JP6810386B2 - Low protein adsorptive material, low protein adsorptive article, low cell adhesion material and low cell adhesion article - Google Patents

Description

The present invention relates to low protein adsorptive materials, low protein adsorptive articles, low cell adherent materials and low cell adherent articles.

A polymer material is used as a material for constituting an instrument for storing blood, an instrument for culturing cells, and the like. Depending on the application, there is a demand for suppressing the adsorption of blood on the polymer material, or there is a demand for adsorbing cells on the polymer material. In order to meet these demands, attempts have been made to control the adsorptivity of blood and cells to polymer materials.

Patent Document 1 describes a biocompatible substrate characterized in that at least a part of the surface thereof is composed of a hydrophobic fluororesin and a hydrophilic fluororesin.

Patent Document 2 includes a medical article that includes a coating that is placed on at least a portion of an implantable medical device, wherein the coating comprises (a) a fluorinated polymer and (b) a biobeneficial polymer. Medical articles are listed.

Patent Document 3 describes a copolymer of tetrafluoroethylene (TFE) and vinyl acetate (VAc) or at least a part of acetate groups contained in the copolymer as a film used for water treatment or medical use. Described is a fluoropolymer film comprising a saponified copolymer and characterized in that the content of tetrafluoroethylene contained in the copolymer is 1 to 70 mol%.

Patent Document 4 describes a copolymer obtained by deprotecting a copolymer of tetrafluoroethylene and t-butyl vinyl ether or vinyl acetate as a hydrophilic material used for an aqueous liquid separation membrane. ..

Patent Document 5 describes a molded product having a low-protein adsorption layer on the surface of the support layer, wherein the low-protein adsorption layer is made of an amphoteric polymer and is covalently bonded to the support layer to form a layer. A low protein adsorptive molded product characterized by the above is described.

Patent Document 6 describes a protein adsorption inhibitor containing a 2-methacryloyloxyethyl phosphorylcholine polymer and / or a copolymer of a 2-methacryloyloxyethyl phosphorylcholine-containing component.

Patent Document 7 describes a cell culture substrate using a polymer composite in which water-swellable clay minerals are finely dispersed in a water-soluble (meth) acrylic acid ester polymer.

Patent Document 8 describes a cell culture well plate in which the inner surface of the well is composed of polymethacrylate and the growth of scaffold-independent cells is easy.

Patent Document 9 describes a culture container for embryoid body formation, which is formed by a cell hypoadhesion treatment or a cell non-adhesion treatment using a water-soluble resin.

Japanese Unexamined Patent Publication No. 4-336072 Japanese Patent Application Laid-Open No. 2007-515208 Japanese Unexamined Patent Publication No. 5-261256 International Publication No. 2012/165503 Japanese Unexamined Patent Publication No. 2004-277652 Japanese Unexamined Patent Publication No. 7-83923 Japanese Unexamined Patent Publication No. 2013-176402 Japanese Unexamined Patent Publication No. 8-322593 Japanese Unexamined Patent Publication No. 2012-210166

INDUSTRIAL APPLICABILITY The present invention is excellent in heat resistance, alkali resistance and molding processability, as well as a novel low protein adsorbing material having excellent heat resistance, alkali resistance and molding processability, and cell adhesion resistance. It is an object of the present invention to provide a novel low cell adhesion material.

The present invention is a low protein adsorptive material, which comprises a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit.

In the low protein adsorptive material of the present invention, the content of the fluorine-containing olefin unit in the fluorine-containing copolymer is preferably 60 to 10 mol%.

In the low protein adsorptive material of the present invention, the alternating ratio of the fluorine-containing olefin unit and the vinyl alcohol unit in the fluorine-containing copolymer is preferably 1 to 75%.

In the low protein adsorptive material of the present invention, the fluorine-containing olefin is preferably at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene.

In the low protein adsorptive material of the present invention, the fluorine-containing copolymer preferably has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit.

The low protein adsorptive material is preferably a coating film.

The present invention is also a coating composition comprising the above-mentioned low protein adsorptive material.

The present invention is also a coating layer, characterized in that it is formed from the low protein adsorptive material described above.

The present invention comprises the above-mentioned low-protein adsorptive material, the above-mentioned coating composition, or the above-mentioned coating layer, and is a low-protein bag, sheet, film, vial, petri dish, or flask. It is also an adsorptive article.

The present invention comprises the above-mentioned low protein-adsorbing material, the above-mentioned coating composition, or the above-mentioned coating layer, and comprises a biopharmacy bag, a biopharmacy sheet, a biopharmacy film, a biopharmacy vial, and a biopharmacy. It is also a low-protein adsorbing article characterized by being a petri dish or a biopharmacy flask.

The present invention is also a low cell adhesion material, which comprises a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit.

In the low cell adhesion material of the present invention, the content of the fluorine-containing olefin unit in the fluorine-containing copolymer is preferably 60 to 10 mol%.

In the low cell adhesion material of the present invention, the alternating ratio of the fluorine-containing olefin unit and the vinyl alcohol unit in the fluorine-containing copolymer is preferably 1 to 75%.

In the low cell adhesion material of the present invention, the fluorine-containing olefin is preferably at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene.

In the low cell adhesion material of the present invention, the fluorine-containing copolymer preferably has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit.

The low cell adhesion material is preferably a coating film.

The present invention is also a coating composition, which comprises the above-mentioned low cell adhesion material.

The present invention is also a coating layer characterized by being formed from the above-mentioned low cell adhesion material.

The present invention comprises the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned coating layer, and is a bag, sheet, film, vial, petri dish, or flask. It is also a cell adherent article.

The present invention comprises the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned coating layer, and comprises a cell culture bag, a cell culture sheet, a cell culture film, a cell culture vial, and cells. It is also a low cell adherent article characterized by being a petri dish for culture or a flask for cell culture.

The present invention is also a low cell adhesion article comprising the above-mentioned low cell adhesion material, the above coating composition, or the above coating layer, and is a culture vessel for embryoid body formation.

The low protein adsorptive material of the present invention does not easily adsorb proteins and is excellent in heat resistance, alkali resistance and molding processability. From the low protein adsorptive material of the present invention, it is possible to obtain an article which is difficult to adsorb proteins and has excellent heat resistance, alkali resistance and molding processability. In addition, the low cell adhesion material of the present invention is resistant to cell adhesion and is excellent in heat resistance, alkali resistance and molding processability. From the low cell adhesion material of the present invention, it is possible to obtain an article in which cells are difficult to adhere and excellent in heat resistance, alkali resistance and molding processability.

The low-protein adsorptive article of the present invention is excellent in heat resistance, alkali resistance and molding processability as it is difficult for proteins to be adsorbed on the surface. In addition, the low cell adhesion article of the present invention is excellent in heat resistance, alkali resistance and molding processability because cells are hard to adhere to the surface.

Hereinafter, the present invention will be specifically described.

The low protein adsorptive material and the low cell adhesion material of the present invention consist of a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit (-CH _2- CH (OH)-). The low protein adsorptivity and low cell adhesion of the fluorine-containing copolymer are properties newly discovered by the present inventors and the like.

Since the low protein adsorptive material and the low cell adhesion material of the present invention are also excellent in heat resistance, they can withstand sterilization treatment performed at high temperature. For example, it can withstand autoclave sterilization performed at 121 ° C. The low protein adsorptive material and the low cell adhesion material of the present invention preferably have a decomposition temperature of more than 121 ° C.

Since the low protein adsorptive material and the low cell adhesion material of the present invention are also excellent in alkali resistance, they can withstand disinfection using an alkaline aqueous solution containing hypochlorous acid. For example, the low protein adsorptive material and the low cell adhesion material of the present invention are immersed in an aqueous solution containing 0.5% by mass of sodium hypochlorite and 0.4% by mass of sodium hydroxide for 24 hours. However, the rate of change in weight is 10% by mass or less.

The low protein adsorptive material and the low cell adhesion material of the present invention are also excellent in molding processability. For example, when the low protein adsorptive material and the low cell adhesion material of the present invention are coating compositions containing an organic solvent, a weak solvent such as alcohol can be used as the organic solvent, so that the material is eroded by a strong solvent. It can also be applied to substrates and undercoats.

The fluorine-containing copolymer preferably contains the fluorine-containing olefin unit in an amount of 60 to 10 mol% of all the monomer units constituting the fluorine-containing copolymer. It is more preferably 40 to 10 mol%, further preferably 35 to 20 mol%, and even more preferably 35 to 25 mol%. When the content of the fluorine-containing olefin unit is within the above range, the protein is more difficult to be adsorbed on the low protein adsorbent material, and the cells are more difficult to adhere to the low cell adhesion material. When the content of the fluorine-containing olefin unit is less than 10 mol%, the water-soluble property of the fluorine-containing copolymer becomes high, the fluorine-containing copolymer easily elutes into water, and the function is less likely to be exhibited. The higher the content of the fluorine-containing olefin unit, the better the heat resistance, alkali resistance and molding processability of the low protein adsorptive material and the low cell adhesion material tend to be.

The fluorine-containing copolymer preferably has a fluorine-containing olefin unit of 60 to 10 mol% and a vinyl alcohol unit of 40 to 90 mol%. As for the content of each monomer unit, the fluorine-containing olefin unit is 40 to 10 mol%, the vinyl alcohol unit is more preferably 60 to 90 mol%, and the fluorine-containing olefin unit is 35 to 20 mol%. The vinyl alcohol unit is more preferably 65 to 80 mol%, the fluoroolefin unit is 35 to 25 mol%, and the vinyl alcohol unit is 65 to 75 mol%. When the content of each monomer unit is in such a range, the protein is more difficult to be adsorbed to the low protein adsorbent material, and the cells are more difficult to adhere to the low cell adhesion material. When the content of the fluorine-containing olefin unit is less than 10 mol%, the water-soluble property of the fluorine-containing copolymer becomes high, the fluorine-containing copolymer easily elutes into water, and the function is less likely to be exhibited. The higher the content of the fluorine-containing olefin unit, the better the heat resistance, alkali resistance and molding processability of the low protein adsorptive material and the low cell adhesion material tend to be.

The fluorine-containing olefin unit represents a polymerization unit based on the fluorine-containing olefin. The fluorine-containing olefin is a monomer having a fluorine atom.

Examples of the fluorine-containing olefin include tetrafluoroethylene [TFE], vinylidene fluoride [VdF], chlorotrifluoroethylene [CTFE], vinyl fluoride, hexafluoropropylene [HFP], hexafluoroisobutene, and CH ₂ = CZ. ¹ _{(CF 2)} (wherein, ^{Z 1} is H, F or Cl, ^{Z 2} is H, F or Cl, n1 is an integer of from 1 to 10.) n1 ^{Z 2} monomer represented by, CF ₂ = CF-ORf ¹ (in the formula, Rf ¹ represents a perfluoroalkyl group having 1 to 8 carbon atoms), perfluoro (alkyl vinyl ether) [PAVE], and CF ₂ = CF-OCH _2. It is preferable that Rf ² is at least one fluorine-containing olefin selected from the group consisting of alkyl perfluorovinyl ether derivatives represented by −Rf ² (in the formula, Rf ² is a perfluoroalkyl group having 1 to 5 carbon atoms). ..

Examples of the monomer represented by CH ₂ = CZ ¹ (CF ₂ ) _n1 Z ² include CH ₂ = CFCF ₃ , CH ₂ = CHCF ₃ , CH ₂ = CFCHF ₂ , CH ₂ = CClCF _{3, and the} like.

Examples of the PAVE include perfluoro (methyl vinyl ether) [PMVE], perfluoro (ethyl vinyl ether) [PEVE], perfluoro (propyl vinyl ether) [PPVE], perfluoro (butyl vinyl ether) and the like.

As the fluorine-containing olefin, at least one selected from the group consisting of TFE, CTFE and HFP is more preferable, and TFE is even more preferable.

The fluorine-containing copolymer preferably has an alternating ratio of fluorine-containing olefin units and vinyl alcohol units of 1 to 75%. When the alternating rate is within the above range, the protein is more difficult to be adsorbed to the low protein adsorbent material, and the cells are more difficult to adhere to the low cell adhesion material. It is more preferably 10 to 60%, further preferably 10 to 35%, and particularly preferably 10 to 20%.

The alternating ratio of the fluorine-containing olefin unit and the vinyl alcohol unit was determined by ¹ H-NMR measurement of the fluorine-containing copolymer using a solvent in which the fluorine-containing copolymer such as deuterated acetone is dissolved, and 3 from the following formula. It can be calculated as the alternating rate of the chain.
Alternate rate (%) = C / (A + B + C) x 100
A: Number of Vs combined with two Vs such as -VVVV- Number of Vs combined with V and T such as -VVV-T-C: -TV- Number of Vs bonded to two Ts like T- (T: Fluorine-containing olefin unit, V: Vinyl alcohol unit)
The number of V units of A, B, and C is calculated from the intensity ratio of H of the main chain bonded to the tertiary carbon of the vinyl alcohol unit (-CH _2- CH (OH)-) measured by ¹ H-NMR. ¹ Estimating the strength ratio of H in the main chain by ¹ H-NMR measurement is carried out with a fluorine-containing copolymer before saponification.

Since the above-mentioned fluorine-containing copolymer is more difficult to adsorb proteins and further difficult to adhere to cells, -CH (OH) -CXY- (in the formula, X and Y are the same or different, and H, respectively. It represents an F or fluoroalkyl group, although at least one of X and Y is preferably an F or fluoroalkyl group) having a fluoroalcohol structure. Further, the fluorine-containing copolymer, -CH (OH) _{-CF 2} - and more preferably has a fluorine alcohol structure represented by.

The fluorine-containing copolymer is further represented by -CH _2- CH (O (C = O) R)-(in the formula, R represents a hydrogen atom or a hydrocarbon group having 1 to 17 carbon atoms). It may have a vinyl ester monomer unit to be used. As described above, it is also one of the preferred embodiments of the present invention that the fluorine-containing copolymer in the present invention has a fluorine-containing olefin unit, a vinyl alcohol unit and a vinyl ester monomer unit. Furthermore, a fluoroolefin / vinyl alcohol / vinyl ester monomer copolymer substantially consisting of only a fluorine-containing olefin unit, a vinyl alcohol unit and a vinyl ester monomer unit is also a preferred embodiment of the present invention. It is one.

The vinyl ester monomer unit is a monomer represented by -CH _2- CH (O (C = O) R)-(in the formula, R represents a hydrogen atom or a hydrocarbon group having 1 to 17 carbon atoms). Although it is a unit, as R in the above formula, an alkyl group having 1 to 11 carbon atoms is preferable, and an alkyl group having 1 to 5 carbon atoms is more preferable. Particularly preferably, it is an alkyl group having 1 to 3 carbon atoms.

Examples of the vinyl ester monomer unit include the following monomer units derived from vinyl ester.
Vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, vinyl valerate, vinyl isovalerate, vinyl caproate, vinyl heptylate, vinyl caprylate, vinyl pivalate, vinyl pelargonate, vinyl caprate, Vinyl laurate, vinyl myristate, vinyl pentadecylate, vinyl partimate, vinyl margarate, vinyl stearate, vinyl octylate, Beova-9 (manufactured by Showa Shell Petroleum Co., Ltd.), Beova-10 (Showa Shell Petroleum Co., Ltd.) ), Vinyl benzoate, vinyl versatic acid.
Among these, monomer units derived from vinyl acetate, vinyl propionate, and vinyl versaticate are preferable. More preferably, it is a vinyl acetate monomer unit, a vinyl propionate monomer unit, and even more preferably a vinyl acetate monomer unit.

When the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit, the content of each monomer unit is 60 to 10 mol% of the fluorine-containing olefin unit, and vinyl alcohol. The unit is preferably 40-90 mol% and the vinyl ester monomer unit is more than 0 mol% and less than 30 mol%. When the content of each monomer unit is within the above range, the protein is more difficult to be adsorbed on the low protein adsorbent material, and the cells are more difficult to adhere to the low cell adhesion material. As for the content of each monomer unit, the fluorine-containing olefin unit is 60 to 10 mol%, the vinyl alcohol unit is 40 to 90 mol%, and the vinyl ester monomer unit is more than 0 and less than 10 mol%. More preferably, the fluorine-containing olefin unit is 40 to 10 mol%, the vinyl alcohol unit is 60 to 90 mol%, and the vinyl ester monomer unit is more than 0 and less than 1.0 mol%. It is more preferable that the fluorine olefin unit is 35 to 20 mol%, the vinyl alcohol unit is 65 to 80 mol%, and the vinyl ester monomer unit is more than 0 and less than 1.0 mol%, and the fluorine-containing olefin unit. Is 35 to 25 mol%, the vinyl alcohol unit is 65 to 75 mol%, and the vinyl ester monomer unit is more than 0 and less than 1.0 mol%, even more preferably. The higher the content of the fluorine-containing olefin unit, the better the heat resistance, alkali resistance and molding processability of the low protein adsorptive material and the low cell adhesion material tend to be.

When the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit and a vinyl ester monomer unit, the alternating ratio between the fluorine-containing olefin unit and the vinyl alcohol unit and the vinyl ester monomer unit is 1 to 75%. Is preferable. When the alternating rate is within the above range, the protein is more difficult to be adsorbed to the low protein adsorbent material, and the cells are more difficult to adhere to the low cell adhesion material. It is more preferably 10 to 60%, further preferably 10 to 35%, and particularly preferably 10 to 20%.

The alternating ratio of the fluorine-containing olefin unit, the vinyl alcohol unit, and the vinyl ester monomer unit was measured by ¹ H-NMR measurement of the fluorine-containing copolymer using a solvent in which the fluorine-containing copolymer such as deuterated acetone is dissolved. It can be calculated as the alternating rate of 3 chains from the following formula.
Alternate rate (%) = C / (A + B + C) x 100
A: Number of Vs combined with two Vs such as -VVVV- Number of Vs combined with V and T such as -VVV-T-C: -TV- Number of Vs bonded to two Ts like T- (T: Fluorine-containing olefin unit, V: Vinyl alcohol unit or vinyl ester monomer unit)
The number of V units of A, B, and C is ¹ vinyl alcohol unit (-CH _2- CH (OH)-) and vinyl ester monomer unit (-CH _2- CH (O (C = O)) measured by H-NMR. It is calculated from the strength ratio of H of the main chain bonded to the tertiary carbon of R)-). ¹ Estimating the strength ratio of H in the main chain by ¹ H-NMR measurement is carried out with a fluorine-containing copolymer before saponification.

The fluorine-containing copolymer may have a monomer unit other than the fluorine-containing olefin unit, the vinyl alcohol unit and the vinyl ester monomer unit as long as the effect of the present invention is not impaired.

Examples of the above-mentioned other monomers include ethylene, propylene, 1-butene, 2-butene, and vinyl chloride as monomers containing no fluorine atom (excluding vinyl alcohol and vinyl ester monomers). At least one fluorine-free ethylenic monomer selected from the group consisting of vinylidene chloride, vinyl ether monomer, and unsaturated carboxylic acid is preferable.

The total content of the other monomer units is preferably 0 to 50 mol%, more preferably 0 to 40 mol%, and 0 to 0 to 40 mol% of all the monomer units of the fluorine-containing copolymer. It is more preferably 30 mol%.

In the present specification, the content of each monomer unit constituting the fluorine-containing copolymer can be calculated by appropriately combining NMR, FT-IR, and elemental analysis according to the type of monomer.

The weight average molecular weight of the fluorine-containing copolymer is not particularly limited, but is preferably 10,000 or more. More preferably, it is 12,000 to 2,000,000, and even more preferably, it is 12,000 to 1,000,000.
The weight average molecular weight can be determined by gel permeation chromatography (GPC).

As will be described later, the fluorine-containing copolymer can be produced by saponifying a copolymer having a fluorine-containing olefin unit and a vinyl ester monomer unit. That is, it is also a preferred embodiment of the present invention that the fluorine-containing copolymer in the present invention is a copolymer obtained by saponifying a copolymer having a fluorine-containing olefin unit and a vinyl ester monomer unit. There is one.

The method for producing the fluorine-containing copolymer in the present invention will be described below.
Usually, the fluorine-containing copolymer in the present invention is produced by copolymerizing a fluorine-containing olefin such as tetrafluoroethylene with a vinyl ester monomer such as vinyl acetate, and then saponifying the obtained copolymer. can do. As a method for polymerizing the above-mentioned fluorine-containing copolymer, it is preferable to carry out the polymerization under the condition that the composition ratio of the fluorine-containing olefin and the vinyl ester monomer is kept substantially constant. That is, the above-mentioned fluoropolymer is polymerized under conditions where the composition ratio of the fluoroolefin and the vinyl ester monomer is kept substantially constant to obtain a copolymer having a fluoroolefin unit and a vinyl ester monomer unit. It is preferably obtained by a production method consisting of a step and a step of saponifying the obtained copolymer to obtain a copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit.

Examples of the vinyl ester monomer include vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, vinyl valerate, vinyl isovalerate, vinyl caproate, vinyl heptylate, vinyl caprylate, vinyl pivalate, and pelargonic acid. Vinyl acetate, vinyl caproate, vinyl laurate, vinyl myristate, vinyl pentadecylate, vinyl partimate, vinyl margarate, vinyl stearate, vinyl octylate, Beova-9 (manufactured by Showa Shell Petroleum Co., Ltd.), Beova- 10 (manufactured by Showa Shell Petroleum Co., Ltd.), vinyl benzoate, vinyl versatic acid and the like can be mentioned. Among them, vinyl acetate, vinyl propionate and vinyl versatic acid are preferably used because they are easily available and inexpensive.
As the vinyl ester monomer, one of these may be used, or two or more of them may be mixed and used.

Examples of the method for copolymerizing the fluorine-containing olefin and the vinyl ester monomer include polymerization methods such as solution polymerization, massive polymerization, emulsion polymerization, and suspension polymerization, which are industrially easy to carry out and thus are emulsion polymerization. , It is preferable to produce by solution polymerization or suspension polymerization, but this is not the case.

In emulsion polymerization, solution polymerization or suspension polymerization, a polymerization initiator, a solvent, a chain transfer agent, a surfactant, a dispersant and the like can be used, and those usually used can be used.

The solvent used in the solution polymerization is preferably one capable of dissolving the fluorine-containing olefin, the vinyl ester monomer, and the synthesized fluorine-containing copolymer, for example, n-butyl acetate, t-butyl acetate, and ethyl acetate. , Esters such as methyl acetate, propyl acetate; ketones such as acetone, methyl ethyl ketone, cyclohexanone; aliphatic hydrocarbons such as hexane, cyclohexane, octane; aromatic hydrocarbons such as benzene, toluene, xylene; methanol, ethanol , Alcohols such as tert-butanol, 2-propanol; cyclic ethers such as tetrahydrofuran and dioxane; fluorine-containing solvents such as HCFC-225; dimethylsulfoxide, dimethylformamide, or a mixture thereof.
Examples of the solvent used in the emulsion polymerization include water, a mixed solvent of water and alcohol, and the like.

Examples of the polymerization initiator include peroxycarbonates such as diisopropylperoxydicarbonate (IPP) and di-n-propylperoxydicarbonate (NPP), and t-butylperoxypivalate (for example, Nichiyu Co., Ltd.). Perbutyl PV) and other oil-soluble radical polymerization initiators, for example, persulfate, perboric acid, perchloric acid, perphosphoric acid, ammonium salt of percarbonate, potassium salt, sodium. A water-soluble radical polymerization initiator such as a salt can be used. Particularly in emulsion polymerization, ammonium persulfate and potassium persulfate are preferable.

As the above-mentioned surfactant, a commonly used surfactant can be used, and for example, a nonionic surfactant, an anionic surfactant, a cationic surfactant and the like can be used. Further, a fluorine-containing surfactant may be used.

Examples of the dispersant used in suspension polymerization include partially saponified polyvinyl acetate, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose and other water-soluble cellulose ethers and acrylic acid-based polymers used in ordinary suspension polymerization. , A water-soluble polymer such as gelatin can be exemplified. Suspension polymerization is carried out under the condition that the ratio of water / monomer is usually 1.5 / 1-3 / 1 by mass, and the dispersant is 0.01 to 0 with respect to 100 parts by mass of the monomer. .1 parts by mass is used. If necessary, a pH buffer such as polyphosphate can also be used.

Examples of the chain transfer agent include hydrocarbons such as ethane, isopentan, n-hexane and cyclohexane; aromatics such as toluene and xylene; ketones such as acetone; and acetate esters such as ethyl acetate and butyl acetate; Alcohols such as methanol and ethanol; mercaptans such as methyl mercaptan; halogenated hydrocarbons such as carbon tetrachloride, chloroform, methylene chloride and methyl chloride can be mentioned.
The amount of the chain transfer agent added may vary depending on the magnitude of the chain transfer constant of the compound used, but is usually used in the range of 0.001 to 10% by mass with respect to the polymerization solvent.

The polymerization temperature may be 0 to 100 ° C. as long as the composition ratio of the fluorine-containing olefin and the vinyl ester monomer during the reaction is substantially constant.

The polymerization pressure may be 0 to 10 MPaG as long as the composition ratio of the fluorine-containing olefin and the vinyl ester monomer during the reaction is substantially constant.

The saponification of an acetate group derived from vinyl acetate has been well known from the past, and can be carried out by a conventionally known method such as alcoholesis or hydrolysis using an acid or an alkali. By this saponification, the acetate group (-OCOCH ₃ ) is converted to a hydroxyl group (-OH). Similarly, other vinyl ester monomers can be saponified by a conventionally known method to obtain a hydroxyl group.

When a copolymer having a fluorine-containing olefin unit and a vinyl ester monomer unit is saponified to obtain the fluorine-containing copolymer in the present invention, the degree of saponification is the content of each monomer unit of the fluorine-containing copolymer in the present invention. The rate may be in the range described above, and specifically, 90% or more is preferable, 95% or more is more preferable, and 99% or more is further preferable.

The degree of saponification is calculated from the following formula by IR measurement or ¹ H-NMR measurement of the fluorine-containing copolymer.
Degree of saponification (%) = D / (D + E) x 100
D: Number of vinyl alcohol units in the fluorine-containing copolymer E: Number of vinyl ester monomer units in the fluorine-containing copolymer

The low protein adsorptive material and the low cell adhesion material may further contain components other than the fluorine-containing copolymer as long as the effects of the present invention are not impaired.

Examples of the other components include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

The blending amount of the other components is preferably 1 to 30% by mass, more preferably 1 to 10% by mass, based on 100% by mass of the fluorine-containing copolymer.

The surface of the low protein adsorptive material and the low cell adhesion material is preferably treated with an aqueous solution containing an organic solvent. By this surface treatment, the hydroxyl group structure is further increased on the surfaces of the low protein adsorptive material and the low cell adherent material, so that protein adsorption and cell adhesion are more strongly suppressed.

The organic solvent that can be used in the surface treatment is not particularly limited as long as it is an organic solvent that is soluble in water and dissolves the fluorine-containing copolymer, but is methanol, ethanol, 2-propanol, acetone, and tetrahydrofuran. , Methylethylketone, dimethylacetamide, dimethylformamide and the like. Of these, methanol, ethanol, 2-propanol and tetrahydrofuran are preferable.

Examples of the method of treatment with the aqueous solution containing the organic solvent include a method of wetting the surfaces of the low protein adsorbent material and the low cell adhesion material with the aqueous solution.

The low protein adsorptive material and the low cell adhesion material are provided by being molded into various shapes depending on the application. The molding method is not particularly limited, and is a spin coating method, a drop casting method, a dip nip method, a spray coating method, a brush coating method, a dipping method, an inkjet printing method, an electrostatic coating method, a compression molding method, an extrusion molding method, and a calendar molding. A method, a transfer molding method, an injection molding method, a roto molding method, a lotining molding method, a heat-induced phase separation method, a non-solvent-induced phase separation method, or the like can be adopted.

Since it can be applied to articles having various shapes, the low protein adsorptive material and the low cell adhesion material are preferably coating films. The coating film refers to a film obtained by applying the fluorine-containing copolymer or a coating composition containing the fluorine-containing copolymer. Examples of the coating film forming method include a spin coating method, a drop casting method, a dip nip method, a spray coating method, a brush coating method, a dipping method, an electrostatic coating method, an inkjet printing method and the like. Among them, the spin coating method, the drop casting method, and the dipping method are preferable from the viewpoint of convenience.

The coating film is preferably obtained by applying a coating composition containing the fluorine-containing copolymer and an organic solvent. As the organic solvent, methanol, ethanol, 2-propanol, 2-butanol, 1-butanol, 1-hexanol, acetone, tetrahydrofuran, methyl ethyl ketone, dimethylacetamide, dimethylformamide and the like can be used. Of these, 2-butanol, 1-butanol, 1-hexanol, and tetrahydrofuran are preferable because a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorine-containing copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran and dimethylformamide are preferable.

Since the coating film can also be obtained from a coating composition using a weak solvent such as alcohol as an organic solvent, it can be easily produced.

When the low protein adsorptive material and the low cell adhesion material are coating films, the film thickness is preferably 0.1 to 50 μm, more preferably 0.5 to 30 μm, and 1.0. It is more preferably ~ 20 μm.

The low protein adsorptive material may have a bovine serum albumin adsorption amount of 200 ng / cm ² or less when contacted with a 0.05 mg / ml bovine serum albumin solution at 23.4 ° C. for 0.5 hours. it can.
The low protein adsorptive material may have an adsorption amount of bovine plasma fibrinogen of 500 ng / cm ² or less when contacted with a 0.05 mg / ml bovine plasma fibrinogen solution at 23.4 ° C. for 0.5 hours. it can.
The low protein-adsorbing material adsorbs bovine serum-derived immunoglobulin G at 500 ng / cm when contacted with a 0.05 mg / ml bovine serum-derived immunoglobulin G solution at 23.4 ° C. for 0.5 hours. It can be ² or less.
The protein-adsorbing material has a surprising effect that the protein is hardly adsorbed even when it is brought into contact with a protein solution of 10 to 0.05 mg / ml. That is, the protein-adsorbing material may be a protein-adsorbing material that is brought into contact with a protein solution of 10 to 0.05 mg / ml.
The adsorption amount of the above protein is measured by the method described in the "protein adsorption test" described later.
A method of preventing the adsorption of the protein by applying the low protein adsorptive material to at least the surface of the article in contact with the protein solution of 10 to 0.05 mg / ml is also a method of using the low white matter adsorptive material. preferable.

Since the low protein adsorptive material has low protein adsorptivity, it can be applied to various articles that are required to avoid protein adsorption. The shape of the article is not particularly limited and may be a bag, a sheet, a film, a vial, a petri dish, or a flask.
A method for preventing protein adsorption on the article, which comprises applying the low protein adsorptive material to the article, is preferable as a method for using the low protein adsorptive material. Examples of the protein include plasma proteins and biopharmacy described later. Examples of the plasma protein include albumin, globulin, fibrinogen and the like. The method of applying the low-protein adsorptive material is not particularly limited as long as it is possible to cover at least a part of the surface of the article with the low-protein adsorptive material. For example, a method of forming a coating film by applying the above-mentioned fluorine-containing copolymer or a coating composition containing the above-mentioned fluorine-containing copolymer can be mentioned. The method for forming the coating film is as described above.

The present invention is also a coating composition comprising the low protein adsorptive material or the low cell adhesion material.

The coating composition may contain an organic solvent. As the organic solvent, methanol, ethanol, 2-propanol, 2-butanol, 1-butanol, 1-hexanol, acetone, tetrahydrofuran, methyl ethyl ketone, dimethylacetamide, dimethylformamide and the like can be used. Of these, 2-butanol, 1-butanol, 1-hexanol, and tetrahydrofuran are preferable because a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorine-containing copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran and dimethylformamide are preferable.

In the above coating composition, a weak solvent such as alcohol can be used as the organic solvent. Therefore, it can be applied to a base material or an undercoat that is eroded by a strong solvent.

The coating composition can be applied to a desired substrate to form a coating film or coating layer. Examples of the coating method include a spin coating method, a drop casting method, a dip nip method, a spray coating method, a brush coating method, a dipping method, an electrostatic coating method, an inkjet printing method and the like. Among them, the spin coating method, the drop casting method, and the dipping method are preferable from the viewpoint of convenience.

The coating film or coating layer formed from the above coating composition preferably has a film thickness of 0.1 to 50 μm, more preferably 0.5 to 30 μm, and more preferably 1.0 to 20 μm. Is more preferable.

The coating composition can be applied to bags, sheets, films, vials, petri dishes, or flasks.

The present invention is also a coating layer characterized by being formed from the low protein adsorptive material or the low cell adhesion material.

The coating layer can be obtained by applying the fluorine-containing copolymer or a coating composition containing the fluorine-containing copolymer. Examples of the coating method include a spin coating method, a drop casting method, a dip nip method, a spray coating method, a brush coating method, a dipping method, an electrostatic coating method, an inkjet printing method and the like. Among them, the spin coating method, the drop casting method, and the dipping method are preferable from the viewpoint of convenience.

The coating layer is preferably obtained by applying a coating composition containing the fluorine-containing copolymer and an organic solvent. As the organic solvent, methanol, ethanol, 2-propanol, 2-butanol, 1-butanol, 1-hexanol, acetone, tetrahydrofuran, methyl ethyl ketone, dimethylacetamide, dimethylformamide and the like can be used. Of these, 2-butanol, 1-butanol, 1-hexanol, and tetrahydrofuran are preferable because a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorine-containing copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran and dimethylformamide are preferable.

Since the coating layer can also be obtained from a coating composition using a weak solvent such as alcohol as an organic solvent, it can be easily produced.

The coating layer preferably has a film thickness of 0.1 to 50 μm, more preferably 0.5 to 30 μm, and even more preferably 1.0 to 20 μm.

The coating layer preferably forms the surface of a bag, sheet, film, vial, petri dish, or flask. Bags, sheets, films, vials, petri dishes, or flasks provided with the coating layer on the surface do not easily adhere proteins or cells.

The present invention comprises the above-mentioned low-protein adsorptive material, the above-mentioned coating composition, or the above-mentioned coating layer, and is a low-protein bag, sheet, film, vial, petri dish, or flask. It is also an adsorptive article.

The present invention comprises the above-mentioned low protein-adsorbing material, the above-mentioned coating composition, or the above-mentioned coating layer, and comprises a biopharmacy bag, a biopharmacy sheet, a biopharmacy film, a biopharmacy vial, and a biopharmacy. It is also a low-protein adsorbing article characterized by being a petri dish or a biopharmacy flask.

If the biopharmacy is easily adsorbed on the device for storing the biopharmacy or the device for using the biopharmacy, the biopharmacy cannot be accurately quantified or accurate analysis becomes difficult. In addition, since biopharmacy is expensive, the economic loss is also large. A biopharmaceutical bag, a biopharmaceutical sheet, a biopharmaceutical film, a biopharmaceutical vial, a biopharmaceutical chalet, or a biopharmaceutical flask is the above-mentioned low protein-adsorbing material, the above-mentioned coating composition, or If it is composed of the above-mentioned coating layer, the biopharmacy is less likely to be adsorbed, and the biopharmacy can be recovered with a high recovery rate.

Examples of biopharmacy include protein drugs, genetically modified viruses, cell therapeutic drugs, nucleic acid drugs and the like.
Examples of the above protein drugs include (1) enzymes of enzymes: interferonase, montebrase, imiglucerase, veragulcerase alpha, agarcidase alpha, agarcidase beta, laronidase, alglucosidase alpha, idur sulfase, galsulfase, lasbricase, dorunase alpha, (2). ) Blood coagulation / fibrinolytic factor proteins: Octocog alpha, Lurioctocog alpha, Eptacog alpha (active form), Nonacog alpha, Turoctocog alpha, Eftrenonacog alpha, Thrombomodulin alpha, (3) Serum proteins: Human serum albumin, (4) Hormonal proteins: human insulin, insulin lispro, insulin aspart, insulin glargine, insulin detemil, insulin glulysine, insulin degludec, insulin degludec and insulin aspart, somatropin, pegbisomant, mechacermin, carperitide, glucagon, holitropin alpha, follitropin Beta, liraglutide, teriparatide, metrereptin, (5) Proteins of vaccines: recombinant precipitated hepatitis B vaccine (derived from yeast), dry cell culture inactivated hepatitis A vaccine, recombinant precipitated divalent human papillomavirus-like particle vaccine (Iraxa) (Derived from Ginuwaba cells), recombinant precipitated tetravalent human papillomavirus-like granule vaccine (derived from yeast), (6) Interferon proteins: interferon alpha (NAMALWA), interferon alpha-2b, interferon alpha (BALL-1), interferon alpha Con-1, Interferon Beta, Interferon Beta-1a, Interferon Beta-1b, Interferon Gamma-1a, Peg Interferon Alpha-2a, Peg Interferon Alpha-2b, (7) Erythropoetin Proteins: Epoetin Alpha, Epoetin Beta, Dalbepoetin Alpha , Epoetin beta pegol, epoetin kappa, (8) cytokine proteins: Philgrastim, pegfilgrastim, lenograstim, naltograstim, selmoloikin, teserokin, trafermin, (9) antibody proteins: muromonab-CD3 , Truss zumab, rituximab, paribi Zumabu, infliximab, basiliximab, tocilizumab, gemtuzumab ozogamicin, bevacizumab, ibritumomab tiuxetan, adalimumab, cetuximab, ranibizumab, omalizumab, eculizumab, panitumumab, Usutekinumabu, golimumab, Kanakinumabu, denosumab, mogamulizumab, certolizumab pegol, ofatumumab, pertuzumab, trastuzumab Emutanshin, Burentsukishima Bubedotin, natalizumab, nibolumab, alemtuzumab, (10) fusion proteins: etanercept, avatacept, lomiprostim, afribercept and the like can be mentioned.

Examples of the nucleic acid drug include antisense, siRNA, decoy nucleic acid, nucleic acid aptamer, ribozyme, miRNA antisense, miRNAmic, and CpG oligodeoxynucleotide.

Since the low cell adhesion material has low cell adhesion, it can be applied to various articles required to avoid cell adhesion. The shape of the article is not particularly limited and may be a bag, a sheet, a film, a vial, a petri dish, or a flask.
A method for preventing cell adhesion to the article, which comprises applying the low cell adhesion material to the article, is preferable as a method for using the low cell adhesion material. The method of applying the low cell adhesion material is not particularly limited, and is not particularly limited as long as at least a part of the surface of the article can be covered with the low cell adhesion material. For example, a method of forming a coating film by applying the above-mentioned fluorine-containing copolymer or a coating composition containing the above-mentioned fluorine-containing copolymer can be mentioned. The method for forming the coating film is as described above.

The present invention comprises the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned coating layer, and is a bag, sheet, film, vial, petri dish, or flask. It is also a cell adherent article.

The present invention comprises the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned coating layer, and comprises a cell culture bag, a cell culture sheet, a cell culture film, a cell culture vial, and cells. It is also a low cell adherent article characterized by being a petri dish for culture or a flask for cell culture.

The present invention is also a low cell adhesion article comprising the above-mentioned low cell adhesion material, the above coating composition, or the above coating layer, which is a culture vessel for embryoid body formation. is there. The embryoid body-forming culture vessel preferably has two or more wells. The shape of each well is not particularly limited, but it is preferable that the well has a bottom having a substantially U-shaped cross section in the vertical direction and a substantially circular opening. Further, the radius of curvature (R') of the inner surface of the bottom is preferably 1.0 mm or more and 3.5 mm or less, and more preferably 3.0 mm or less. The diameter of the opening is preferably 4.0 to 11.0 mm. The volume of each well can be 80-500 μL. The embryoid body-forming culture vessel preferably has at least the inner surface of the well made of the low cell adhesion material, the coating composition described above, or the coating layer described above.

If cells easily adhere to the equipment used to culture cells, the recovery rate of the cultured cells will decrease, the cells cannot be recovered in the proliferating shape, or the properties of the cells will change. Relax. The cell culture bag, cell culture sheet, cell culture film, cell culture vial, cell culture chalet or cell culture flask are the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned. When it is composed of the coating layer of No. 1, it is difficult for cells to adhere, and the cells obtained by culturing can be recovered at a high recovery rate and in a preferable state of the shape and properties of the cells.

Examples of the above cells include stem cells such as hematopoietic stem cells, nerve stem cells, mesenchymal hepatocytes, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, and embryonic stem cells, cells obtained by differentiating stem cells into target cells, or immune system. Cells, blood cell lineage cells, nerve cells, vascular endothelial cells, fibroblasts, epithelial cells, keratinized cells, corneal cells, osteoblasts, cartilage cells, fat cells, epidermal cells, hepatocytes, pancreatic β cells, myocardial cells, Living body-derived cells such as bone marrow cells, sheep membrane cells, and umbilical cord blood cells, or NIH3T3 (NIH3T3) cells, 3T3-L1 (3T3-L1) cells, 3T3-E1 (3T3-E1) cells, Hela ( Healer cells, PC-12 cells, P19 cells, CHO (Chinese hamster egg mother) cells, COS cells, HEK cells, Hep-G2 (Hep-G2) Hepzy-two cells, L929 (ernine-to-nine) cells, C2C12 (sea-to-sie-zelb) cells, Daudi cells, Jurkat cells, KG-1a (cage-one-a) cells, CTLL-2 ( Established cells such as CTL2 cells, NS-1 cells, MALT-4 cells, HUT78 cells, MT-4 cells, or Examples thereof include various hybridoma cell lines that are antibody-producing cells, and cells obtained by genetically modifying these cells.

Even in the case of culturing adherent cells, which are cells that easily adhere to a polymer material, the low cell adhesion article of the present invention can suppress the adhesion of the cultured cells.

Examples of the cells include embryoid bodies (EB) such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).

Embryoid body formation is a widely adopted method because it is effective in inducing differentiation of pluripotent stem cells such as ES cells in vitro. For embryoid body formation, it is important to culture ES cells and iPS cells in a floating state without adhering them to the culture vessel, and it is difficult for embryoid bodies to be formed by adhesive culture using a normal culture vessel. When the embryoid body forming incubator is composed of the low cell adhesion material described above, the coating composition described above, or the coating layer described above, the embryoid body is uniform, efficient, and of high quality. Can be formed.

Next, the present invention will be described with reference to examples, but the present invention is not limited to such examples.

Each numerical value of the example was measured by the following method.

[Measurement of fluorine-containing olefin unit content by fluorine content]
10 mg of the sample was burned by the oxygen flask combustion method, the decomposed gas was absorbed by 20 ml of deionized water, and the fluorine ion concentration in the absorption liquid was measured by the fluorine selective electrode method (mass%). The fluorine-containing olefin unit content (mol%) in the polymer was calculated from the fluorine content of the polymer before saponification.

[Measurement of alternating rate by NMR (nuclear magnetic resonance)]
^{1 1} H-NMR measurement conditions: 400 MHz (tetramethylsilane = 0 ppm)

[Molecular weight and molecular weight distribution]
The average molecular weight was calculated from the data measured by flowing tetrahydrofuran (THF) as a solvent at a flow rate of 1 ml / min by gel permeation chromatography (GPC).
RI was used as the detector, and a polystyrene standard sample was used as the calibration curve sample, and the measurement was performed at a flow velocity of 1 ml / min and a sample injection amount of 200 μL.

[Measurement of saponification by IR analysis]
It was measured at room temperature with a Fourier transform infrared spectrophotometer.

[Melting point (Tm)]
Using a DSC (Differential Scanning Calorimetry), the temperature corresponding to the maximum value in the heat of fusion curve when the temperature was raised (second run) under the condition of 10 ° C./min was defined as Tm (° C.).

[Decomposition temperature]
The decomposition temperature was defined as the temperature of the inflection point showing a large weight loss in the thermal decomposition curve in the measurement of TGA (calorific value measuring device). Specifically, in the TGA curve, the decomposition temperature was determined by a method of obtaining an intersection by drawing an auxiliary line before and after a large weight loss (intersection method).

[Quartz crystal microbalance method (QCM-D)]
Using QCM-D (QCM-D300 manufactured by Meiwaforsis Co., Ltd.), the amount of protein adsorbed on the coating film of P-1 to 4 was determined. As the QCM chip, one manufactured by qsense (QSX 301, Au) was used. The QCM chip was first coated with PET and then coated with P-1 to P-4, which was used for the measurement. The details of the PET coating method for the QCM chip are shown in Comparative Example 1.

To determine the amount of protein adsorbed on the coating film of P-1 to P-1, the frequency obtained from the experiment after 30 minutes of adsorption time was calculated from the Sauerbrey formula introduced in the analysis software QTools, Real mass [ng / cm ² ] Was analyzed and calculated. Details of the experiment will be described in Example 1.

The polymers used in the examples are shown in Table 1.

Evaluation of Alkali Resistance 2.3 g of a polymer solution consisting of 13% by mass of polymer P-2 and 87% by mass of methanol was prepared, developed on a petri dish having a diameter of about 58 mm, and dried to obtain a polymer film. The polymer film was immersed in an aqueous solution containing 100 mg, 0.5% by mass of sodium hypochlorite and 0.4% by mass of sodium hydroxide, and kept at room temperature for 24 hours. As a result of measuring the weight change rate, it was reduced by only about 1%, and sufficient alkali resistance was confirmed. In addition, there was no visual change in appearance.

Example 1
A 0.1% by mass methanol solution of polymer P-1 was spin-coated on one side reaction surface of a PET-coated QCM chip (disk-shaped: 1.4 cm in diameter) at room temperature. Specifically, 30 μL of the above solution was dropped onto the QCM chip and rotated at 2000 rpm for 60 seconds. Then, it dried under reduced pressure with a rotary vacuum pump at room temperature for 3 hours to obtain a coating film (QCM chip).

The obtained coating membrane was incorporated into QCM-D, and a protein adsorption test (derived from bovine serum albumin and fibrinogen bovine plasma) on the coating membrane was evaluated by the following method. The results of the experiment are shown in Table 2.

[Protein adsorption test]
The QCM chip coated with P-1 was mounted on the QCM-D by superimposing it on the PET-coated QCM chip by the above method, and stabilized in phosphate buffered saline (PBS) in an environment of 23.4 ° C. It was.
After confirming that the frequency baselines are parallel, inject 0.5 mL of a phosphate buffered saline solution containing protein (protein concentration 0.05 mg / mL), and adjust the amount of frequency change for an adsorption time of 30 minutes. It was measured. As the protein, bovine serum albumin (BSA) or bovine plasma fibrinogen (BPF) was used.

The amount of protein adsorbed on the coating film was calculated by analyzing the Real mass [ng / cm ² ] from the Sauerbury formula introduced in the analysis software QTools using the frequency obtained from the experiment after the adsorption time of 30 minutes.

Examples 2-4
A coating film of polymers P-2 to P-4 was obtained in the same manner as in Example 1. A protein adsorption test was carried out on the obtained coating film in the same manner as in Example 1.

Comparative Example 1 (PET)
QCM in a 1.0% by mass solution of polyethylene terephthalate (PET) (dissolved in a mixed solvent of trifluoroacetic acid, dichloromethane and 1,1,2,2 tetrachloroethane (mixing ratio 1/4/45)) at room temperature The chip (disk-shaped: 1.4 cm in diameter) was spin-coated on one side reaction surface. Specifically, 30 μL of the above solution was dropped onto the QCM chip and rotated at 2000 rpm for 60 seconds. Then, it was dried under reduced pressure with a rotary vacuum pump at 50 ° C. for 3 hours to obtain a coating film.
A protein adsorption test was carried out on the obtained coating film in the same manner as in Example 1.

Example 5
A 0.1 mass% methanol solution of polymer P-1 was spin coated onto a PET film (1.0 cm × 1.0 cm) at room temperature. Specifically, 30 μL of the above solution was dropped onto a PET film and rotated at 2000 rpm for 60 seconds. Then, it was dried under reduced pressure with a rotary vacuum pump for 3 hours at room temperature to obtain a coating film.
The cell adhesion test of the obtained coating film was evaluated by the following method. The results are shown in Table 3.

[Cell adhesion test]
A coating film spin-coated with P-1 on a PET film (1.0 cm × 1.0 cm) was fixed to the bottom of a 24-well cell culture plate with a small amount of silicone adhesive. After washing 3 times with ultrapure water, it was immersed in PBS under a 37 ° C. environment for 12 hours.

Mouse fibroblasts (NIH3T3) were cultured in Dulbecco's modified Eagle's medium (DMEM containing FCS) containing 10% fetal bovine serum (FCS) in an environment of 5% CO ₂ and 37 ° C. The cultured NIH3T3 cells were washed once with sterile PBS, 1 mL of 0.02% ethylenediaminetetraacetic acid (EDTA) solution and 1 mL of 0.25% trypsin solution were added, and the cells were peeled off from the cell culture plate. The cell suspension was centrifuged to collect NIH3T3 cells, and then resuspended in DMEM containing FCS to obtain a solution of 61.2 × 10 ⁴ cells / mL.

The cell suspension was prepared in a medium at 1 × 10 ⁴ cells / cm ² for each well of the cell culture plate on which the coating membrane was fixed, and seeded on the coating membrane. After culturing in an environment of 5% CO ₂ and 37 ° C. for 1 hour, the coating membrane was washed 3 times with PBS and adherent cells on the coating membrane were immobilized with 4% paraformaldehyde PBS solution. After washing with ultrapure water three times, it was dried under reduced pressure with a rotary vacuum pump. Adherent cells were stained with a crystal violet PBS staining solution having a concentration of 1% by mass, and the number of adherent cells was counted by arbitrarily observing three fields of view using an optical microscope.

Examples 6-8
A coating film of polymer P-2 to P-4 was obtained in the same manner as in Example 5. The cell adhesion test was carried out on the obtained coating film in the same manner as in Example 5.

Comparative Example 2 (PET)
A cell adhesion test was carried out in the same manner as in Example 5 using a PET film (1.0 cm × 1.0 cm).

Examples 9-12 and Comparative Example 3
A protein adsorption test was carried out in the same manner as in Examples 1 to 4 and Comparative Example 1 except that bovine serum-derived immunoglobulin G (IgG) was used as the protein. The results are shown in Table 4.

Claims (11)

It is formed from a low protein-adsorbing material composed of a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit and a content of the fluorine-containing olefin unit of 35 to 32 mol% .
The coating layer is characterized in that the fluorine-containing olefin is at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene . The coating layer according to claim 1, wherein the alternating ratio of the fluorine-containing olefin unit and the vinyl alcohol unit in the fluorine-containing copolymer is 1 to 75%. The coating layer according to claim 1 or 2, wherein the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit. A low protein adsorptive article comprising the coating layer according to claim 1, 2 or 3 , which is a bag, sheet, film, vial, petri dish, or flask. A biopharmacy bag, a biopharmacy sheet, a biopharmacy film, a biopharmacy vial, a biopharmacy petri dish, or a biopharmacy flask, comprising the coating layer according to claim 1, 2 or 3. A characteristic low protein adsorptive article. It consists of a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit.
It is formed from a low cell adhesion material having a fluorine-containing olefin unit content of 35 to 32 mol% in the fluorine-containing copolymer .
The coating layer is characterized in that the fluorine-containing olefin is at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene . The coating layer according to claim 6 , wherein the alternating ratio of the fluorine-containing olefin unit and the vinyl alcohol unit in the fluorine-containing copolymer is 1 to 75%. The coating layer according to claim 6 or 7, wherein the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit. A low cell adhesion article comprising the coating layer according to claim 6, 7 or 8 , which is a bag, sheet, film, vial, petri dish, or flask. The coating layer according to claim 6, 7 or 8 , which is a cell culture bag, a cell culture sheet, a cell culture film, a cell culture vial, a cell culture petri dish, or a cell culture flask. A characteristic low cell adherent article. A low cell adhesion article comprising the coating layer according to claim 6, 7 or 8, which is a culture vessel for embryoid body formation.