polymer of a monomer having a norbornene structure, or a ring-opening polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydrogenated product thereof; an addition polymer of a monomer having a norbornene structure, or an addition polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydrogenated product thereof; and the like.
  • a norbornene-based polymer is a polymer that contains 50% by mass or more, preferably 60% by mass or more, of monomer units having a norbornene skeleton relative to the total monomer units constituting the norbornene-based polymer. More specifically, norbornene-based polymers are obtained by polymerizing norbornene-based monomers, which are monomers having a norbornene skeleton, and are broadly divided into those obtained by ring-opening polymerization and those obtained by addition polymerization.
  • Examples of materials obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers capable of ring-opening copolymerization with the norbornene-based monomers, and hydrogenated products thereof.
  • Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith.
  • a ring-opening polymer hydrogenate of a norbornene monomer is preferred.
  • Norbornene monomers that can be used in the synthesis of norbornene polymers include bicyclo[2.2.1]hept-2-ene (commonly known as norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2.2.1]hept-2-ene, and 5-ethylidene-bicyclo[2.2.1]hept-2-ene.
  • bicyclo[2.2.1]hept-2-ene commonly known as norbornene
  • 5-methyl-bicyclo[2.2.1]hept-2-ene 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene
  • 5-ethyl-bicyclo[2.2.1]hept-2-ene 5-ethylidene-bicyclo[2.2.1]hept-2-ene
  • Bicyclic monomers such as hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene; Tricyclic monomers such as tricyclo[ 4.3.01,6.12,5 ]deca-3,7-diene (commonly known as dicyclopentadiene), 2 -methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene; Tetracyclo[4.4.0.1 2,5 .
  • tetracyclic monomers such as 1,4 - methano-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro- 1,4,4a ,9a-tetrahydrofluorene, and 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene; and the like.
  • monomers capable of ring-opening copolymerization with norbornene monomers include monocyclic cycloolefin monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene.
  • These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.
  • Other monomers capable of addition copolymerization with norbornene monomers include ⁇ -olefin monomers having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cycloolefin monomers, such as cyclobutene, cyclopentene, cyclohexene, cyclooctene, and tetracyclo [ 9.2.1.02,10.03,8 ]tetradeca-3,5,7,12-tetraene (also called 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); and non-conjugated diene monomers, such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene.
  • ⁇ -olefin monomers having 2 to 20 carbon atoms such as ethylene, propy
  • an ⁇ -olefin monomer is preferable, and ethylene is more preferable.
  • These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.
  • the ring-opening polymer of a norbornene monomer or the ring-opening polymer of a norbornene monomer and another monomer capable of ring-opening copolymerization therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst.
  • a catalyst consisting of a halide of a metal such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst consisting of a halide or an acetylacetone compound of a metal such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used.
  • the hydrogenated ring-opening polymer of a norbornene monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.
  • Addition polymers of norbornene-based monomers, or addition polymers of norbornene-based monomers and other monomers copolymerizable therewith, can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst.
  • a catalyst consisting of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.
  • Monocyclic olefin polymer for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used.
  • cyclic conjugated diene polymer for example, a polymer obtained by 1,2- or 1,4-addition polymerization of a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene, or a hydrogenated product thereof can be used.
  • Vinyl alicyclic hydrocarbon polymer examples include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof.
  • the vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers with other monomers that are copolymerizable therewith.
  • the "hydrogenated styrene-conjugated diene block copolymer” is not included in the "vinyl alicyclic hydrocarbon polymer".
  • the weight average molecular weight calculated as polyisoprene, measured by gel permeation chromatography of a cyclohexane solution (a toluene solution if the polymer does not dissolve) is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and even more preferably 10,000 to 100,000.
  • the weight average molecular weight is within this range, mechanical strength and moldability are well balanced, making this an ideal range.
  • the content of the cycloolefin polymer in the resin is preferably 99.50 parts by mass or more, more preferably 99.70 parts by mass or more, and even more preferably 99.80 parts by mass or more, and preferably 99.99 parts by mass or less, and more preferably 99.98 parts by mass or less, relative to 100 parts by mass of the resin. If the content of the cycloolefin polymer is within the above range relative to 100 parts by mass of the resin, the denaturation of the protein attached to the culture surface can be further suppressed, and the adsorption of the protein to the culture surface can be increased.
  • the hydrogenated styrene-conjugated diene block copolymer is a hydrogenated styrene block-conjugated diene block copolymer having a styrene block mainly composed of repeating units derived from a styrene-based monomer and a diene block mainly composed of repeating units derived from a conjugated diene monomer.
  • the hydrogenated styrene-conjugated diene block copolymer for example, a commercially available product can be used.
  • the hydrogenated styrene-conjugated diene block copolymer may be obtained by obtaining a styrene-conjugated diene block copolymer according to a known method, and then hydrogenating the carbon-carbon unsaturated bonds in the main chain and side chain of the conjugated diene block of the obtained styrene-conjugated diene block copolymer.
  • the hydrogenation method of the unsaturated bonds and the reaction form are not particularly limited, and may be performed according to a known method.
  • the content of the hydrogenated styrene-conjugated diene block copolymer in the resin is preferably 0.01 parts by mass or more, more preferably 0.02 parts by mass or more, preferably 0.5 parts by mass or less, more preferably 0.3 parts by mass or less, and even more preferably 0.2 parts by mass or less, relative to 100 parts by mass of the total amount of the resin. If the content of the hydrogenated styrene-conjugated diene block copolymer is within the above range relative to 100 parts by mass of the resin, the denaturation of proteins attached to the culture surface can be further suppressed, and the adsorption of proteins to the culture surface can be increased.
  • the resin composition used to form the culture surface of the cell culture vessel of the present invention may further contain components other than the above-mentioned resins (other components).
  • the other components include compounding agents commonly used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent whitening agents.
  • the other components may be used alone or in combination of two or more in any ratio.
  • antioxidants include phenol-based antioxidants, organic phosphite-based antioxidants, and sulfur-based antioxidants.
  • antioxidants include phenol-based antioxidants, organic phosphite-based antioxidants, and sulfur-based antioxidants.
  • ultraviolet absorbers include benzophenone-based ultraviolet absorbers and benzotriazole-based ultraviolet absorbers.
  • light stabilizers include hindered amine-based stabilizers. Among these, from the viewpoints of further suppressing the denaturation of proteins attached to the culture surface and increasing the adsorption of proteins to the culture surface, it is preferable that the resin composition contains an antioxidant.
  • the content of the antioxidant in the resin composition is preferably 0.05 parts by mass or more, more preferably 0.1 parts by mass or more, and preferably 2 parts by mass or less, more preferably 1 part by mass or less, and even more preferably 0.5 parts by mass or less, per 100 parts by mass of resin. If the content of the antioxidant is within the above range per 100 parts by mass of resin, the denaturation of proteins attached to the culture surface can be further suppressed, and the adsorption of proteins to the culture surface can be increased.
  • the glass transition temperature of the resin composition may be appropriately selected depending on the intended use, but is usually 50 to 300° C., preferably 80 to 280° C., more preferably 90 to 250° C., and even more preferably 90 to 200° C. When the glass transition temperature is within this range, heat resistance and moldability are highly balanced, which is preferable. In the present invention, the glass transition temperature of the resin composition is measured based on JIS K 7121.
  • the mixing method for obtaining a resin composition containing the above-mentioned resin and optionally other components is not particularly limited, and can be performed using a known melt kneader such as a single screw extruder, a twin screw extruder, a Banbury mixer, a kneader, a feeder ruder, etc. After mixing, the mixture can be extruded into a rod shape according to a conventional method, and cut to an appropriate length with a strand cutter to form pellets.
  • ⁇ Area ratio of 1680 cm -1 band ⁇ Area ratio of 1680 cm -1 band>
  • the ratio of the area of the band having a peak at a frequency of 1680 cm -1 to the total area of all bands (area ratio of the 1680 cm -1 band) must be 10% or more
  • the area ratio of the 1680 cm -1 band is preferably 11% or more, more preferably 12% or more, and even more preferably 13% or more.
  • the area ratio of the 1680 cm -1 band is less than 10%, the degree of denaturation of the protein attached to the culture surface increases.
  • the upper limit of the area ratio of the 1680 cm -1 band is not particularly limited, and can be, for example, 30% or less, or 25% or less.
  • the area ratio of the 1680 cm band can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the presence or absence of surface treatment of the culture surface, the type and conditions of the surface treatment, etc. Specifically, the area ratio of the 1680 cm band can be increased by not performing a surface treatment, or by shortening the treatment time when a heat treatment is performed as the surface treatment.
  • the cell culture vessel of the present invention preferably has an XPS spectrum of the culture surface that does not have a peak derived from a ⁇ - ⁇ * bond.
  • the presence or absence of a peak derived from a ⁇ - ⁇ * bond in the XPS spectrum of the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface. Specifically, by using a resin that does not have an aromatic ring, an XPS spectrum that does not have a peak derived from a ⁇ - ⁇ * bond can be obtained.
  • the abundance ratio of oxygen-containing functional groups on the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, the presence or absence of surface treatment on the culture surface, the type and conditions of the surface treatment, etc. Specifically, the abundance ratio of oxygen-containing functional groups on the culture surface can be reduced by not performing surface treatment, or by shortening the treatment time when heat treatment is performed as the surface treatment.
  • the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is preferably 2.0% or more, more preferably 2.5% or more, even more preferably 3.0% or more, preferably 10.0% or less, more preferably 7.5% or less, even more preferably 5.0% or less, even more preferably 3.9% or less, and particularly preferably 2.6% or less. If the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is 2.0% or more, the adsorption of protein to the culture surface can be increased.
  • the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is 10.0%% or less, the denaturation of protein attached to the culture surface can be further suppressed.
  • the ratio of the amount of oxygen element to the amount of carbon element on the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, the presence or absence of a surface treatment on the culture surface, the type and conditions of the surface treatment, etc. Specifically, the ratio of the amount of oxygen element to the amount of carbon element on the culture surface can be reduced by not performing a surface treatment, or by shortening the treatment time when a heat treatment is performed as the surface treatment.
  • the protein adsorption rate of the culture surface is preferably 60% or more, more preferably 65% or more, and even more preferably 70% or more. If the protein adsorption rate of the culture surface is 60% or more, the protein adsorption property of the culture surface can be well ensured. Furthermore, the upper limit of the protein adsorption rate of the culture surface is not particularly limited, and can be, for example, 100% or less, or 90% or less. In the present invention, the protein adsorption rate of the culture surface can be measured by the method described in the Examples of this specification.
  • the protein adsorption rate of the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, whether or not the culture surface is surface-treated, the type and conditions of the surface treatment, etc.
  • the method for producing the cell culture vessel of the present invention is not particularly limited, but the cell culture vessel of the present invention can be produced, for example, through a step of molding a resin composition containing the above-mentioned resin and, optionally, other components, to obtain a culture surface (molding step), and optionally a step of applying a surface treatment to the culture surface (surface treatment step).
  • the method of molding the resin composition is not particularly limited, and can be appropriately selected from known molding methods according to the desired shape of the culture surface.
  • known molding methods include, for example, extrusion molding, injection molding, inflation molding, blow molding, extrusion blow molding, injection blow molding, press molding, vacuum molding, powder slush molding, calendar molding, foam molding, thermoforming, etc.
  • injection molding it is preferable to use injection molding as the molding method.
  • the temperature of the mold used for injection molding (hereinafter, sometimes simply referred to as "mold temperature") is preferably (Tg-40)°C or higher and preferably (Tg-20)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition. If the mold temperature is within the above-mentioned range, the denaturation of the protein attached to the culture surface can be further suppressed and the adsorption of the protein to the culture surface can be increased.
  • the temperature of the cylinder (hereinafter, sometimes simply referred to as "cylinder temperature") provided in the injection molding machine is preferably (Tg+120)°C or higher and preferably (Tg+170)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition. If the cylinder temperature is within the above-mentioned range, the denaturation of the protein attached to the culture surface can be further suppressed and the adsorption of the protein to the culture surface can be increased.
  • the type of surface treatment applied to the culture surface is not particularly limited and can be appropriately selected from known surface treatment methods.
  • Such known surface treatment methods include heat treatment, plasma treatment, vacuum ultraviolet treatment, corona treatment, ozone treatment, etc. Among these, heat treatment is preferred from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface.
  • the treatment temperature in the heat treatment is preferably (Tg-10)°C or lower, and more preferably (Tg-15)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition.
  • the lower limit of the treatment temperature in the heat treatment is not particularly limited, and can be, for example, (Tg-40)°C or higher, or (Tg-30)°C or higher.
  • the treatment time in the heat treatment may be set appropriately depending on the treatment temperature. From the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, the treatment time in the heat treatment is preferably 50 hours or less, and more preferably 40 hours or less.
  • the lower limit of the treatment time in the heat treatment is not particularly limited, and can be, for example, 10 hours or more, or 20 hours or more.
  • the cell culture vessel of the present invention may be produced through any step (other step) other than the above-mentioned molding step and surface treatment step, for example, a step of pre-drying the resin composition prior to the molding step (pre-drying step), a step of assembling the cell culture vessel by combining the culture surface with other members (assembly step), a step of sterilizing the cell culture vessel (sterilization step), etc.
  • ⁇ X-ray photoelectron spectroscopy measurement> The bottom surface (culture surface) of the dishes prepared in the examples and comparative examples was used as the measurement object, and the photoelectrons emitted from the surface when irradiated with soft X-rays in an ultra-vacuum using X-ray photoelectron spectroscopy (XPS) were detected with an analyzer to obtain elemental information on the culture surface from the binding energy of bound electrons in the material.
  • XPS X-ray photoelectron spectroscopy
  • Abundance ratio of oxygen-containing functional groups (S2+S3)/S1 [Presence or absence of peaks derived from ⁇ - ⁇ * bonds]
  • the binding energy range in which the C1s main peak appears was scanned with high resolution (measurement range: 275 eV to 300 eV).
  • ⁇ Area ratio of 1680 cm -1 band > 1.
  • Protein Processing RetroNectin (manufactured by Takara Bio, model number "T100B") was dissolved in heavy water ( D2O , manufactured by Kanto Chemical) to a concentration of 10 ⁇ g/mL to obtain a protein solution.
  • heavy water D2O , manufactured by Kanto Chemical
  • 2.5 mL of the protein solution was placed in the dish (diameter 35 mm) prepared in the Examples and Comparative Examples, and kept at 37°C for 45 minutes. Next, the protein solution in the dish was removed, and 3 mL of heavy water was added to wash the dish. Thereafter, the washing water in the dish was removed, and 3 mL of new heavy water was added for washing.
  • the amide I region (1600-1700 cm -1 ) of the measured spectrum was band-decomposed.
  • one of the bands used for band decomposition was a band having a peak at 1680 cm -1 .
  • the total area of all bands obtained by band resolution was defined as SA
  • the area of the band having a peak at 1680 cm ⁇ 1 was defined as SB
  • Measurement method ATR method Number of integrations: 32 Resolution: 16 cm -1 Background correction: before sample measurement Spectral intensity: absorbance Function (distribution function) representing band shape: Gaussian function Peak detection sensitivity: low sensitivity Noise level in peak fitting: 0.01 Baseline correction in peak fitting: Linear interpolation ⁇ protein adsorption rate> 1.
  • Protein Processing RetroNectin manufactured by Takara Bio, model number "T100B” was dissolved in a phosphate buffered saline (PBS) solution (manufactured by Fujifilm Wako Pure Chemical Industries, PBS (-)) to a concentration of 10 ⁇ g/mL to obtain a protein solution.
  • PBS phosphate buffered saline
  • the ruthenium stained part (gray) and the gold colloid part (white) were binarized as bright parts, and the parts other than the ruthenium stained part and the gold colloid part (black) were binarized as dark parts, and the ratio (%) of bright parts to all pixels of the SEM observation image was determined as the protein adsorption rate.
  • the higher the protein adsorption rate the better the protein adsorption of the cell culture vessel. Note that if the protein adsorption rate is 60% or more, the protein adsorption of the culture surface is sufficiently ensured.
  • Example 1 Preparation of cell culture vessel (dish)> [Pre-drying and molding]
  • a resin composition having the following composition glass transition temperature (Tg): 163°C, hereinafter referred to as "COP1"
  • Tg-20 glass transition temperature
  • COP1 glass transition temperature
  • Mold temperature (Tg-30) °C Injection pressure: 70MPa
  • the bottom surface (culture surface) of the resulting dish was measured for the ratio of oxygen element content to carbon element content, the abundance ratio of oxygen-containing functional groups, the presence or absence of a peak derived from ⁇ - ⁇ * bonds, the area ratio of the 1680 cm -1 band of recombinant human fibronectin fragment, and the protein adsorption rate. The results are shown in Table 1.
  • Example 2 ⁇ Preparation of cell culture vessel> [Pre-drying and molding] Preliminary drying and molding were carried out in the same manner as in Example 1 to prepare a dish (diameter 35 mm). [Heat treatment] The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 140° C. for 24 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
  • Example 3 Except for changing the heat treatment time from 24 hours to 30 hours, a cell culture vessel was produced in the same manner as in Example 2. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
  • Example 4 A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition having the following composition (Tg: 136° C., hereinafter referred to as “COP2”) was used instead of COP1. Evaluation was then performed in the same manner as in Example 1. The results are shown in Table 1. - Composition of COP2 - Cycloolefin polymer (hydrogenated ring-opened polymer of norbornene monomer, Mw: 35,000) 99.9 parts Antioxidant 0.135 parts Hydrogenated styrene-conjugated diene block copolymer 0.1 parts
  • Example 5 Preparation of cell culture vessel> [Pre-drying and molding] Preliminary drying and molding were carried out in the same manner as in Example 4 to prepare a dish (diameter 35 mm). [Heat treatment] The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 115° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
  • Example 6 A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition having the following composition (Tg: 100° C., hereinafter referred to as “COP3”) was used instead of COP1. Evaluation was then performed in the same manner as in Example 1. The results are shown in Table 1. - Composition of COP3 - Cycloolefin polymer (hydrogenated ring-opened polymer of norbornene monomer, Mw: 29,400) 99.98 parts Antioxidant 0.48 parts Hydrogenated styrene-conjugated diene block copolymer 0.02 parts
  • Example 7 ⁇ Preparation of cell culture vessel> [Pre-drying and molding] Preliminary drying and molding were carried out in the same manner as in Example 6 to prepare a dish (diameter 35 mm). [Heat treatment] The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 80° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
  • Example 8 A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition containing an addition copolymer of norbornene and ethylene (Tg: 138° C., hereinafter referred to as “COC1”) was used instead of COP1. Then, evaluation was performed in the same manner as in Example 1. The results are shown in Table 2.
  • Example 9 ⁇ Preparation of cell culture vessel> [Pre-drying and molding] Preliminary drying and molding were carried out in the same manner as in Example 8 to prepare a dish (diameter 35 mm). [Heat treatment] The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 115° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
  • the present invention provides a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.

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Abstract

The purpose of the present invention is to provide a cell culturing container that can suppress denaturing of protein attached to a culturing surface. A cell culturing container according to the present invention is provided with a culturing surface formed by using a resin composition containing a resin. In an infrared absorption spectrum of a recombinant human fibronectin fragment attached to the culturing surface, the area of a band having a peak at a vibration frequency of 1680 cm-1 obtained through band decomposition performed for a vibration frequency range of 1600-1700 cm-1 <sp /> is 10% or more when the total area of all bands obtained through the band decomposition is defined as 100%.

Description

細胞培養容器Cell Culture Vessels
　本発明は、細胞培養容器に関するものである。 The present invention relates to a cell culture vessel.
　生命科学の分野では、ｉＰＳ細胞など動物体細胞を使った研究が盛んに行われている。細胞は、培地（血清など細胞に必要な栄養素が入った細胞を培養するための液体）とともに、ディッシュやウェルプレートなどの細胞培養容器に入れられ、約３７℃で数日から数週間の期間にわたって培養される。 In the field of life sciences, research using animal somatic cells such as iPS cells is being actively conducted. The cells are placed in cell culture containers such as dishes or well plates together with culture medium (a liquid for culturing cells that contains nutrients necessary for the cells, such as serum), and cultured at around 37°C for a period of several days to several weeks.
　ここで、培養細胞は接着細胞と浮遊細胞に大別される。そして、接着細胞は、細胞膜表面に存在するインテグリンと、細胞培養容器表面に付着したフィブロネクチンなどの細胞外マトリックス構成タンパク質との間の結合を介して、細胞培養容器表面に付着した状態で増殖する。 Here, cultured cells are broadly divided into adherent cells and suspension cells. Adherent cells grow while attached to the surface of the cell culture vessel via binding between integrins present on the cell membrane surface and extracellular matrix proteins such as fibronectin attached to the surface of the cell culture vessel.
　そこで、近年では、細胞培養容器の細胞接着性の向上を目的として、細胞培養容器に用いるポリマー材料の改良が試みられている。
　例えば、特許文献１には、接触表面と内部とからなり、当該接触表面が所定のパラメータによって定量化される粗さを有する、ポリマー支持体が開示されている。そして、特許文献１によれば、当該ポリマー支持体の接触表面は、細胞接着、細胞増殖、及び細胞回収率が向上している。 Therefore, in recent years, attempts have been made to improve the polymer materials used for cell culture vessels in order to improve the cell adhesiveness of the cell culture vessels.
For example, Patent Document 1 discloses a polymer support having a contact surface and an interior, the contact surface having a roughness quantified by a predetermined parameter, and according to Patent Document 1, the contact surface of the polymer support has improved cell adhesion, cell proliferation, and cell recovery rate.
特表２０１８－５３７５５６号公報Special table 2018-537556 publication
　しかしながら、本発明者の検討によると、上記従来のポリマー支持体からなる細胞培養容器には、当該細胞培養容器の培養面に付着したタンパク質が変性しやすいという問題があることが明らかになった。 However, the inventors' investigations revealed that cell culture vessels made of the above-mentioned conventional polymer supports have a problem in that proteins attached to the culture surface of the cell culture vessel are easily denatured.
　そこで、本発明は、培養面に付着したタンパク質の変性を抑制し得る細胞培養容器の提供を目的とする。 The present invention aims to provide a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.
　本発明者は、上記課題を解決することを目的として鋭意検討を行った。そして、本発明者は、樹脂組成物を用いてなる培養面を備え、且つ、当該培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルのアミドＩ領域（１６００ｃｍ-1～１７００ｃｍ-1）をバンド分解したときに、すべてのバンドの合計面積に対する１６８０ｃｍ-1にピークを有するバンドの面積の比率が所定値以上である細胞培養容器を用いれば、培養面に付着したタンパク質の変性を良好に抑制可能であること新たにを見出し、本発明を完成させた。 The present inventors have conducted extensive research to solve the above problems, and have newly discovered that the denaturation of proteins attached to the culture surface can be effectively suppressed by using a cell culture vessel that is provided with a culture surface made of a resin composition, and in which, when the amide I region (1600 cm -1 to 1700 cm -1 ) of the infrared absorption spectrum of a recombinant human fibronectin fragment attached to the culture surface is band-decomposed, the ratio of the area of the band having a peak at 1680 cm -1 to the total area of all bands is equal to or greater than a predetermined value, thereby completing the present invention.
　即ち、この発明は、上記課題を有利に解決することを目的とするものであり、本発明によれば、下記（１）～（４）の細胞培養容器が提供される。 In other words, the present invention aims to advantageously solve the above problems, and provides the following cell culture vessels (1) to (4).
（１）樹脂を含む樹脂組成物を用いてなる培養面を備える細胞培養容器であって、前記培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルにおいて、振動数１６００ｃｍ-1以上１７００ｃｍ-1以下の領域のバンド分解により得られる振動数１６８０ｃｍ-1にピークを有するバンドの面積が、前記バンド分解により得られるすべてのバンドの合計面積を１００％として、１０％以上である、細胞培養容器。
　樹脂を含む樹脂組成物を用いてなる培養面を備え、且つ、当該培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルの振動数１６００ｃｍ-1以上１７００ｃｍ-1以下の領域をバンド分解したときに、すべてのバンドの合計面積に対する１６８０ｃｍ-1にピークを有するバンドの面積の比率（以下、「１６８０ｃｍ-1バンドの面積比率」と略記する場合がある。）が上記値以上である細胞培養容器によれば、培養面に付着したタンパク質の変性を良好に抑制することができる。
　なお、本発明において、「培養面」とは、培養中の細胞が接する細胞培養容器表面を意味する。
　また、本発明において、「組み換えヒトフィブロネクチンフラグメント」とは、ヒトフィブロネクチンの細胞接着ドメイン（Ｃ－ｄｏｍａｉｎ）、ヘパリン結合ドメイン（Ｈ－ｄｏｍａｉｎ）およびＣＳ－１部位の３種類の機能性ドメインを含む組換えタンパク質（リコンビナントヒトフィブロネクチン　ＣＨ－２９６）を意味する。「リコンビナントヒトフィブロネクチン　ＣＨ－２９６」は、Ｒｅｃｔｒｏｎｅｃｔｉｎ（登録商標、タカラバイオ社製）として市販されており、そのアミノ酸配列は、国際公開第２０１８／０２１５４３号に配列番号１８として開示されている。
　そして、本発明において、「培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトル」は、本明細書の実施例に記載の方法を用いて測定することができる。
　さらに、本発明において、「１６８０ｃｍ-1バンドの面積比率」は、本明細書の実施例に記載の方法に従って算出することができる。 (1) A cell culture vessel having a culture surface formed using a resin composition containing a resin, wherein in an infrared absorption spectrum of a recombinant human fibronectin fragment attached to the culture surface, the area of a band having a peak at a frequency of 1680 cm - 1 obtained by band decomposition in the frequency range of 1600 cm -1 or more and 1700 cm -1 or less is 10% or more, with the total area of all bands obtained by the band decomposition being 100%.
A cell culture vessel having a culture surface made of a resin composition containing a resin, and in which, when the infrared absorption spectrum of a recombinant human fibronectin fragment attached to the culture surface is band-decomposed in the frequency range of 1600 cm -1 to 1700 cm -1 , the ratio of the area of the band having a peak at 1680 cm -1 to the total area of all bands (hereinafter sometimes abbreviated as "area ratio of the 1680 cm -1 band") is equal to or greater than the above value, can effectively suppress the denaturation of proteins attached to the culture surface.
In the present invention, the "culture surface" refers to the surface of a cell culture vessel with which cells are in contact during culture.
In the present invention, the term "recombinant human fibronectin fragment" refers to a recombinant protein (recombinant human fibronectin CH-296) containing three functional domains, namely, the cell adhesion domain (C-domain), the heparin-binding domain (H-domain), and the CS-1 site of human fibronectin. "Recombinant human fibronectin CH-296" is commercially available as Rectronectin (registered trademark, manufactured by Takara Bio Inc.), and its amino acid sequence is disclosed as SEQ ID NO: 18 in WO 2018/021543.
In the present invention, the "infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface" can be measured by the method described in the Examples of this specification.
Furthermore, in the present invention, the "area ratio of the 1680 cm -1 band" can be calculated according to the method described in the Examples of this specification.
（２）前記培養面のＸ線光電子分光法スペクトルが、π－π*結合に由来するピークを有さず、前記Ｘ線光電子分光法スペクトルに基づいて得られる、前記培養面におけるＣ－Ｏ基の量に対するＣ＝Ｏ基およびＯ－Ｃ＝Ｏ基の合計量の比が０．４５以下である、上記（１）に記載の細胞培養容器。
　培養面のＸ線光電子分光法スペクトル（以下、「ＸＰＳスペクトル」と略記する場合がある。）がπ－π*結合に由来するピークを有さず、且つ、培養面におけるＣ－Ｏ基の量に対するＣ＝Ｏ基およびＯ－Ｃ＝Ｏ基の合計量の比（以下、「酸素含有官能基の存在比」と略記する場合がある。）が上記値以下である細胞培養容器によれば、培養面に付着したタンパク質の変性を更に抑制することができる。
　なお、本発明において、「培養面のＸＰＳスペクトル」は、本明細書の実施例に記載の方法を用いて測定することができる。
　また、本発明において、「π－π*結合に由来するピーク」とは、ＸＰＳスペクトルの２９１ｅＶ近傍に観測される（π－π*）サテライトピークを意味する。
　そして、本発明において、「酸素含有官能基の存在比」は、本明細書の実施例に記載の方法に従って算出することができる。 (2) The cell culture vessel according to (1) above, wherein the X-ray photoelectron spectroscopy spectrum of the culture surface does not have a peak derived from a π-π * bond, and the ratio of the total amount of C═O groups and O—C═O groups to the amount of C—O groups on the culture surface, as obtained based on the X-ray photoelectron spectroscopy spectrum, is 0.45 or less.
According to a cell culture vessel in which the X-ray photoelectron spectroscopy spectrum (hereinafter sometimes abbreviated as "XPS spectrum") of the culture surface does not have a peak derived from a π-π * bond and the ratio of the total amount of C═O groups and O—C═O groups to the amount of C—O groups on the culture surface (hereinafter sometimes abbreviated as "abundance ratio of oxygen-containing functional groups") is equal to or less than the above value, the denaturation of proteins attached to the culture surface can be further suppressed.
In the present invention, the "XPS spectrum of the culture surface" can be measured by the method described in the Examples of this specification.
In the present invention, the "peak derived from a π-π * bond" means a (π-π * ) satellite peak observed in the vicinity of 291 eV in the XPS spectrum.
In the present invention, the "abundance ratio of oxygen-containing functional groups" can be calculated according to the method described in the Examples of this specification.
（３）Ｘ線光電子分光法スペクトルに基づいて得られる、前記培養面における炭素元素量に対する酸素元素量の割合が２．０％以上である、上記（１）または（２）に記載の細胞培養容器。
　培養面における炭素元素量に対する酸素元素量の割合が上記値以上である細胞培養容器によれば、培養面へのタンパク質の吸着性を高めることができる。
　なお、本発明において、「培養面における炭素元素量に対する酸素元素量の割合」は、本明細書の実施例に記載の方法に従って測定することができる。 (3) A cell culture vessel according to (1) or (2) above, in which the ratio of the amount of oxygen element to the amount of carbon element on the culture surface, as obtained based on an X-ray photoelectron spectroscopy spectrum, is 2.0% or more.
A cell culture vessel having a ratio of the amount of oxygen element to the amount of carbon element on the culture surface equal to or greater than the above value can enhance the adsorptivity of proteins to the culture surface.
In the present invention, the "ratio of the amount of oxygen element to the amount of carbon element on the culture surface" can be measured according to the method described in the Examples of this specification.
（４）前記樹脂がノルボルネン系単量体の開環重合体水素化物を含む、上記（１）～（３）の何れかに記載の細胞培養容器。
　ノルボルネン系単量体の開環重合体水素化物を含む樹脂組成物を用いてなる培養面を備える細胞培養容器によれば、培養面に付着したタンパク質の変性を更に抑制することができる。 (4) The cell culture vessel according to any one of (1) to (3) above, wherein the resin contains a hydrogenated ring-opening polymer of a norbornene monomer.
According to a cell culture vessel having a culture surface formed using a resin composition containing a ring-opening polymer hydrogenated product of a norbornene-based monomer, the denaturation of proteins attached to the culture surface can be further suppressed.
　本発明によれば、培養面に付着したタンパク質の変性を抑制し得る細胞培養容器を提供することができる。 The present invention provides a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.
　以下、本発明の実施形態について詳細に説明する。
　ここで、本発明の細胞培養容器は、その内部で細胞を培養することを目的としたものであれば特に限定されない。そして、本発明の細胞培養容器の具体例としては、ディッシュ、プレート、マイクロ流路チップ、バッグ、チューブ、スキャホールド、カップ、ジャー・ファーメンター等が挙げられる。 Hereinafter, an embodiment of the present invention will be described in detail.
Here, the cell culture vessel of the present invention is not particularly limited as long as it is intended to culture cells therein. Specific examples of the cell culture vessel of the present invention include dishes, plates, microchannel chips, bags, tubes, scaffolds, cups, jar fermenters, etc.
　また、細胞培養容器に収容される細胞も特に限定されず、目的に応じて任意に選択することができる。具体的には、ＣＨＯ細胞、ＶＥＲＯ細胞、ＮＩＨ３Ｔ３細胞、ＨＥＫ２９３細胞、ヒトｉＰＳ細胞より分化された神経細胞、心筋細胞、肝臓細胞等などの接着細胞が好ましく挙げられる。
　なお、細胞は、１種類を単独で用いてもよく、２種類以上を任意の比率で組み合わせて用いてもよい。 The cells to be contained in the cell culture vessel are not particularly limited and can be selected arbitrarily depending on the purpose.Specific examples of the cells include adherent cells such as CHO cells, VERO cells, NIH3T3 cells, HEK293 cells, nerve cells differentiated from human iPS cells, cardiomyocytes, and liver cells.
The cells may be used alone or in combination of two or more types in any ratio.
（細胞培養容器）
　本発明の細胞培養容器は、少なくとも培養面が樹脂を含む樹脂組成物を用いて形成される。例えば、本発明の細胞培養容器がマルチウェルプレートである場合、ウェル内壁面のうち、培養中の細胞が接する部分が樹脂組成物を用いて形成されていればよい。また、本発明の細胞培養容器が複数のフィルムからなる積層構造を有するバッグである場合、最内層のフィルム（バッグ内面）が、樹脂組成物を用いて形成されていればよい。あるいは、細胞培養容器全体が、樹脂組成物を用いて形成されていてもよい。
　ここで、本発明の細胞培養容器は、培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルにおける１６８０ｃｍ-1バンドの面積比率が１０％以上であることを特徴とする。 (Cell culture vessel)
At least the culture surface of the cell culture vessel of the present invention is formed using a resin composition containing a resin. For example, when the cell culture vessel of the present invention is a multi-well plate, the part of the well inner wall surface that contacts the cells being cultured may be formed using the resin composition. When the cell culture vessel of the present invention is a bag having a laminated structure made of multiple films, the innermost film (the inner surface of the bag) may be formed using the resin composition. Alternatively, the entire cell culture vessel may be formed using the resin composition.
Here, the cell culture vessel of the present invention is characterized in that the area ratio of the 1680 cm -1 band in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is 10% or more.
　そして、本発明の細胞培養容器は、培養面に付着した組み換えヒトフィブロネクチンフラグメントの１６８０ｃｍ-1バンドの面積比率が１０％以上であるため、培養面に付着したタンパク質の変性が抑制されている。かかる本発明の細胞培養容器を用いることで、上記の効果が得られる理由は定かではないが、以下の通りであると推察される。 In addition, in the cell culture vessel of the present invention, the area ratio of the 1680 cm band of the recombinant human fibronectin fragment attached to the culture surface is 10% or more, so that denaturation of the protein attached to the culture surface is suppressed. Although the reason why the above-mentioned effects are obtained by using such a cell culture vessel of the present invention is not clear, it is presumed to be as follows.
　まず、本発明の細胞培養容器は、培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルの振動数１６００ｃｍ-1以上１７００ｃｍ-1以下の領域をバンド分解したときに、すべてのバンドの合計面積に対する振動数１６８０ｃｍ-1にピークを有するバンドの面積の比率（１６８０ｃｍ-1バンドの面積比率）が１０％以上である。
　ここで、タンパク質の赤外吸収スペクトルにおいて、振動数１６００～１７００ｃｍ-1の領域はアミドＩ領域と呼ばれ、タンパク質の二次構造を反映することが知られている。そして、組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルでは、１６８０ｃｍ-1にピークを有するバンドがβシート構造に帰属されることが本発明者の検討により新たに明らかになった。したがって、「１６８０ｃｍ-1バンドの面積比率」とは、組み換えヒトフィブロネクチンフラグメント分子中のβシートの含有率（％）に関連する値と見なすことができる。そして、本発明の細胞培養容器は、１６８０ｃｍ-1バンドの面積比率が１０％以上であることで、培養面に付着した組み換えヒトフィブロネクチンフラグメントがβシート構造に富む構造、すなわち、天然状態に近い構造を維持しているといえる。換言すると、本発明の細胞培養容器は、培養面に付着した組み換えヒトフィブロネクチンフラグメントの１６８０ｃｍ-1バンドの面積比率が１０％以上であるため、培養面に付着したタンパク質の変性が十分に抑制されていると考えられる。
　したがって、本発明の細胞培養容器を用いれば、培養面に付着したタンパク質の変性を抑制することができる。 First, in the cell culture vessel of the present invention, when the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is band-resolved in the frequency region of 1600 cm -1 to 1700 cm -1 , the ratio of the area of the band having a peak at a frequency of 1680 cm -1 to the total area of all bands (area ratio of the 1680 cm -1 band) is 10% or more.
Here, in the infrared absorption spectrum of a protein, the region with a frequency of 1600 to 1700 cm −1 is called the amide I region, and is known to reflect the secondary structure of the protein. In addition, the present inventors have newly discovered that in the infrared absorption spectrum of a recombinant human fibronectin fragment, a band having a peak at 1680 cm −1 is assigned to a β-sheet structure. Therefore, the “area ratio of the 1680 cm −1 band” can be regarded as a value related to the content (%) of β-sheet in the recombinant human fibronectin fragment molecule. In addition, since the area ratio of the 1680 cm −1 band of the cell culture vessel of the present invention is 10% or more, it can be said that the recombinant human fibronectin fragment attached to the culture surface maintains a structure rich in β-sheet structure, that is, a structure close to the native state. In other words, since the area ratio of the 1680 cm −1 band of the recombinant human fibronectin fragment attached to the culture surface of the cell culture vessel of the present invention is 10% or more, it is considered that the denaturation of the protein attached to the culture surface is sufficiently suppressed.
Therefore, by using the cell culture vessel of the present invention, it is possible to suppress the denaturation of proteins attached to the culture surface.
＜樹脂組成物＞
　本発明の細胞培養容器が備える培養面の形成に用いる樹脂組成物は、樹脂を含み、任意に、樹脂以外の成分（その他の成分）を更に含有する。 <Resin Composition>
The resin composition used to form the culture surface of the cell culture vessel of the present invention contains a resin, and optionally further contains components other than the resin (other components).
＜＜樹脂＞＞
　ここで、樹脂組成物に含まれる樹脂としては、特に限定されず、例えば、シクロオレフィンポリマー、ポリエチレンテレフタレート（ＰＥＴ）、ポリスチレン（ＰＳ）、アクリル樹脂、ポリカーボネート、スチレン－共役ジエンブロック共重合体水素化物等の樹脂を用いることができる。なお、これらの樹脂は、１種単独で用いてもよいし、２種以上を任意の比率で組み合わせて用いてもよい。
　上述した樹脂の中でも、培養面に付着したタンパク質の変性を更に抑制する観点から、シクロオレフィンポリマーを用いることが好ましく、シクロオレフィンポリマーとスチレン－共役ジエンブロック共重合体水素化物の双方を用いることがより好ましい。 <<Resin>>
Here, the resin contained in the resin composition is not particularly limited, and examples of resins that can be used include cycloolefin polymers, polyethylene terephthalate (PET), polystyrene (PS), acrylic resins, polycarbonates, hydrogenated styrene-conjugated diene block copolymers, etc. These resins may be used alone or in combination of two or more kinds in any ratio.
Among the above-mentioned resins, from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, it is preferable to use a cycloolefin polymer, and it is more preferable to use both a cycloolefin polymer and a hydrogenated styrene-conjugated diene block copolymer.
［シクロオレフィンポリマー］
　ここで、シクロオレフィンポリマーとは、主鎖および／または側鎖に脂環構造を有する樹脂である。シクロオレフィンポリマーが有する脂環構造としては、飽和環状炭化水素（シクロアルカン）構造、不飽和環状炭化水素（シクロアルケン）構造などが挙げられる。また、シクロオレフィンポリマーの具体例としては、ノルボルネン系重合体、単環の環状オレフィン系重合体、環状共役ジエン系重合体、ビニル脂環式炭化水素系重合体、および、これらの水素化物などが挙げられる。これらの中でも、培養面に付着したタンパク質の変性を更に抑制する観点から、ノルボルネン系重合体およびその水素化物が好ましい。 [Cycloolefin polymer]
Here, the cycloolefin polymer is a resin having an alicyclic structure in the main chain and/or side chain. Examples of the alicyclic structure of the cycloolefin polymer include a saturated cyclic hydrocarbon (cycloalkane) structure and an unsaturated cyclic hydrocarbon (cycloalkene) structure. Specific examples of the cycloolefin polymer include norbornene-based polymers, monocyclic olefin-based polymers, cyclic conjugated diene-based polymers, vinyl alicyclic hydrocarbon-based polymers, and hydrogenated versions of these. Among these, norbornene-based polymers and hydrogenated versions of these are preferred from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface.
―ノルボルネン系重合体―
　ノルボルネン系重合体としては、例えば、ノルボルネン構造を有する単量体の開環重合体、もしくはノルボルネン構造を有する単量体と任意の単量体との開環重合体、またはそれらの水素化物；ノルボルネン構造を有する単量体の付加重合体、もしくはノルボルネン構造を有する単量体と任意の単量体との付加重合体、またはそれらの水素化物；等を挙げることができる。 --Norbornene-based polymers--
Examples of the norbornene-based polymer include a ring-opening polymer of a monomer having a norbornene structure, or a ring-opening polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydrogenated product thereof; an addition polymer of a monomer having a norbornene structure, or an addition polymer of a monomer having a norbornene structure and an arbitrary monomer, or a hydrogenated product thereof; and the like.
　ここで、ノルボルネン系重合体は、ノルボルネン骨格を有する単量体単位を、ノルボルネン系重合体を構成する全単量体単位に対して５０質量％以上、好ましくは６０質量％以上含む重合体である。より具体的には、ノルボルネン系重合体は、ノルボルネン骨格を有する単量体であるノルボルネン系単量体を重合してなるものであり、開環重合によって得られるものと、付加重合によって得られるものに大別される。 Here, a norbornene-based polymer is a polymer that contains 50% by mass or more, preferably 60% by mass or more, of monomer units having a norbornene skeleton relative to the total monomer units constituting the norbornene-based polymer. More specifically, norbornene-based polymers are obtained by polymerizing norbornene-based monomers, which are monomers having a norbornene skeleton, and are broadly divided into those obtained by ring-opening polymerization and those obtained by addition polymerization.
　開環重合によって得られるものとしては、ノルボルネン系単量体の開環重合体、ノルボルネン系単量体とこれと開環共重合可能なその他の単量体との開環重合体、および、これらの水素化物等が挙げられる。
　付加重合によって得られるものとしては、ノルボルネン系単量体の付加重合体およびノルボルネン系単量体とこれと共重合可能なその他の単量体との付加重合体等が挙げられる。
　これらの中でも、培養面に付着したタンパク質の変性を更に抑制する観点から、ノルボルネン系単量体の開環重合体水素化物が好ましい。 Examples of materials obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers capable of ring-opening copolymerization with the norbornene-based monomers, and hydrogenated products thereof.
Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith.
Among these, from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, a ring-opening polymer hydrogenate of a norbornene monomer is preferred.
　ノルボルネン系重合体の合成に使用可能なノルボルネン系単量体としては、ビシクロ［２．２．１］ヘプタ－２－エン（慣用名ノルボルネン）、５－メチル－ビシクロ［２．２．１］ヘプタ－２－エン、５，５－ジメチル－ビシクロ［２．２．１］ヘプタ－２－エン、５－エチル－ビシクロ［２．２．１］ヘプタ－２－エン、５－エチリデン－ビシクロ［２．２．１］ヘプタ－２－エン、５－ビニル－ビシクロ［２．２．１］ヘプタ－２－エン、５－プロペニルビシクロ［２．２．１］ヘプタ－２－エン、５－メトキシカルボニル－ビシクロ［２．２．１］ヘプタ－２－エン、５－シアノビシクロ［２．２．１］ヘプタ－２－エン、５－メチル－５－メトキシカルボニル－ビシクロ［２．２．１］ヘプタ－２－エン等の２環式単量体；
　トリシクロ［４．３．０1,6．１2,5］デカ－３，７－ジエン（慣用名ジシクロペンタジエン）、２－メチルジシクロペンタジエン、２，３－ジメチルジシクロペンタジエン、２，３－ジヒドロキシジシクロペンタジエン等の３環式単量体；
　テトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン（テトラシクロドデセン）、テトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－メチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－エチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－エチリデンテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８，９－ジメチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－エチル－９－メチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－エチリデン－９－メチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、８－メチル－８－カルボキシメチルテトラシクロ［４．４．０．１2,5．１7,10］－３－ドデセン、７，８－ベンゾトリシクロ［４．３．０．１2,5］デカ－３－エン（慣用名メタノテトラヒドロフルオレン：１，４－メタノ－１，４，４ａ，９ａ－テトラヒドロフルオレンともいう）、１，４－メタノ－８－メチル－１，４，４ａ，９ａ－テトラヒドロフルオレン、１，４－メタノ－８－クロロ－１，４，４ａ，９ａ－テトラヒドロフルオレン、１，４－メタノ－８－ブロモ－１，４，４ａ，９ａ－テトラヒドロフルオレン等の４環式単量体；等が挙げられる。 Norbornene monomers that can be used in the synthesis of norbornene polymers include bicyclo[2.2.1]hept-2-ene (commonly known as norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2.2.1]hept-2-ene, and 5-ethylidene-bicyclo[2.2.1]hept-2-ene. Bicyclic monomers such as hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene;
Tricyclic monomers such as tricyclo[ 4.3.01,6.12,5 ]deca-3,7-diene (commonly known as dicyclopentadiene), 2 -methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene;
Tetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8-methyltetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8-ethyltetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 2,5 . 1 7,10 ] -3 - dodecene , 8-ethyl-9-methyltetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 2,5 . 1 7,10 ]-3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 2,5 . tetracyclic monomers such as 1,4 - methano-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro- 1,4,4a ,9a-tetrahydrofluorene, and 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene; and the like.
　ノルボルネン系単量体と開環共重合可能なその他の単量体としては、シクロヘキセン、シクロヘプテン、シクロオクテン、１，４－シクロヘキサジエン、１，５－シクロオクタジエン、１，５－シクロデカジエン、１，５，９－シクロドデカトリエン、１，５，９，１３－シクロヘキサデカテトラエン等の単環のシクロオレフィン系単量体が挙げられる。
　これらの単量体は、置換基を１種または２種以上有していてもよい。置換基としては、アルキル基、アルキレン基、アリール基、シリル基、アルコキシカルボニル基、アルキリデン基等が挙げられる。 Other monomers capable of ring-opening copolymerization with norbornene monomers include monocyclic cycloolefin monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene.
These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.
　ノルボルネン系単量体と付加共重合可能なその他の単量体としては、エチレン、プロピレン、１－ブテン、１－ペンテン、１－ヘキセン等の炭素数２～２０のα－オレフィン系単量体；シクロブテン、シクロペンテン、シクロヘキセン、シクロオクテン、テトラシクロ［９．２．１．０2,10．０3,8］テトラデカ－３，５，７，１２－テトラエン（３ａ，５，６，７ａ－テトラヒドロ－４，７－メタノ－１Ｈ－インデンとも言う）等のシクロオレフィン系単量体；１，４－ヘキサジエン、４－メチル－１，４－ヘキサジエン、５－メチル－１，４－ヘキサジエン、１，７－オクタジエン等の非共役ジエン系単量体；等が挙げられる。 Other monomers capable of addition copolymerization with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cycloolefin monomers, such as cyclobutene, cyclopentene, cyclohexene, cyclooctene, and tetracyclo [ 9.2.1.02,10.03,8 ]tetradeca-3,5,7,12-tetraene (also called 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); and non-conjugated diene monomers, such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene.
　これらの中でも、ノルボルネン系単量体と付加共重合可能なその他の単量体としては、α－オレフィン系単量体が好ましく、エチレンがより好ましい。
　これらの単量体は、置換基を１種または２種以上有していてもよい。置換基としては、アルキル基、アルキレン基、アリール基、シリル基、アルコキシカルボニル基、アルキリデン基等が挙げられる。 Among these, as the other monomer capable of addition copolymerization with the norbornene monomer, an α-olefin monomer is preferable, and ethylene is more preferable.
These monomers may have one or more kinds of substituents, such as an alkyl group, an alkylene group, an aryl group, a silyl group, an alkoxycarbonyl group, and an alkylidene group.
　ノルボルネン系単量体の開環重合体、またはノルボルネン系単量体とこれと開環共重合可能なその他の単量体との開環重合体は、単量体成分を、公知の開環重合触媒の存在下で重合して得ることができる。開環重合触媒としては、例えば、ルテニウム、オスミウムなどの金属のハロゲン化物と、硝酸塩またはアセチルアセトン化合物、および還元剤とからなる触媒、あるいは、チタン、ジルコニウム、タングステン、モリブデンなどの金属のハロゲン化物またはアセチルアセトン化合物と、有機アルミニウム化合物とからなる触媒を用いることができる。
　ノルボルネン系単量体の開環重合体水素化物は、通常、上記開環重合体の重合溶液に、ニッケル、パラジウムなどの遷移金属を含む公知の水素化触媒を添加し、炭素－炭素不飽和結合を水素化することにより得ることができる。 The ring-opening polymer of a norbornene monomer, or the ring-opening polymer of a norbornene monomer and another monomer capable of ring-opening copolymerization therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst consisting of a halide of a metal such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst consisting of a halide or an acetylacetone compound of a metal such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used.
The hydrogenated ring-opening polymer of a norbornene monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.
　ノルボルネン系単量体の付加重合体、またはノルボルネン系単量体とこれと共重合可能なその他の単量体との付加重合体は、単量体成分を、公知の付加重合触媒の存在下で重合して得ることができる。付加重合触媒としては、例えば、チタン、ジルコニウムまたはバナジウム化合物と有機アルミニウム化合物とからなる触媒を用いることができる。 Addition polymers of norbornene-based monomers, or addition polymers of norbornene-based monomers and other monomers copolymerizable therewith, can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. As the addition polymerization catalyst, for example, a catalyst consisting of a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.
―単環の環状オレフィン系重合体―
　単環の環状オレフィン系重合体としては、例えば、シクロヘキセン、シクロヘプテン、シクロオクテンなどの、単環の環状オレフィン系単量体の付加重合体を用いることができる。 - Monocyclic olefin polymer -
As the monocyclic olefin polymer, for example, an addition polymer of a monocyclic olefin monomer such as cyclohexene, cycloheptene, or cyclooctene can be used.
―環状共役ジエン系重合体―
　環状共役ジエン系重合体としては、例えば、シクロペンタジエン、シクロヘキサジエンなどの環状共役ジエン系単量体を１，２－または１，４－付加重合した重合体およびその水素化物などを用いることができる。 -Cyclic conjugated diene polymer-
As the cyclic conjugated diene polymer, for example, a polymer obtained by 1,2- or 1,4-addition polymerization of a cyclic conjugated diene monomer such as cyclopentadiene or cyclohexadiene, or a hydrogenated product thereof can be used.
―ビニル脂環式炭化水素重合体―
　ビニル脂環式炭化水素重合体としては、例えば、ビニルシクロヘキセン、ビニルシクロヘキサンなどのビニル脂環式炭化水素系単量体の重合体およびその水素化物；などが挙げられる。ビニル脂環式炭化水素重合体は、これらの単量体と共重合可能な他の単量体との共重合体であってもよい。
　なお、本発明において、「スチレン－共役ジエンブロック共重合体水素化物」は「ビニル脂環式炭化水素重合体」に含まれないものとする。 - Vinyl alicyclic hydrocarbon polymer -
Examples of the vinyl alicyclic hydrocarbon polymer include polymers of vinyl alicyclic hydrocarbon monomers such as vinylcyclohexene and vinylcyclohexane, and hydrogenated products thereof. The vinyl alicyclic hydrocarbon polymer may be a copolymer of these monomers with other monomers that are copolymerizable therewith.
In the present invention, the "hydrogenated styrene-conjugated diene block copolymer" is not included in the "vinyl alicyclic hydrocarbon polymer".
　シクロオレフィンポリマーの分子量に格別な制限はないが、シクロヘキサン溶液（重合体が溶解しない場合はトルエン溶液）のゲル・パーミエーション・クロマトグラフィーで測定したポリイソプレン換算の重量平均分子量で、通常５，０００以上であり、好ましくは５，０００～５００，０００、より好ましくは８，０００～２００，０００、更に好ましくは１０，０００～１００，０００である。重量平均分子量がこの範囲内であるときに、機械的強度と成形加工性とが高度にバランスし、好適である。 There are no particular limitations on the molecular weight of the cycloolefin polymer, but the weight average molecular weight, calculated as polyisoprene, measured by gel permeation chromatography of a cyclohexane solution (a toluene solution if the polymer does not dissolve) is usually 5,000 or more, preferably 5,000 to 500,000, more preferably 8,000 to 200,000, and even more preferably 10,000 to 100,000. When the weight average molecular weight is within this range, mechanical strength and moldability are well balanced, making this an ideal range.
　そして、樹脂中のシクロオレフィンポリマーの含有量は、樹脂の全量１００質量部に対して、９９．５０質量部以上であることが好ましく、９９．７０質量部以上であることがより好ましく、９９．８０質量部以上であることが更に好ましく、９９．９９質量部以下であることが好ましく、９９．９８質量部以下であることがより好ましい。シクロオレフィンポリマーの含有量が樹脂１００質量部に対して上記範囲内であれば、培養面に付着したタンパク質の変性を更に抑制するとともに、培養面へのタンパク質の吸着性を高めることができる。 The content of the cycloolefin polymer in the resin is preferably 99.50 parts by mass or more, more preferably 99.70 parts by mass or more, and even more preferably 99.80 parts by mass or more, and preferably 99.99 parts by mass or less, and more preferably 99.98 parts by mass or less, relative to 100 parts by mass of the resin. If the content of the cycloolefin polymer is within the above range relative to 100 parts by mass of the resin, the denaturation of the protein attached to the culture surface can be further suppressed, and the adsorption of the protein to the culture surface can be increased.
［スチレン－共役ジエンブロック共重合体水素化物］
　スチレン－共役ジエンブロック共重合体水素化物は、スチレン系単量体由来の繰り返し単位を主成分とするスチレンブロックと、共役ジエン単量体由来の繰り返し単位を主成分とするジエンブロックとを有するスチレンブロック－共役ジエンブロック共重合体の水素化物である。 [Hydrogenated styrene-conjugated diene block copolymer]
The hydrogenated styrene-conjugated diene block copolymer is a hydrogenated styrene block-conjugated diene block copolymer having a styrene block mainly composed of repeating units derived from a styrene-based monomer and a diene block mainly composed of repeating units derived from a conjugated diene monomer.
　ここで、スチレン－共役ジエンブロック共重合体水素化物としては、例えば市販のものを用いることができる。また、スチレン－共役ジエンブロック共重合体水素化物は、公知の方法に従ってスチレン－共役ジエンブロック共重合体を得た後、得られたスチレン－共役ジエンブロック共重合体の共役ジエンブロックの主鎖および側鎖の炭素－炭素不飽和結合を水素化して得てもよい。なお、不飽和結合の水素化方法や反応形態などは特に限定されず、公知の方法に従って行えばよい。 Here, as the hydrogenated styrene-conjugated diene block copolymer, for example, a commercially available product can be used. Alternatively, the hydrogenated styrene-conjugated diene block copolymer may be obtained by obtaining a styrene-conjugated diene block copolymer according to a known method, and then hydrogenating the carbon-carbon unsaturated bonds in the main chain and side chain of the conjugated diene block of the obtained styrene-conjugated diene block copolymer. The hydrogenation method of the unsaturated bonds and the reaction form are not particularly limited, and may be performed according to a known method.
　そして、樹脂中のスチレン－共役ジエンブロック共重合体水素化物の含有量は、樹脂の全量１００質量部に対して、０．０１質量部以上であることが好ましく、０．０２質量部以上であることがより好ましく、０．５質量部以下であることが好ましく、０．３質量部以下であることがより好ましく、０．２質量部以下であることが更に好ましい。スチレン－共役ジエンブロック共重合体水素化物の含有量が樹脂１００質量部に対して上記範囲内であれば、培養面に付着したタンパク質の変性を更に抑制するとともに、培養面へのタンパク質の吸着性を高めることができる。 The content of the hydrogenated styrene-conjugated diene block copolymer in the resin is preferably 0.01 parts by mass or more, more preferably 0.02 parts by mass or more, preferably 0.5 parts by mass or less, more preferably 0.3 parts by mass or less, and even more preferably 0.2 parts by mass or less, relative to 100 parts by mass of the total amount of the resin. If the content of the hydrogenated styrene-conjugated diene block copolymer is within the above range relative to 100 parts by mass of the resin, the denaturation of proteins attached to the culture surface can be further suppressed, and the adsorption of proteins to the culture surface can be increased.
＜＜その他の成分＞＞
　本発明の細胞培養容器が備える培養面の形成に用いる樹脂組成物は、上述した樹脂以外の成分（その他の成分）を更に含有することができる。その他の成分としては、熱可塑性樹脂材料で通常用いられている配合剤、例えば、軟質重合体、酸化防止剤、紫外線吸収剤、光安定剤、近赤外線吸収剤、離型剤、染料や顔料等の着色剤、可塑剤、帯電防止剤、蛍光増白剤などが挙げられる。なお、その他の成分は、１種を単独で用いてもよく、２種以上を任意の比率で組み合わせて用いてもよい。 <<Other ingredients>>
The resin composition used to form the culture surface of the cell culture vessel of the present invention may further contain components other than the above-mentioned resins (other components). Examples of the other components include compounding agents commonly used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent whitening agents. The other components may be used alone or in combination of two or more in any ratio.
　酸化防止剤としては、フェノール系酸化防止剤、有機ホスファイト系酸化防止剤、硫黄系酸化防止剤などが挙げられる。紫外線吸収剤としては、ベンゾフェノン系紫外線吸収剤、ベンゾトリアゾール系紫外線吸収剤などが挙げられる。光安定剤としては、ヒンダードアミン系安定剤などが挙げられる。中でも、培養面に付着したタンパク質の変性を更に抑制するとともに培養面へのタンパク質の吸着性を高める観点から、樹脂組成物は、酸化防止剤を含むことが好ましい。 Examples of antioxidants include phenol-based antioxidants, organic phosphite-based antioxidants, and sulfur-based antioxidants. Examples of ultraviolet absorbers include benzophenone-based ultraviolet absorbers and benzotriazole-based ultraviolet absorbers. Examples of light stabilizers include hindered amine-based stabilizers. Among these, from the viewpoints of further suppressing the denaturation of proteins attached to the culture surface and increasing the adsorption of proteins to the culture surface, it is preferable that the resin composition contains an antioxidant.
　樹脂組成物中における酸化防止剤の含有量は、樹脂１００質量部に対して０．０５質量部以上であることが好ましく、０．１質量部以上であることがより好ましく、２質量部以下であることが好ましく、１質量部以下であることがより好ましく、０．５質量部以下であることが更に好ましい。酸化防止剤の含有量が樹脂１００質量部に対して上記範囲内であれば、培養面に付着したタンパク質の変性を更に抑制するとともに、培養面へのタンパク質の吸着性を高めることができる。 The content of the antioxidant in the resin composition is preferably 0.05 parts by mass or more, more preferably 0.1 parts by mass or more, and preferably 2 parts by mass or less, more preferably 1 part by mass or less, and even more preferably 0.5 parts by mass or less, per 100 parts by mass of resin. If the content of the antioxidant is within the above range per 100 parts by mass of resin, the denaturation of proteins attached to the culture surface can be further suppressed, and the adsorption of proteins to the culture surface can be increased.
　樹脂組成物のガラス転移温度は、使用目的に応じて適宜選択すればよいが、通常５０～３００℃、好ましくは８０～２８０℃、より好ましくは９０～２５０℃、更に好ましくは９０～２００℃である。ガラス転移温度がこの範囲内であるときに、耐熱性と成形加工性とが高度にバランスし、好適である。
　本発明において、樹脂組成物のガラス転移温度は、ＪＩＳ　Ｋ　７１２１に基づいて測定されたものである。 The glass transition temperature of the resin composition may be appropriately selected depending on the intended use, but is usually 50 to 300° C., preferably 80 to 280° C., more preferably 90 to 250° C., and even more preferably 90 to 200° C. When the glass transition temperature is within this range, heat resistance and moldability are highly balanced, which is preferable.
In the present invention, the glass transition temperature of the resin composition is measured based on JIS K 7121.
＜＜樹脂組成物の調製方法＞＞
　上述した樹脂と、任意にその他の成分とを含む樹脂組成物を得る際の混合方法は、特に限定されず、例えば、単軸押出機、二軸押出機、バンバリーミキサー、ニーダー、フィーダールーダー等の既知の溶融混練機を用いて行うことができる。混合後は、常法に従って、棒状に押出し、ストランドカッターで適当な長さに切ることで、ペレット化することができる。 <<Method for preparing resin composition>>
The mixing method for obtaining a resin composition containing the above-mentioned resin and optionally other components is not particularly limited, and can be performed using a known melt kneader such as a single screw extruder, a twin screw extruder, a Banbury mixer, a kneader, a feeder ruder, etc. After mixing, the mixture can be extruded into a rod shape according to a conventional method, and cut to an appropriate length with a strand cutter to form pellets.
＜１６８０ｃｍ-1バンドの面積比率＞
　ここで、本発明の細胞培養容器は、培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルの振動数１６００ｃｍ-1以上１７００ｃｍ-1以下の領域をバンド分解したときに、すべてのバンドの合計面積に対する振動数１６８０ｃｍ-1にピークを有するバンドの面積の比率（１６８０ｃｍ-1バンドの面積比率）が１０％以上であることが必要であり、１６８０ｃｍ-1バンドの面積比率は、１１％以上であることが好ましく、１２％以上であることがより好ましく、１３％以上であることが更に好ましい。１６８０ｃｍ-1バンドの面積比率が１０％未満であると、培養面に付着したタンパク質の変性度が増加する。また、１６８０ｃｍ-1バンドの面積比率の上限は、特に限定されず、例えば、３０％以下とすることができ、２５％以下とすることができる。
　なお、１６８０ｃｍ-1バンドの面積比率は、例えば、培養面の形成に用いる樹脂組成物中の樹脂の種類、培養面の表面処理の有無、表面処理の種類や条件などを変更することにより調整することができる。具体的には、表面処理を行わないこと、または、表面処理として熱処理を行う場合には処理時間を短くすることで、１６８０ｃｍ-1バンドの面積比率を大きくすることができる。 <Area ratio of 1680 cm -1 band>
Here, in the cell culture vessel of the present invention, when the region of frequencies from 1600 cm -1 to 1700 cm -1 in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is decomposed into bands, the ratio of the area of the band having a peak at a frequency of 1680 cm -1 to the total area of all bands (area ratio of the 1680 cm -1 band) must be 10% or more, and the area ratio of the 1680 cm -1 band is preferably 11% or more, more preferably 12% or more, and even more preferably 13% or more. If the area ratio of the 1680 cm -1 band is less than 10%, the degree of denaturation of the protein attached to the culture surface increases. In addition, the upper limit of the area ratio of the 1680 cm -1 band is not particularly limited, and can be, for example, 30% or less, or 25% or less.
The area ratio of the 1680 cm band can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the presence or absence of surface treatment of the culture surface, the type and conditions of the surface treatment, etc. Specifically, the area ratio of the 1680 cm band can be increased by not performing a surface treatment, or by shortening the treatment time when a heat treatment is performed as the surface treatment.
＜π－π*結合に由来するピーク＞
　そして、本発明の細胞培養容器は、培養面に付着したタンパク質の変性を更に抑制する観点から、培養面のＸＰＳスペクトルがπ－π*結合に由来するピークを有さないことが好ましい。
　なお、培養面のＸＰＳスペクトルにおけるπ－π*結合に由来するピークの有無は、例えば、培養面の形成に用いる樹脂組成物中の樹脂の種類を変更することにより調整することができる。具体的には、樹脂として芳香環を有さない樹脂を用いることで、π－π*結合に由来するピークを有さないＸＰＳスペクトルを得ることができる。 <Peaks derived from π-π * bonds>
From the viewpoint of further suppressing denaturation of proteins attached to the culture surface, the cell culture vessel of the present invention preferably has an XPS spectrum of the culture surface that does not have a peak derived from a π-π * bond.
The presence or absence of a peak derived from a π-π * bond in the XPS spectrum of the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface. Specifically, by using a resin that does not have an aromatic ring, an XPS spectrum that does not have a peak derived from a π-π * bond can be obtained.
＜酸素含有官能基の存在比＞
　また、本発明の細胞培養容器は、培養面におけるＣ－Ｏ基の量に対するＣ＝Ｏ基およびＯ－Ｃ＝Ｏ基の合計量の比（酸素含有官能基の存在比）が、０．４５以下であることが好ましく、０．４０以下であることがより好ましく、０．３５以下であることが更に好ましく、０．３３以下であることが特に好ましい。培養面における酸素含有官能基の存在比が０．４５以下であれば、培養面に付着したタンパク質の変性を更に抑制することができる。また、培養面における酸素含有官能基の存在比の下限は、特に限定されず、例えば、０．０５以上とすることができ、０．１０以上とすることができる。
　なお、培養面における酸素含有官能基の存在比は、例えば、培養面の形成に用いる樹脂組成物中の樹脂の種類、樹脂組成物から培養面を形成する際の成形条件、培養面の表面処理の有無、表面処理の種類や条件などを変更することにより調整することができる。具体的には、表面処理を行わないこと、または、表面処理として熱処理を行う場合には処理時間を短くすることで、培養面における酸素含有官能基の存在比を低下させることができる。 <Ratio of Oxygen-Containing Functional Groups>
Furthermore, in the cell culture vessel of the present invention, the ratio of the total amount of C=O groups and O-C=O groups to the amount of C-O groups on the culture surface (abundance ratio of oxygen-containing functional groups) is preferably 0.45 or less, more preferably 0.40 or less, even more preferably 0.35 or less, and particularly preferably 0.33 or less. If the abundance ratio of oxygen-containing functional groups on the culture surface is 0.45 or less, denaturation of proteins attached to the culture surface can be further suppressed. Furthermore, the lower limit of the abundance ratio of oxygen-containing functional groups on the culture surface is not particularly limited, and can be, for example, 0.05 or more, or 0.10 or more.
The abundance ratio of oxygen-containing functional groups on the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, the presence or absence of surface treatment on the culture surface, the type and conditions of the surface treatment, etc. Specifically, the abundance ratio of oxygen-containing functional groups on the culture surface can be reduced by not performing surface treatment, or by shortening the treatment time when heat treatment is performed as the surface treatment.
＜炭素元素量に対する酸素元素量の割合＞
　ここで、本発明の細胞培養容器は、培養面における炭素元素量に対する酸素元素量の割合が、２．０％以上であることが好ましく、２．５％以上であることがより好ましく、３．０％以上であることが更に好ましく、１０．０％以下であることが好ましく、７．５％以下であることがより好ましく、５．０％以下であることが更に好ましく、３．９％以下であることがより一層好ましく、２．６％以下であることが特に好ましい。培養面における炭素元素量に対する酸素元素量の割合が２．０％以上であれば、培養面へのタンパク質の吸着性を高めることができる。一方、培養面における炭素元素量に対する酸素元素量の割合が１０．０％％以下であれば、培養面に付着したタンパク質の変性を更に抑制することができる。
　なお、培養面における炭素元素量に対する酸素元素量の割合は、例えば、培養面の形成に用いる樹脂組成物中の樹脂の種類、樹脂組成物から培養面を形成する際の成形条件、培養面の表面処理の有無、表面処理の種類や条件などを変更することにより調整することができる。具体的には、表面処理を行わないこと、または、表面処理として熱処理を行う場合には処理時間を短くすることで、培養面における炭素元素量に対する酸素元素量の割合を低下させることができる。 <Ratio of oxygen element content to carbon element content>
Here, in the cell culture vessel of the present invention, the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is preferably 2.0% or more, more preferably 2.5% or more, even more preferably 3.0% or more, preferably 10.0% or less, more preferably 7.5% or less, even more preferably 5.0% or less, even more preferably 3.9% or less, and particularly preferably 2.6% or less. If the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is 2.0% or more, the adsorption of protein to the culture surface can be increased. On the other hand, if the ratio of the amount of oxygen element to the amount of carbon element on the culture surface is 10.0%% or less, the denaturation of protein attached to the culture surface can be further suppressed.
The ratio of the amount of oxygen element to the amount of carbon element on the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, the presence or absence of a surface treatment on the culture surface, the type and conditions of the surface treatment, etc. Specifically, the ratio of the amount of oxygen element to the amount of carbon element on the culture surface can be reduced by not performing a surface treatment, or by shortening the treatment time when a heat treatment is performed as the surface treatment.
＜タンパク質吸着率＞
　そして、本発明の細胞培養容器は、培養面のタンパク質吸着率が、６０％以上であることが好ましく、６５％以上であることがより好ましく、７０％以上であることが更に好ましい。培養面のタンパク質吸着率が６０％以上であれば、培養面のタンパク質吸着性を良好に確保することができる。また、培養面のタンパク質吸着率の上限は特に限定されず、例えば、１００％以下とすることができ、９０％以下とすることができる。
　なお、本発明において、培養面のタンパク質吸着率は、本明細書の実施例に記載の方法を用いて測定することができる。
　また、培養面のタンパク質吸着率は、例えば、培養面の形成に用いる樹脂組成物中の樹脂の種類、樹脂組成物から培養面を形成する際の成形条件、培養面の表面処理の有無、表面処理の種類や条件などを変更することにより調整することができる。 <Protein adsorption rate>
In the cell culture vessel of the present invention, the protein adsorption rate of the culture surface is preferably 60% or more, more preferably 65% or more, and even more preferably 70% or more. If the protein adsorption rate of the culture surface is 60% or more, the protein adsorption property of the culture surface can be well ensured. Furthermore, the upper limit of the protein adsorption rate of the culture surface is not particularly limited, and can be, for example, 100% or less, or 90% or less.
In the present invention, the protein adsorption rate of the culture surface can be measured by the method described in the Examples of this specification.
In addition, the protein adsorption rate of the culture surface can be adjusted, for example, by changing the type of resin in the resin composition used to form the culture surface, the molding conditions when forming the culture surface from the resin composition, whether or not the culture surface is surface-treated, the type and conditions of the surface treatment, etc.
＜細胞培養容器の製造方法＞
　本発明の細胞培養容器を製造する方法は、特に限定されないが、本発明の細胞培養容器は、例えば、上述した樹脂と、任意にその他の成分とを含む樹脂組成物を成形して培養面を得る工程（成形工程）と、任意に培養面に表面処理を施す工程（表面処理工程）とを経て製造され得る。 <Method of manufacturing cell culture vessel>
The method for producing the cell culture vessel of the present invention is not particularly limited, but the cell culture vessel of the present invention can be produced, for example, through a step of molding a resin composition containing the above-mentioned resin and, optionally, other components, to obtain a culture surface (molding step), and optionally a step of applying a surface treatment to the culture surface (surface treatment step).
＜＜成形工程＞＞
　ここで、樹脂組成物を成形する方法としては、特に限定されず、培養面の所望の形状に応じて既知の成形方法から適宜選択しうる。このような既知の成形方法としては、例えば、押出成形、射出成形、インフレーション成形、ブロー成形、押出ブロー成形、射出ブロー成形、プレス成形、真空成形、パウダースラッシュ成形、カレンダー成形、発泡成形、熱成形等が挙げられる。これらの中でも、成形方法として、射出成形を用いることが好ましい。 <<Molding process>>
Here, the method of molding the resin composition is not particularly limited, and can be appropriately selected from known molding methods according to the desired shape of the culture surface. Such known molding methods include, for example, extrusion molding, injection molding, inflation molding, blow molding, extrusion blow molding, injection blow molding, press molding, vacuum molding, powder slush molding, calendar molding, foam molding, thermoforming, etc. Among these, it is preferable to use injection molding as the molding method.
　そして、射出成形によって培養面を形成する場合、射出成形に用いる金型の温度（以下、単に「金型温度」と略記する場合がある。）は、樹脂組成物のガラス転移温度をＴｇ（℃）として、（Ｔｇ－４０）℃以上であることが好ましく、（Ｔｇ－２０）℃以下であることが好ましい。金型温度が上述した範囲内であれば、培養面に付着したタンパク質の変性を更に抑制するとともに、培養面へのタンパク質の吸着性を高めることができる。また、射出成形によって培養面を形成する場合、射出成形機が備えるシリンダーの温度（以下、単に「シリンダー温度」と略記する場合がある。）は、樹脂組成物のガラス転移温度をＴｇ（℃）として、（Ｔｇ＋１２０）℃以上であることが好ましく、（Ｔｇ＋１７０）℃以下であることが好ましい。シリンダー温度が上述した範囲内であれば、培養面に付着したタンパク質の変性を更に抑制するとともに、培養面へのタンパク質の吸着性を高めることができる。 When the culture surface is formed by injection molding, the temperature of the mold used for injection molding (hereinafter, sometimes simply referred to as "mold temperature") is preferably (Tg-40)°C or higher and preferably (Tg-20)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition. If the mold temperature is within the above-mentioned range, the denaturation of the protein attached to the culture surface can be further suppressed and the adsorption of the protein to the culture surface can be increased. Furthermore, when the culture surface is formed by injection molding, the temperature of the cylinder (hereinafter, sometimes simply referred to as "cylinder temperature") provided in the injection molding machine is preferably (Tg+120)°C or higher and preferably (Tg+170)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition. If the cylinder temperature is within the above-mentioned range, the denaturation of the protein attached to the culture surface can be further suppressed and the adsorption of the protein to the culture surface can be increased.
＜＜表面処理工程＞＞
　培養面に施す表面処理の種類としては、特に限定されず、既知の表面処理方法から適宜選択しうる。このような既知の表面処理方法としては、熱処理、プラズマ処理、真空紫外線処理、コロナ処理、オゾン処理等が挙げられる。これらの中でも、培養面に付着したタンパク質の変性を更に抑制する観点から、熱処理が好ましい。 <<Surface treatment process>>
The type of surface treatment applied to the culture surface is not particularly limited and can be appropriately selected from known surface treatment methods. Such known surface treatment methods include heat treatment, plasma treatment, vacuum ultraviolet treatment, corona treatment, ozone treatment, etc. Among these, heat treatment is preferred from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface.
　ここで、熱処理における処理温度は、培養面に付着したタンパク質の変性を更に抑制する観点から、樹脂組成物のガラス転移温度をＴｇ（℃）として、（Ｔｇ－１０）℃以下とすることが好ましく、（Ｔｇ－１５）℃以下とすることがより好ましい。なお、熱処理における処理温度の下限は、特に限定されず、例えば、（Ｔｇ－４０）℃以上とすることができ、（Ｔｇ－３０）℃以上とすることができる。 Here, from the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, the treatment temperature in the heat treatment is preferably (Tg-10)°C or lower, and more preferably (Tg-15)°C or lower, where Tg (°C) is the glass transition temperature of the resin composition. The lower limit of the treatment temperature in the heat treatment is not particularly limited, and can be, for example, (Tg-40)°C or higher, or (Tg-30)°C or higher.
　また、熱処理における処理時間は、処理温度に応じて適宜設定すればよい。培養面に付着したタンパク質の変性を更に抑制する観点からは、熱処理における処理時間は、５０時間以下とすることが好ましく、４０時間以下とすることがより好ましい。なお、熱処理における処理時間の下限は、特に限定されず、例えば、１０時間以上とすることができ、２０時間以上とすることができる。 The treatment time in the heat treatment may be set appropriately depending on the treatment temperature. From the viewpoint of further suppressing the denaturation of proteins attached to the culture surface, the treatment time in the heat treatment is preferably 50 hours or less, and more preferably 40 hours or less. The lower limit of the treatment time in the heat treatment is not particularly limited, and can be, for example, 10 hours or more, or 20 hours or more.
＜＜その他の工程＞＞
　また、本発明の細胞培養容器は、任意に、上述した成形工程および表面処理工程以外の工程（その他の工程）を経て製造してもよい。その他の工程としては、例えば、成形工程に先んじて樹脂組成物を予備乾燥する工程（予備乾燥工程）、培養面と他の部材を組み合わせて細胞培養容器を組み立てる工程（組み立て工程）、細胞培養容器に滅菌処理を施す工程（滅菌工程）などが挙げられる。 <<Other processes>>
The cell culture vessel of the present invention may be produced through any step (other step) other than the above-mentioned molding step and surface treatment step, for example, a step of pre-drying the resin composition prior to the molding step (pre-drying step), a step of assembling the cell culture vessel by combining the culture surface with other members (assembly step), a step of sterilizing the cell culture vessel (sterilization step), etc.
　以下、本発明について実施例に基づき具体的に説明するが、本発明はこれら実施例に限定されるものではない。なお、以下の説明において、量を表す「％」および「部」は、特に断らない限り、質量基準である。
　そして、実施例および比較例において、炭素元素量に対する酸素元素量の割合、酸素含有官能基の存在比、π－π*結合に由来するピークの有無、培養面に付着した組み換えヒトフィブロネクチンフラグメントの１６８０ｃｍ-1バンドの面積比率、およびタンパク質吸着率は、下記の方法で評価した。 The present invention will be specifically described below based on examples, but the present invention is not limited to these examples. In the following description, "%" and "parts" expressing amounts are based on mass unless otherwise specified.
In the examples and comparative examples, the ratio of the amount of oxygen element to the amount of carbon element, the abundance ratio of oxygen-containing functional groups, the presence or absence of a peak derived from π-π * bonds, the area ratio of the 1680 cm -1 band of the recombinant human fibronectin fragment attached to the culture surface, and the protein adsorption rate were evaluated by the following methods.
＜Ｘ線光電子分光法の測定＞
　実施例および比較例で作製したディッシュの底面（培養面）を測定対象として、Ｘ線光電子分光法（ＸＰＳ）を用いて超真空中で軟Ｘ線を照射した際に表面から放出される光電子をアナライザーで検出することにより、物質中の束縛電子の結合エネルギーから培養面の元素情報を取得した。測定条件は以下の通りである。
　装置：アルバック・ファイ社製、製品名「Ｑｕａｎｔｅｒａ　ＳＸＭ」
　励起Ｘ線：単色化ＡｌＫα12線（１４８６．６ｅＶ）
　Ｘ線直径：２００μｍ
　光電子検出角度：４５°
　横軸補正：Ｃ１ｓメインピークを２８４．６ｅＶに合わせた
［炭素元素量に対する酸素元素量の割合］
　ＸＰＳのワイドスキャンモードを用い、培養面に存在する元素種を同定した（測定範囲：０ｅＶ～１１００ｅＶ）。そして、同定された各元素のピーク面積を積分し、元素別の感度係数で補正後、培養面における炭素元素量に対する酸素元素量の割合（％）を算出した。
［酸素含有官能基の存在比］
　ＸＰＳのナロースキャンモードを用い、Ｃ１ｓメインピークが現れる結合エネルギー範囲を高分解能で走査した（測定範囲：２７５ｅＶ～３００ｅＶ）。得られたスペクトルをピーク分割し、個々のピーク位置（結合エネルギーの値）から、Ｃ－Ｏ、Ｃ＝ＯおよびＯ－Ｃ＝Ｏにそれぞれ対応するピークを同定した。そして、Ｃ－Ｏに対応するピークの面積をＳ１、Ｃ＝Ｏに対応するピークの面積をＳ２、Ｏ－Ｃ＝Ｏに対応するピークの面積をＳ３として、下記式から、培養面におけるＣ－Ｏ基の量に対するＣ＝Ｏ基およびＯ－Ｃ＝Ｏ基の合計量の比（酸素含有官能基の存在比）を算出した。
　　酸素含有官能基の存在比＝（Ｓ２＋Ｓ３）／Ｓ１
［π－π*結合に由来するピークの有無］
　ＸＰＳのナロースキャンモードを用い、Ｃ１ｓメインピークが現れる結合エネルギー範囲を高分解能で走査した（測定範囲：２７５ｅＶ～３００ｅＶ）。得られたスペクトルにおいて、２９１ｅＶ近傍に、π－π*結合に由来するピークが観察されるか否かを確認した。
＜１６８０ｃｍ-1バンドの面積比率＞
［１．タンパク質処理］
　ＲｅｔｒｏＮｅｃｔｉｎ（タカラバイオ社製、型番「Ｔ１００Ｂ」）を１０μｇ／ｍＬになるように重水（Ｄ2Ｏ、関東化学社製）に溶解し、タンパク質溶液を得た。上記タンパク質溶液を実施例および比較例で作製したディッシュ（直径３５ｍｍ）に２.５ｍＬ入れ、３７℃で４５分間保持した。次に、上記ディッシュ内のタンパク質溶液を除去した後に、重水を３ｍＬ入れてディッシュを洗浄した。その後、ディッシュ内の洗浄水を除去し、新しい重水を３ｍＬ入れ洗浄した。上記の洗浄操作を３回実施した後、洗浄水を除去し、ディッシュを大気環境下において目視で液体が観測できなくなるまで乾燥させた。
［２．ＦＴ－ＩＲ測定およびバンド分解］
　上記タンパク質処理後のディッシュの底面（培養面）を、ＦＴ－ＩＲ装置（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製、製品名「Ｎｉｃｌｅｔ　６７００」）のＡＴＲアタッチメントのゲルマニウムプリズムに密着させ、赤外吸収スペクトル測定を行った。測定および解析には、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社製のソフトウェア「ＯＭＮＩＣ」を使用した。取得した実測スペクトルのアミドＩ領域（１６００～１７００ｃｍ-1）を二次微分し、二次微分スペクトルを求めた。次に、二次微分スペクトル中の負のピーク（谷）の位置に基づき、実測スペクトルのアミドＩ領域（１６００～１７００ｃｍ-1）をバンド分解した。この際、バンド分解に用いるバンドの１つを、１６８０ｃｍ-1にピークを有するバンドとした。そして、バンド分解により得られたすべてのバンドの合計面積をＳＡ、１６８０ｃｍ-1にピークを有するバンドの面積をＳＢとして、下記式により、「１６８０ｃｍ-1バンドの面積比率」を算出した。
　　１６８０ｃｍ-1バンドの面積比率（％）＝（ＳＢ／ＳＡ）×１００
なお、測定条件および解析条件は以下のとおりである。
　測定法：ＡＴＲ法
　積算回数：３２回
　分解能：１６ｃｍ-1
　バックグラウンド補正：サンプル測定前
　スペクトル強度：吸光度
　バンド形状を表す関数（分布関数）：Ｇａｕｓｓｉａｎ関数
　ピーク検出感度：低感度
　ピークフィッティングにおけるノイズレベル：０．０１
　ピークフィッティングにおけるベースライン補正：１次（線形）補間
＜タンパク質吸着率＞
［１．タンパク質処理］
　ＲｅｔｒｏＮｅｃｔｉｎ（タカラバイオ社製、型番「Ｔ１００Ｂ」）を１０μｇ／ｍＬになるようにリン酸緩衝生理食塩水（ＰＢＳ）溶液（富士フイルム和光純薬社製、ＰＢＳ（－））に溶解し、タンパク質溶液を得た。上記タンパク質溶液を実施例および比較例で作製したディッシュ（直径３５ｍｍ）に２．５ｍＬ入れ、３７℃で４５分間保持した。次に、上記ディッシュ内のタンパク質溶液を除去し、３．０ｍＬの滅菌水を用いて洗浄を１回実施した。その後、上記ディッシュに金コロイド溶液（バイオラッド社製、型番「＃１７０６５２７」）を２ｍＬ添加し、２５℃で４５分間保持した。次に、上記ディッシュ内の金コロイド溶液を除去した後に、滅菌水を３ｍＬ入れてディッシュを洗浄した。その後、ディッシュ内の洗浄水を除去し、再度滅菌水を３ｍＬ入れてディッシュを洗浄した。最後に、洗浄水を除去し、ディッシュを大気環境下において目視で液体が観測できなくなるまで乾燥させた。
［２．ルテニウム染色処理］
　ガラス製デシケータ内に四酸化ルテニウムと上記タンパク質処理後のディッシュを入れ、気相環境下で３０分間ルテニウム染色を行った。
［３．ＳＥＭ撮影］
　上記ルテニウム染色処理後のディッシュ底面（培養面）の中央付近の任意の視野を、電界放出形操作電子顕微鏡（ＦＥ－ＳＥＭ）（日立ハイテク社製、製品名「ＳＵ８２２０」）を用いて、二次電子像、加速電圧０．７ｋＶの条件で、倍率５万倍にて観察し、走査電子顕微鏡（ＳＥＭ）観察画像を取得した。
［４．画像解析］
　上記ＳＥＭ観察画像を画像処理ソフトＩｍａｇｅＪ（オープンソース）で読み込み、画像データを８ｂｉｔ化した。ルテニウム染色部分（灰色）および金コロイド部分（白色）を明部とし、ルテニウム染色部分および金コロイド部分以外の部分（黒色）を暗部として二値化し、ＳＥＭ観察画像の全画素中の明部の割合（％）をタンパク質吸着率とした。タンパク質吸着率が大きいほど、細胞培養容器はタンパク質吸着性に優れていることを示す。なお、タンパク質吸着率が６０％以上であれば、培養面のタンパク質吸着性が十分に確保されている。 <X-ray photoelectron spectroscopy measurement>
The bottom surface (culture surface) of the dishes prepared in the examples and comparative examples was used as the measurement object, and the photoelectrons emitted from the surface when irradiated with soft X-rays in an ultra-vacuum using X-ray photoelectron spectroscopy (XPS) were detected with an analyzer to obtain elemental information on the culture surface from the binding energy of bound electrons in the material. The measurement conditions were as follows.
Equipment: ULVAC-PHI, product name "Quantera SXM"
Excitation X-ray: Monochromated AlKα 12 ray (1486.6 eV)
X-ray diameter: 200 μm
Photoelectron detection angle: 45°
Horizontal axis correction: C1s main peak was adjusted to 284.6 eV [ratio of oxygen element amount to carbon element amount]
The elemental species present on the culture surface were identified using the wide scan mode of XPS (measurement range: 0 eV to 1100 eV). The peak area of each identified element was integrated and corrected with the sensitivity coefficient for each element, after which the ratio (%) of the amount of oxygen element to the amount of carbon element on the culture surface was calculated.
[Ratio of oxygen-containing functional groups]
Using the narrow scan mode of XPS, the binding energy range where the C1s main peak appears was scanned at high resolution (measurement range: 275 eV to 300 eV). The obtained spectrum was divided into peaks, and the peaks corresponding to C-O, C=O, and O-C=O were identified from the individual peak positions (binding energy values). Then, the area of the peak corresponding to C-O was designated S1, the area of the peak corresponding to C=O was designated S2, and the area of the peak corresponding to O-C=O was designated S3. The ratio of the total amount of C=O groups and O-C=O groups to the amount of C-O groups on the culture surface (abundance ratio of oxygen-containing functional groups) was calculated from the following formula.
Abundance ratio of oxygen-containing functional groups=(S2+S3)/S1
[Presence or absence of peaks derived from π-π * bonds]
Using the narrow scan mode of XPS, the binding energy range in which the C1s main peak appears was scanned with high resolution (measurement range: 275 eV to 300 eV). In the obtained spectrum, it was confirmed whether a peak derived from a π-π * bond was observed near 291 eV.
<Area ratio of 1680 cm -1 band>
1. Protein Processing
RetroNectin (manufactured by Takara Bio, model number "T100B") was dissolved in heavy water ( D2O , manufactured by Kanto Chemical) to a concentration of 10 μg/mL to obtain a protein solution. 2.5 mL of the protein solution was placed in the dish (diameter 35 mm) prepared in the Examples and Comparative Examples, and kept at 37°C for 45 minutes. Next, the protein solution in the dish was removed, and 3 mL of heavy water was added to wash the dish. Thereafter, the washing water in the dish was removed, and 3 mL of new heavy water was added for washing. After carrying out the above washing operation three times, the washing water was removed, and the dish was dried in an air environment until liquid could no longer be observed with the naked eye.
2. FT-IR Measurement and Band Resolution
The bottom surface (culture surface) of the dish after the protein treatment was attached to the germanium prism of the ATR attachment of an FT-IR device (manufactured by Thermo Fisher Scientific, product name "Niclet 6700") and infrared absorption spectrum measurement was performed. For measurement and analysis, "OMNIC" software manufactured by Thermo Fisher Scientific was used. The amide I region (1600-1700 cm -1 ) of the acquired measured spectrum was subjected to second differentiation to obtain a second derivative spectrum. Next, based on the position of the negative peak (valley) in the second derivative spectrum, the amide I region (1600-1700 cm -1 ) of the measured spectrum was band-decomposed. At this time, one of the bands used for band decomposition was a band having a peak at 1680 cm -1 . The total area of all bands obtained by band resolution was defined as SA, the area of the band having a peak at 1680 cm −1 was defined as SB, and the “area ratio of the 1680 cm −1 band” was calculated according to the following formula.
Area ratio of 1680 cm −1 band (%)=(SB/SA)×100
The measurement and analysis conditions are as follows.
Measurement method: ATR method Number of integrations: 32 Resolution: 16 cm -1
Background correction: before sample measurement Spectral intensity: absorbance Function (distribution function) representing band shape: Gaussian function Peak detection sensitivity: low sensitivity Noise level in peak fitting: 0.01
Baseline correction in peak fitting: Linear interpolation <protein adsorption rate>
1. Protein Processing
RetroNectin (manufactured by Takara Bio, model number "T100B") was dissolved in a phosphate buffered saline (PBS) solution (manufactured by Fujifilm Wako Pure Chemical Industries, PBS (-)) to a concentration of 10 μg/mL to obtain a protein solution. 2.5 mL of the protein solution was placed in a dish (diameter 35 mm) prepared in the Examples and Comparative Examples, and held at 37°C for 45 minutes. Next, the protein solution in the dish was removed, and washing was performed once with 3.0 mL of sterile water. Then, 2 mL of a gold colloid solution (manufactured by Bio-Rad, model number "#1706527") was added to the dish, and held at 25°C for 45 minutes. Next, after removing the gold colloid solution in the dish, 3 mL of sterile water was added to wash the dish. Then, the washing water in the dish was removed, and 3 mL of sterile water was added again to wash the dish. Finally, the washing water was removed, and the dish was dried in an air environment until liquid could no longer be observed with the naked eye.
[2. Ruthenium dyeing treatment]
Ruthenium tetroxide and the above-mentioned protein-treated dish were placed in a glass desiccator, and ruthenium staining was carried out for 30 minutes in a gas phase environment.
[3. SEM photography]
An arbitrary field of view near the center of the bottom surface (culture surface) of the dish after the above-mentioned ruthenium staining treatment was observed at a magnification of 50,000 times under conditions of a secondary electron image and an accelerating voltage of 0.7 kV using a field emission scanning electron microscope (FE-SEM) (manufactured by Hitachi High-Technologies Corporation, product name "SU8220"), and a scanning electron microscope (SEM) observation image was obtained.
4. Image Analysis
The SEM observation image was read by the image processing software ImageJ (open source), and the image data was converted to 8 bits. The ruthenium stained part (gray) and the gold colloid part (white) were binarized as bright parts, and the parts other than the ruthenium stained part and the gold colloid part (black) were binarized as dark parts, and the ratio (%) of bright parts to all pixels of the SEM observation image was determined as the protein adsorption rate. The higher the protein adsorption rate, the better the protein adsorption of the cell culture vessel. Note that if the protein adsorption rate is 60% or more, the protein adsorption of the culture surface is sufficiently ensured.
（実施例１）
＜細胞培養容器（ディッシュ）の作製＞
［予備乾燥および成形］
　下記の組成を有する樹脂組成物（ガラス転移温度（Ｔｇ）：１６３℃、以下「ＣＯＰ１」と称する。）を、雰囲気温度：（Ｔｇ－２０）℃、乾燥時間：４時間の条件で真空乾燥した（予備乾燥）。
―ＣＯＰ１の組成―
　シクロオレフィンポリマー（ノルボルネン系単量体の開環重合体水素化物、重量平均分子量（Ｍｗ）：３４，０００）９９．９部
　酸化防止剤０．２部
　スチレン－共役ジエンブロック共重合体水素化物０．１部
この予備乾燥後のＣＯＰ１を用いて、以下の条件で射出成形を行い、細胞培養容器としてのディッシュ（直径３５ｍｍ）を作製した。
―射出成形の条件―
　射出成形機：ファナック社製、製品名「ロボショットα－Ｓ５０ｉＡ」
　金型：ディッシュ（直径３５ｍｍ）用の金型
　シリンダー温度：（Ｔｇ＋１４０）℃
　金型温度：（Ｔｇ－３０）℃
　射出圧：７０ＭＰａ
得られたディッシュの底面（培養面）について、炭素元素量に対する酸素元素量の割合、酸素含有官能基の存在比、π－π*結合に由来するピークの有無、組み換えヒトフィブロネクチンフラグメントの１６８０ｃｍ-1バンドの面積比率、およびタンパク質吸着率を測定した。結果を表１に示す。 Example 1
<Preparation of cell culture vessel (dish)>
[Pre-drying and molding]
A resin composition having the following composition (glass transition temperature (Tg): 163°C, hereinafter referred to as "COP1") was vacuum dried (pre-dried) under the conditions of an atmospheric temperature of (Tg-20)°C and a drying time of 4 hours.
- Composition of COP1 -
Cycloolefin polymer (hydrogenated ring-opened polymer of norbornene monomer, weight average molecular weight (Mw): 34,000) 99.9 parts Antioxidant 0.2 parts Hydrogenated styrene-conjugated diene block copolymer 0.1 parts This pre-dried COP1 was used for injection molding under the following conditions to produce a dish (diameter 35 mm) as a cell culture vessel.
- Injection molding conditions -
Injection molding machine: FANUC, product name "ROBOSHOT α-S50iA"
Mold: Mold for dish (diameter 35 mm) Cylinder temperature: (Tg + 140) ° C.
Mold temperature: (Tg-30) °C
Injection pressure: 70MPa
The bottom surface (culture surface) of the resulting dish was measured for the ratio of oxygen element content to carbon element content, the abundance ratio of oxygen-containing functional groups, the presence or absence of a peak derived from π-π * bonds, the area ratio of the 1680 cm -1 band of recombinant human fibronectin fragment, and the protein adsorption rate. The results are shown in Table 1.
（実施例２）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　実施例１と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［熱処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、１４０℃で２４時間の熱処理を行った。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表１に示す。 Example 2
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 1 to prepare a dish (diameter 35 mm).
[Heat treatment]
The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 140° C. for 24 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
（実施例３）
　熱処理の処理時間を２４時間から３０時間に変更したこと以外は、実施例２と同様にして、細胞培養容器を作製した。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表１に示す。 Example 3
Except for changing the heat treatment time from 24 hours to 30 hours, a cell culture vessel was produced in the same manner as in Example 2. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
（実施例４）
　ＣＯＰ１に代えて下記の組成を有する樹脂組成物（Ｔｇ：１３６℃、以下「ＣＯＰ２」と称する。）を使用したこと以外は、実施例１と同様にして、細胞培養容器を作製した。そして、実施例１と同様にして評価を行った。結果を表１に示す。
―ＣＯＰ２の組成―
　シクロオレフィンポリマー（ノルボルネン系単量体の開環重合体水素化物、Ｍｗ：３５，０００）９９．９部
　酸化防止剤０．１３５部
　スチレン－共役ジエンブロック共重合体水素化物０．１部 Example 4
A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition having the following composition (Tg: 136° C., hereinafter referred to as “COP2”) was used instead of COP1. Evaluation was then performed in the same manner as in Example 1. The results are shown in Table 1.
- Composition of COP2 -
Cycloolefin polymer (hydrogenated ring-opened polymer of norbornene monomer, Mw: 35,000) 99.9 parts Antioxidant 0.135 parts Hydrogenated styrene-conjugated diene block copolymer 0.1 parts
（実施例５）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　実施例４と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［熱処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、１１５℃で３０時間の熱処理を行った。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表１に示す。 Example 5
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 4 to prepare a dish (diameter 35 mm).
[Heat treatment]
The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 115° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
（実施例６）
　ＣＯＰ１に代えて下記の組成を有する樹脂組成物（Ｔｇ：１００℃、以下「ＣＯＰ３」と称する。）を使用したこと以外は、実施例１と同様にして、細胞培養容器を作製した。そして、実施例１と同様にして評価を行った。結果を表１に示す。
―ＣＯＰ３の組成―
　シクロオレフィンポリマー（ノルボルネン系単量体の開環重合体水素化物、Ｍｗ：２９，４００）９９．９８部
　酸化防止剤０．４８部
　スチレン－共役ジエンブロック共重合体水素化物０．０２部 Example 6
A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition having the following composition (Tg: 100° C., hereinafter referred to as “COP3”) was used instead of COP1. Evaluation was then performed in the same manner as in Example 1. The results are shown in Table 1.
- Composition of COP3 -
Cycloolefin polymer (hydrogenated ring-opened polymer of norbornene monomer, Mw: 29,400) 99.98 parts Antioxidant 0.48 parts Hydrogenated styrene-conjugated diene block copolymer 0.02 parts
（実施例７）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　実施例６と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［熱処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、８０℃で３０時間の熱処理を行った。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表１に示す。 (Example 7)
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 6 to prepare a dish (diameter 35 mm).
[Heat treatment]
The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 80° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 1.
（実施例８）
　ＣＯＰ１に代えてノルボルネンとエチレンの付加共重合体を含む樹脂組成物（Ｔｇ：１３８℃、以下「ＣＯＣ１」と称する。）を使用したこと以外は、実施例１と同様にして、細胞培養容器を作製した。そして、実施例１と同様にして評価を行った。結果を表２に示す。 (Example 8)
A cell culture vessel was produced in the same manner as in Example 1, except that a resin composition containing an addition copolymer of norbornene and ethylene (Tg: 138° C., hereinafter referred to as “COC1”) was used instead of COP1. Then, evaluation was performed in the same manner as in Example 1. The results are shown in Table 2.
（実施例９）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　実施例８と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［熱処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、１１５℃で３０時間の熱処理を行った。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表２に示す。 Example 9
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 8 to prepare a dish (diameter 35 mm).
[Heat treatment]
The thus obtained dish (diameter 35 mm) was subjected to a heat treatment at 115° C. for 30 hours. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
（比較例１）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　実施例１と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［プラズマ処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、低圧プラズマ装置（泉工業株式会社製）を用いて、電圧１００Ｖ、真空度２００Ｐａにて６０秒間プラズマ処理を行った。そして、プラズマ処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表２に示す。 (Comparative Example 1)
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 1 to prepare a dish (diameter 35 mm).
[Plasma treatment]
The dish (diameter 35 mm) obtained as described above was subjected to plasma treatment for 60 seconds using a low pressure plasma device (manufactured by Izumi Kogyo Co., Ltd.) at a voltage of 100 V and a vacuum degree of 200 Pa. Then, the bottom surface (culture surface) of the dish after the plasma treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
（比較例２）
　熱処理の処理時間を３０時間から９６時間に変更したこと以外は、実施例７と同様にして、細胞培養容器を作製した。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表２に示す。 (Comparative Example 2)
Except for changing the heat treatment time from 30 hours to 96 hours, a cell culture vessel was produced in the same manner as in Example 7. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
（比較例３）
　熱処理の処理時間を３０時間から１４４時間に変更したこと以外は、実施例９と同様にして、細胞培養容器を作製した。そして、熱処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表２に示す。 (Comparative Example 3)
Except for changing the heat treatment time from 30 hours to 144 hours, a cell culture vessel was produced in the same manner as in Example 9. Then, the bottom surface (culture surface) of the dish after the heat treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
（比較例４）
＜細胞培養容器の作製＞
［予備乾燥および成形］
　ＣＯＰ１に代えてポリスチレン（Ｃｏｒｎｉｎｇ社製、製品名「Ｆａｌｃｏｎ」、Ｔｇ：１００℃）を使用したこと以外は、実施例１と同様にして、予備乾燥および成形を行い、ディッシュ（直径３５ｍｍ）を作製した。
［プラズマ処理］
　上記のようにして得られたディッシュ（直径３５ｍｍ）に対して、低圧プラズマ装置（泉工業株式会社製）を用いて、電圧１００Ｖ、真空度２００Ｐａにて６０秒間プラズマ処理を行った。そして、プラズマ処理後のディッシュの底面（培養面）について、実施例１と同様にして評価を行った。結果を表２に示す。 (Comparative Example 4)
<Preparation of cell culture vessel>
[Pre-drying and molding]
Preliminary drying and molding were carried out in the same manner as in Example 1, except that polystyrene (manufactured by Corning, product name "Falcon", Tg: 100°C) was used instead of COP1, to prepare a dish (diameter 35 mm).
[Plasma treatment]
The dish (diameter 35 mm) obtained as described above was subjected to plasma treatment for 60 seconds using a low pressure plasma device (manufactured by Izumi Kogyo Co., Ltd.) at a voltage of 100 V and a vacuum degree of 200 Pa. Then, the bottom surface (culture surface) of the dish after the plasma treatment was evaluated in the same manner as in Example 1. The results are shown in Table 2.
　なお、以下に示す表１および２中、
「ＰＳ」は、ポリスチレンを示し、
「プラズマ」は、プラズマ処理を示し、
「Ｔｇ」は、ガラス転移温度を示す。 In Tables 1 and 2 shown below,
"PS" indicates polystyrene;
"Plasma" refers to plasma treatment;
"Tg" refers to the glass transition temperature.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表１および２より、樹脂組成物を用いてなる培養面を備え、且つ、当該培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルにおける１６８０ｃｍ-1バンドの面積比率が所定値以上である細胞培養容器を用いた実施例１～９では、培養面に付着したタンパク質の変性を十分に抑制できていることが分かる。
　一方、培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルにおける１６８０ｃｍ-1バンドの面積比率が所定値未満である細胞培養容器を用いた比較例１～４では、培養面に付着したタンパク質の変性を十分に抑制できていないことが分かる。 As can be seen from Tables 1 and 2, in Examples 1 to 9, which use cell culture vessels having a culture surface made of a resin composition and in which the area ratio of the 1680 cm -1 band in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface is equal to or greater than a predetermined value, the denaturation of proteins attached to the culture surface can be sufficiently suppressed.
On the other hand, in Comparative Examples 1 to 4, which used cell culture vessels in which the area ratio of the 1680 cm -1 band in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface was less than a specified value, it was found that the denaturation of proteins attached to the culture surface was not sufficiently suppressed.
　本発明によれば、培養面に付着したタンパク質の変性を抑制し得る細胞培養容器を提供することができる。 The present invention provides a cell culture vessel that can suppress the denaturation of proteins attached to the culture surface.

Claims (4)

  1. 　樹脂を含む樹脂組成物を用いてなる培養面を備える細胞培養容器であって、
    　前記培養面に付着した組み換えヒトフィブロネクチンフラグメントの赤外吸収スペクトルにおいて、振動数１６００ｃｍ-1以上１７００ｃｍ-1以下の領域のバンド分解により得られる振動数１６８０ｃｍ-1にピークを有するバンドの面積が、前記バンド分解により得られるすべてのバンドの合計面積を１００％として、１０％以上である、細胞培養容器。     A cell culture vessel having a culture surface made of a resin composition containing a resin,
    A cell culture vessel, in which in the infrared absorption spectrum of the recombinant human fibronectin fragment attached to the culture surface, the area of a band having a peak at a frequency of 1680 cm- 1 obtained by band decomposition in the frequency range of 1600 cm - 1 to 1700 cm - 1 is 10% or more, with the total area of all bands obtained by the band decomposition being 100%.
  2. 　前記培養面のＸ線光電子分光法スペクトルが、π－π*結合に由来するピークを有さず、
    　前記Ｘ線光電子分光法スペクトルに基づいて得られる、前記培養面におけるＣ－Ｏ基の量に対するＣ＝Ｏ基およびＯ－Ｃ＝Ｏ基の合計量の比が０．４５以下である、請求項１に記載の細胞培養容器。     The X-ray photoelectron spectroscopy spectrum of the culture surface does not have a peak derived from a π-π * bond;
    The cell culture vessel according to claim 1, wherein the ratio of the total amount of C═O groups and O—C═O groups to the amount of C—O groups on the culture surface, based on the X-ray photoelectron spectroscopy spectrum, is 0.45 or less.
  3. 　Ｘ線光電子分光法スペクトルに基づいて得られる、前記培養面における炭素元素量に対する酸素元素量の割合が２．０％以上である、請求項１に記載の細胞培養容器。 The cell culture vessel according to claim 1, wherein the ratio of the amount of oxygen element to the amount of carbon element on the culture surface, as obtained based on an X-ray photoelectron spectroscopy spectrum, is 2.0% or more.
  4. 　前記樹脂がノルボルネン系単量体の開環重合体水素化物を含む、請求項１～３の何れかに記載の細胞培養容器。 The cell culture vessel according to any one of claims 1 to 3, wherein the resin contains a ring-opening polymer hydride of a norbornene-based monomer.
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* Cited by examiner, † Cited by third party
Title
HIRANO TAKAAKI, ADACHI SATORU, ICHIMURA NAOYA, KASAI AKIRA, KOBAYASHI MASASHI, OKUDA TAKASHI, OGAWA RYOHEI, KAGIYA GO: "Culturing Chinese hamster ovary cells on cyclo olefin polymer triggers epithelial‐mesenchymal transition and spheroid formation, which increases the foreign gene expression driven by the Moloney murine leukemia virus long terminal repeat promoter", BIOTECHNOLOGY PROGRESS, vol. 37, no. 4, US, pages 1 - 10, XP093218485, ISSN: 8756-7938, DOI: 10.1002/btpr.3159 *
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