JPWO2016104596A1 - Low protein adsorbing material, low protein adsorbing article, low cell adhering material and low cell adhering article - Google Patents

Abstract

Provided is a novel low protein adsorptive material which is difficult to adsorb proteins and is excellent in heat resistance, alkali resistance and molding processability. A low protein adsorptive material comprising a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit.

Description

The present invention relates to a low protein adsorbing material, a low protein adsorbing article, a low cell adhering material, and a low cell adhering article.

Polymer materials are used as materials constituting instruments for storing blood, instruments for culturing cells, and the like. Depending on the application, there is a demand for suppressing the adsorption of blood to the polymer material, or 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 in which at least a part of the surface is composed of a hydrophobic fluororesin and a hydrophilic fluororesin.

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

In Patent Document 3, as a film used for water treatment or medical use, a copolymer of tetrafluoroethylene (TFE) and vinyl acetate (VAc) or at least a part of an acetate group contained in the copolymer is included. There is described a fluorine-containing copolymer film comprising a saponified copolymer and having a tetrafluoroethylene content of 1 to 70 mol% contained in the copolymer.

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

Patent Document 5 discloses a molded product having a low protein adsorption layer on the surface of a support layer, the low protein adsorption layer is made of an amphiphilic polymer, and is bonded to the support layer by a covalent bond to form a layer. A low protein adsorptive molded product characterized in that is described.

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

Patent Document 7 describes a cell culture substrate using a polymer composite in which a water-swellable clay mineral is 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 anchorage-independent cells is easy.

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

Japanese Patent Laid-Open No. 4-336072 Special table 2007-515208 JP-A-5-261256 International Publication No. 2012/165503 JP 2004-277852 A JP-A-7-83923 JP 2013-176402 A JP-A-8-322593 JP 2012-210166 A

The present invention makes it difficult for proteins to adsorb, a novel low protein adsorbing material excellent in heat resistance, alkali resistance and molding processability and cells, and excellent in heat resistance, alkali resistance and molding processability. An object is to provide a novel low cell adhesion material.

The present invention is a low protein adsorptive material comprising 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 fluorine-containing olefin unit content in the fluorine-containing copolymer is preferably 60 to 10 mol%.

In the low protein adsorptive material of the present invention, it is preferable that the alternating rate of the fluorinated olefin unit and the vinyl alcohol unit in the fluorinated copolymer is 1 to 75%.

In the low protein adsorptive material of the present invention, the fluorinated 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 also provides a coating composition comprising the above-described low protein adsorptive material.

The present invention also provides a coating layer formed from the above-described low protein adsorptive material.

The present invention is a low protein comprising the above-described low protein adsorbing material, the above coating composition, or the above coating layer, and being a bag, sheet, film, vial, petri dish, or flask. It is also an absorptive article.

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

The present invention is also a low cell adhesion material characterized by comprising 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 fluorine-containing olefin unit content in the fluorine-containing copolymer is preferably 60 to 10 mol%.

In the low cell adhesion material of the present invention, it is preferable that the alternating rate of the fluorinated olefin unit and the vinyl alcohol unit in the fluorinated copolymer is from 1 to 75%.

In the low cell adhesion material of the present invention, the fluorinated 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 also provides a coating composition comprising the above-mentioned low cell adhesion material.

The present invention is also a coating layer 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-adhesive 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, a cell It is also a low cell adhesion 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-mentioned coating composition, or the above-mentioned coating layer and being a culture container for embryoid body formation.

The low protein adsorptive material of the present invention hardly adsorbs protein, and is excellent in heat resistance, alkali resistance and moldability. From the low protein adsorptive material of the present invention, it is possible to obtain an article that is difficult to adsorb proteins and is excellent in heat resistance, alkali resistance and molding processability. Further, the low cell adhesion material of the present invention is less likely to adhere to cells 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 that is difficult to adhere to cells and is excellent in heat resistance, alkali resistance and molding processability.

The low protein adsorptive article of the present invention hardly adsorbs protein on the surface, and is excellent in heat resistance, alkali resistance and molding processability. In addition, the low cell adhesion article of the present invention is less susceptible to cell adhesion on the surface and is excellent in heat resistance, alkali resistance and molding processability.

Hereinafter, the present invention will be specifically described.

The low protein adsorptive material and the low cell adhesion material of the present invention are composed of a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit (—CH ₂ —CH (OH) —). The low protein adsorption property and low cell adhesion property of the fluorine-containing copolymer are characteristics newly found by the present inventors.

Since the low protein adsorptive material and the low cell adhesion material of the present invention are excellent in heat resistance, they can withstand sterilization performed at high temperatures. For example, it can withstand autoclave sterilization performed at 121 ° C. It is preferable that the low protein adsorbing material and the low cell adhesion material of the present invention have a decomposition temperature exceeding 121 ° C.

Since the low protein adsorbing material and the low cell adhesion material of the present invention are excellent in alkali resistance, they can withstand disinfection using an alkaline aqueous solution containing hypochlorous acid. For example, when 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 weight change rate is 10% by mass or less.

The low protein adsorptive material and the low cell adhesion material of the present invention are excellent in moldability. For example, when the low protein adsorptive material and the low cell adhesion material of the present invention are a coating composition containing an organic solvent, a weak solvent such as alcohol can be used as the organic solvent, so that it is eroded by the 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 monomer units constituting the fluorine-containing copolymer. More preferably, it is 40-10 mol%, More preferably, it is 35-20 mol%, More preferably, it is 35-25 mol%. When the content of the fluorinated olefin unit is within the above range, proteins are more difficult to adsorb to the low protein adsorbing material, and cells are less likely to adhere to the low cell adhering material. When the content of the fluorinated olefin unit is less than 10 mol%, the water-soluble property of the fluorinated copolymer is increased, and the fluorinated copolymer is easily eluted in water, and the function is hardly exhibited. When the content of the fluorine-containing olefin unit is large, the heat resistance, alkali resistance, and molding processability of the low protein adsorbing material and the low cell adhesion material tend to be excellent.

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 a content rate of each monomer unit, it is more preferable that a fluorine-containing olefin unit is 40-10 mol%, a vinyl alcohol unit is 60-90 mol%, and a fluorine-containing olefin unit is 35-20 mol%. The vinyl alcohol unit is more preferably 65 to 80 mol%, the fluorinated olefin unit is 35 to 25 mol%, and the vinyl alcohol unit is more preferably 65 to 75 mol%. When the content of each monomer unit is in such a range, proteins are more difficult to adsorb to the low protein adsorbing material, and cells are less likely to adhere to the low cell adhering material. When the content of the fluorinated olefin unit is less than 10 mol%, the water-soluble property of the fluorinated copolymer is increased, and the fluorinated copolymer is easily eluted in water, and the function is hardly exhibited. When the content of the fluorine-containing olefin unit is large, the heat resistance, alkali resistance, and molding processability of the low protein adsorbing material and the low cell adhesion material tend to be excellent.

The said fluorine-containing olefin unit represents the polymerization unit based on a 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, CH ₂ = CZ. ¹ (CF ₂ ) _n1 Z ² (wherein Z ¹ is H, F or Cl, Z ² is H, F or Cl, and n1 is an integer of 1 to 10), CF ₂ = CF-ORf ¹ (wherein Rf ¹ represents a perfluoroalkyl group having 1 to 8 carbon atoms) and CF ₂ = CF-OCH ₂ -rf ² (wherein, Rf ² is a perfluoroalkyl group having 1 to 5 carbon atoms) selected from the group consisting of alkyl perfluorovinyl ether derivative represented by the It is preferably be at least one kind of fluorine-containing olefin.

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 and CH ₂ = CClCF ₃ .

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 said fluorine-containing olefin, at least 1 sort (s) selected from the group which consists of TFE, CTFE, and HFP is more preferable, and TFE is still more preferable.

The fluorine-containing copolymer preferably has an alternating rate of fluorine-containing olefin units and vinyl alcohol units of 1 to 75%. When the alternating rate is within the above range, proteins are more difficult to adsorb to the low protein adsorbing material, and cells are less likely to adhere to the low cell adhering material. More preferably, it is 10-60%, More preferably, it is 10-35%, Most preferably, it is 10-20%.

The alternating rate of the fluorinated olefin unit and the vinyl alcohol unit was determined by performing ¹ H-NMR measurement of the fluorinated copolymer using a solvent in which the fluorinated copolymer such as heavy acetone is dissolved, and calculating from the following formula: It can be calculated as the alternating rate of chaining.
Alternating rate (%) = C / (A + B + C) × 100
A: The number of V combined with two Vs such as -VVVV- B: The number of V combined with V and T as -VVT- C: -TV- Number of V bonded to two T 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) —) of ¹ H-NMR measurement. The estimation of the strength ratio of H in the main chain by ¹ H-NMR measurement is carried out with the fluorinated copolymer before saponification.

Since the fluorine-containing copolymer is more difficult to adsorb proteins and more difficult to adhere to cells, -CH (OH) -CXY- (wherein X and Y are the same or different, and H, F or a fluoroalkyl group, provided that at least one of X and Y is F or a fluoroalkyl group). The fluorine-containing copolymer more preferably has a fluorine alcohol structure represented by —CH (OH) —CF ₂ —.

The fluorine-containing copolymer is further represented by —CH ₂ —CH (O (C═O) R) — (wherein R represents a hydrogen atom or a hydrocarbon group having 1 to 17 carbon atoms). It may have a vinyl ester monomer unit. Thus, it is also one of the preferred embodiments of the present invention that the fluorinated copolymer in the present invention has a fluorinated olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit. Furthermore, it is a fluorine-containing olefin / vinyl alcohol / vinyl ester monomer copolymer consisting essentially of a fluorine-containing olefin unit, a vinyl alcohol unit and a vinyl ester monomer unit. One.

The vinyl ester monomer unit is a monomer represented by —CH ₂ —CH (O (C═O) R) — (wherein R represents a hydrogen atom or a hydrocarbon group having 1 to 17 carbon atoms). Although it is a unit, as R in the said formula, a C1-C11 alkyl group is preferable and a C1-C5 alkyl group is more preferable. Particularly preferred is an alkyl group having 1 to 3 carbon atoms.

Examples of the vinyl ester monomer unit include monomer units derived from the following vinyl esters.
Vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, vinyl valelate, vinyl isovalerate, vinyl caproate, vinyl heptylate, vinyl caprylate, vinyl pivalate, vinyl pelargonate, vinyl caprate, Vinyl laurate, vinyl myristate, vinyl pentadecylate, vinyl palmitate, vinyl margarate, vinyl stearate, vinyl octylate, Veova-9 (manufactured by Showa Shell Sekiyu KK), Veova-10 (Showa Shell Sekiyu KK) )), Vinyl benzoate, vinyl versatate.
Among these, monomer units derived from vinyl acetate, vinyl propionate, and vinyl versatate are preferable. More preferred are vinyl acetate monomer units and vinyl propionate monomer units, and even more preferred are vinyl acetate monomer units.

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 It is preferable that the unit is 40 to 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, proteins are more difficult to adsorb to the low protein adsorbing material, and cells are less likely to adhere to the low cell adhering material. The content of each monomer unit is such that 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%, the vinyl ester monomer unit is more than 0 and less than 1.0 mol%. More preferably, the fluorine olefin unit is 35 to 20 mol%, the vinyl alcohol unit is 65 to 80 mol%, the vinyl ester monomer unit is more than 0 and less than 1.0 mol%. Is 35 to 25 mol%, vinyl alcohol units are 65 to 75 mol%, It is even more preferred more mer units is from greater than 0 to less than 1.0 mol%. When the content of the fluorine-containing olefin unit is large, the heat resistance, alkali resistance, and molding processability of the low protein adsorbing material and the low cell adhesion material tend to be excellent.

When the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit, the alternating rate of the fluorine-containing olefin unit, the vinyl alcohol unit, and the vinyl ester monomer unit is 1 to 75%. It is preferable. When the alternating rate is within the above range, proteins are more difficult to adsorb to the low protein adsorbing material, and cells are less likely to adhere to the low cell adhering material. More preferably, it is 10-60%, More preferably, it is 10-35%, Most preferably, it is 10-20%.

The alternating rate of the fluorinated olefin unit, the vinyl alcohol unit, and the vinyl ester monomer unit is determined by performing ¹ H-NMR measurement of the fluorinated copolymer using a solvent in which the fluorinated copolymer such as heavy acetone is dissolved, It can be calculated as an alternating rate of three chains from the following formula.
Alternating rate (%) = C / (A + B + C) × 100
A: The number of V combined with two Vs such as -VVVV- B: The number of V combined with V and T as -VVT- C: -TV- Number of Vs bonded to two Ts such as T- (T: fluorinated olefin unit, V: vinyl alcohol unit or vinyl ester monomer unit)
The number of V units of A, B, and C is the vinyl alcohol unit (—CH ₂ —CH (OH) —) and vinyl ester monomer unit (—CH ₂ —CH (O (C═O)) of ¹ H-NMR measurement. Calculated from the intensity ratio of H of the main chain bonded to the tertiary carbon of R)-). The estimation of the strength ratio of H in the main chain by ¹ H-NMR measurement is carried out with the fluorinated copolymer before saponification.

The said fluorine-containing copolymer may have other monomer units other than a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit in the range which does not impair the effect of this invention.

As said other monomer, as a monomer which does not contain a fluorine atom (however, except vinyl alcohol and a vinyl ester monomer), for example, ethylene, propylene, 1-butene, 2-butene, vinyl chloride, Preference is given to at least one fluorine-free ethylenic monomer selected from the group consisting of vinylidene chloride, vinyl ether monomers and unsaturated carboxylic acids.

The total content of the other monomer units is preferably 0 to 50 mol%, more preferably 0 to 40 mol%, based on all monomer units of the fluorine-containing copolymer, More preferably, it is 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 depending on 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 12,000-2,000,000, More preferably, it is 12,000-1,000,000.
The weight average molecular weight can be determined by gel permeation chromatography (GPC).

As 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, 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. One.

Below, the manufacturing method of the fluorine-containing copolymer in this invention is demonstrated.
Usually, the fluorine-containing copolymer in the present invention is produced by copolymerizing a fluorine-containing olefin such as tetrafluoroethylene and a vinyl ester monomer such as vinyl acetate, and then saponifying the obtained copolymer. can do. As a method for polymerizing the fluorine-containing copolymer, it is preferable to carry out the polymerization under a condition in which the composition ratio of the fluorine-containing olefin and the vinyl ester monomer is kept substantially constant. That is, the above-mentioned fluorine-containing copolymer is polymerized under a condition in which the composition ratio of the fluorine-containing olefin and the vinyl ester monomer is kept almost constant to obtain a copolymer having a fluorine-containing olefin unit and a vinyl ester monomer unit. It is preferably obtained by a production method comprising 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 monomers include vinyl formate, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl isobutyrate, vinyl valerate, vinyl isovalerate, vinyl caproate, vinyl heptylate, vinyl caprylate, vinyl pivalate, pelargon. Vinyl acetate, vinyl caprate, vinyl laurate, vinyl myristate, vinyl pentadecylate, vinyl palmitate, vinyl margarate, vinyl stearate, vinyl octylate, Veova-9 (manufactured by Showa Shell Sekiyu KK), Veova 10 (manufactured by Showa Shell Sekiyu KK), vinyl benzoate, vinyl versatate, and the like. Of these, vinyl acetate, vinyl propionate, and vinyl versatate are preferably used because they are easily available and inexpensive.
As said vinyl ester monomer, 1 type of these may be used and 2 or more types may be mixed and used.

Examples of the method of copolymerizing the fluorinated olefin and the vinyl ester monomer include solution polymerization, bulk polymerization, emulsion polymerization, suspension polymerization and the like, and emulsion polymerization is easy because it is industrially easy to implement. However, it is preferable to produce by solution polymerization or suspension polymerization, but not limited thereto.

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 a solvent capable of dissolving the fluorine-containing olefin, the vinyl ester monomer, and the fluorine-containing copolymer to be synthesized. For example, n-butyl acetate, t-butyl acetate, ethyl acetate Esters such as methyl acetate and propyl acetate; Ketones such as acetone, methyl ethyl ketone and cyclohexanone; Aliphatic hydrocarbons such as hexane, cyclohexane and octane; Aromatic hydrocarbons such as benzene, toluene and xylene; Methanol and ethanol , Tert-butanol, alcohols such as 2-propanol; cyclic ethers such as tetrahydrofuran and dioxane; fluorine-containing solvents such as HCFC-225; dimethyl sulfoxide, 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 diisopropyl peroxydicarbonate (IPP) and di-n-propyl peroxydicarbonate (NPP), and t-butyl peroxypivalate (for example, NOF Corporation). Oil-soluble radical polymerization initiators typified by peroxyesters such as perbutyl PV), for example, persulfuric acid, perboric acid, perchloric acid, perphosphoric acid, ammonium percarbonate, potassium salt, sodium Water-soluble radical polymerization initiators such as salts can be used. Particularly in emulsion polymerization, ammonium persulfate and potassium persulfate are preferred.

As the surfactant, a commonly used surfactant can be used. For example, a nonionic surfactant, an anionic surfactant, a cationic surfactant and the like can be used. Moreover, you may use a fluorine-containing surfactant.

Examples of the dispersant used in suspension polymerization include partially saponified polyvinyl acetate, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and other water-soluble cellulose ethers used in ordinary suspension polymerization, and acrylic acid polymers. And water-soluble polymers such as gelatin. The suspension polymerization is carried out under the condition that the ratio of water / monomer is usually 1.5 / 1 to 3/1 in terms of mass ratio, and the dispersant is 0.01 to 0 with respect to 100 parts by mass of the monomer. .1 part by weight is used. If necessary, a pH buffer such as polyphosphate can be used.

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

The polymerization temperature may be in a range where the composition ratio during the reaction of the fluorinated olefin and the vinyl ester monomer is substantially constant, and may be 0 to 100 ° C.

The polymerization pressure may be in a range in which the composition ratio during the reaction of the fluorinated olefin and the vinyl ester monomer is substantially constant, and may be 0 to 10 MPaG.

Saponification of an acetate group derived from vinyl acetate is well known in the art, and can be performed by a conventionally known method such as alcoholysis or hydrolysis using an acid or alkali. By this saponification, the acetate group (—OCOCH ₃ ) is converted into a hydroxyl group (—OH). Similarly, other vinyl ester monomers can be saponified by a conventionally known method to obtain a hydroxyl group.

The degree of saponification in the case of obtaining a fluorine-containing copolymer in the present invention by saponifying a copolymer having a fluorine-containing olefin unit and a vinyl ester monomer unit is the content of each monomer unit of the fluorine-containing copolymer in the present invention. It is sufficient that the rate is in the range described above, specifically 90% or more is preferable, 95% or more is more preferable, and 99% or more is still more preferable.

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

The low protein adsorptive material and the low cell adhesion material may further contain other 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, polyvinyl pyrrolidone, and polyethylene glycol.

The blending amount of the other component is preferably 1 to 30% by mass and more preferably 1 to 10% by mass with respect to 100% by mass of the fluorine-containing copolymer.

The surface of the low protein adsorbing material and the low cell adhering material is preferably treated with an aqueous solution containing an organic solvent. By this surface treatment, the hydroxyl structure further increases on the surface of the low protein adsorbing material and the low cell adhering 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 methanol, ethanol, 2-propanol, acetone, tetrahydrofuran , Methyl ethyl ketone, dimethylacetamide, dimethylformamide and the like. Of these, methanol, ethanol, 2-propanol and tetrahydrofuran are preferred.

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

The low protein adsorbing material and the low cell adhering material are provided by being molded into various shapes depending on the application. The molding method is not particularly limited, and spin coating method, drop casting method, dip nip method, spray coating method, brush coating method, dipping method, ink jet printing method, electrostatic coating method, compression molding method, extrusion molding method, calendar molding method. Methods, transfer molding methods, injection molding methods, lotto molding methods, lotining molding methods, thermally induced phase separation methods, non-solvent induced phase separation methods, and the like can be employed.

Since it can be applied to articles of various shapes, the low protein adsorbing material and the low cell adhering material are preferably coating films. The said coating film means the film | membrane obtained by apply | coating the coating composition containing the said fluorine-containing copolymer or the said fluorine-containing copolymer. Examples of the method for forming the coating film include spin coating, drop casting, dip nip, spray coating, brush coating, dipping, electrostatic coating, and inkjet printing. Of these, spin coating, drop casting, and dipping are preferred from the standpoint of simplicity.

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 in that a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorinated copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran, and dimethylformamide are preferable.

Since the said coating film can be obtained also from the coating composition which uses weak solvents, such as alcohol, as an organic solvent, it can be manufactured easily.

When the low protein adsorbing 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, 1.0 More preferably, it is ˜20 μm.

The low protein adsorptive material may have an adsorption amount of bovine serum albumin 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 hour. it can.
The low protein adsorptive material may have a bovine plasma fibrinogen adsorption amount 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 hour. it can.
The low protein adsorptive material has an adsorption amount of bovine serum-derived immunoglobulin G of 500 ng / cm when contacted with 0.05 mg / ml bovine serum-derived immunoglobulin G solution at 23.4 ° C. for 0.5 hour. ² or less.
The protein-adsorbing material has a surprising effect that almost no protein is 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 protein adsorption amount is measured by the method described in “Protein adsorption test” described later.
A method for preventing adsorption of the protein by applying a low protein adsorbent material to at least the surface of an article in contact with a protein solution of 10 to 0.05 mg / ml is also used as a method for using the low white matter adsorbent material. preferable.

Since the low protein adsorptive material has low protein adsorptivity, it can be applied to various articles 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.
The method for preventing protein adsorption to the article, comprising the step of 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 biopharmaceuticals described below. Examples of the plasma protein include albumin, globulin, and fibrinogen. The method for applying the low protein adsorptive 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 protein adsorptive material. For example, a method of forming a coating film by applying the fluorine-containing copolymer or a coating composition containing the 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 adsorbing 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 in that a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorinated copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran, and dimethylformamide are preferable.

The said coating composition can use weak solvents, such as alcohol, as an organic solvent. Therefore, the present invention 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 spin coating, drop casting, dip nip, spray coating, brush coating, dipping, electrostatic coating, and inkjet printing. Of these, spin coating, drop casting, and dipping are preferred from the standpoint of simplicity.

The coating film or coating layer formed from the coating composition preferably has a film thickness of 0.1 to 50 μm, more preferably 0.5 to 30 μm, and 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 formed from the low protein adsorbing material or the low cell adhesion material.

The said coating layer is obtained by apply | coating the coating composition containing the said fluorine-containing copolymer or the said fluorine-containing copolymer. Examples of the coating method include spin coating, drop casting, dip nip, spray coating, brush coating, dipping, electrostatic coating, and inkjet printing. Of these, spin coating, drop casting, and dipping are preferred from the standpoint of simplicity.

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 in that a transparent and uniform coating film can be easily obtained. From the viewpoint of solubility of the fluorinated copolymer, methanol, ethanol, 2-propanol, tetrahydrofuran, and dimethylformamide are preferable.

Since the said coating layer can be obtained also from the coating composition which uses weak solvents, such as alcohol, as an organic solvent, it can be manufactured easily.

The coating layer preferably has a thickness of 0.1 to 50 μm, more preferably 0.5 to 30 μm, and still 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 having the coating layer on the surface are less likely to adhere proteins or cells.

The present invention is a low protein comprising the above-described low protein adsorbing material, the above coating composition, or the above coating layer, and being a bag, sheet, film, vial, petri dish, or flask. It is also an absorptive article.

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

If the biopharmaceutical is easily adsorbed to an instrument for storing the biopharmaceutical or an instrument for using the biopharmaceutical, the biopharmaceutical cannot be accurately quantified or accurate analysis becomes difficult. In addition, since biopharmaceuticals are expensive, there is a great economic loss. A biopharmaceutical bag, a biopharmaceutical sheet, a biopharmaceutical film, a biopharmaceutical vial, a biopharmaceutical petri dish, or a biopharmaceutical flask is the above-described low protein adsorptive material, the above-described coating composition, or When the coating layer is composed of the above-described coating layer, the biopharmaceutical is hardly adsorbed, and the biopharmaceutical can be recovered at a high recovery rate.

Examples of biopharmaceuticals include protein drugs, genetically modified viruses, cellular therapeutic drugs, and nucleic acid drugs.
The above protein drugs include: (1) Enzymes proteins: Arterase, Montebrase, Imiglucerase, Veraglucerase alpha, Agarcidase alpha, Agarcidase beta, Laronidase, Alglucosidase alpha, Idursulfase, Galsulfase, Rasburicase, Dornase alpha, (2 ) Proteins of blood coagulation and fibrinolytic factors: Octocog alpha, Luriooctogog 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, i Surin detemir, insulin gurlysine, insulin degludec, insulin degludec and insulin aspart, somatropin, pegvisomant, mecasermine, carperitide, glucagon, follitropin alpha, follitropin beta, liraglutide, teriparatide, metreleptin, (5) vaccine proteins: recombinant precipitated B Hepatitis B vaccine (derived from yeast), dry cell culture inactivated hepatitis A vaccine, recombinant precipitated bivalent human papillomavirus-like particle vaccine (derived from Iraqus guinea waba cells), recombinant precipitated 4-valent human papilloma virus-like granule vaccine (derived from yeast) (6) Proteins of interferons: interferon alpha (NAMALWA), interferon alpha-2b, interferon alpha (BA L-1), interferon alphacon-1, interferon beta, interferon beta-1a, interferon beta-1b, interferon gamma-1a, peginterferon alpha-2a, peginterferon alpha-2b, (7) proteins of erythropoietins: epoetin Alpha, epoetin beta, darbepoetin alfa, epoetin beta pegol, epoetin kappa, (8) proteins of cytokines: filgrastim, pegfilgrastim, lenograstim, nartograstim, sermoleukin, teseleukin, trafermin, (9) antibody Class of proteins: muromonab-CD3, trastuzumab, rituximab, palivizumab, infliximab, basiliximab, tosiri Mabu, gemtuzumab ozogamicin, bevacizumab, ibritumomab tiuxetan, adalimumab, cetuximab, ranibizumab, omalizumab, eculizumab, panitumumab, Usutekinumabu, golimumab, Kanakinumabu, denosumab, mogamulizumab, certolizumab pegol, ofatumumab, pertuzumab, trastuzumab Emutanshin, brentuximab Bedochin, natalizumab, nivolumab And alemtuzumab, (10) fusion proteins: etanercept, abatacept, romiplostim, aflibercept and the like.

Examples of nucleic acid pharmaceuticals include antisense, siRNA, decoy nucleic acid, nucleic acid aptamer, ribozyme, miRNA antisense, miRNAmimic, 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.
The method for preventing cell adhesion to the article, comprising the step of 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 as long as it is a method capable of covering at least a part of the surface of the article with the low cell adhesion material. For example, a method of forming a coating film by applying the fluorine-containing copolymer or a coating composition containing the 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-adhesive 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, a cell It is also a low cell adhesion article characterized by being a petri dish for culture or a flask for cell culture.

The present invention also relates to 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 container for embryoid body formation. is there. The culture container for embryoid body formation preferably has two or more wells. Although the shape of each well is not particularly limited, it is preferable that the cross section in the vertical direction has a substantially U-shaped bottom portion and a substantially circular opening. Furthermore, the curvature radius (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. In the culture container for embryoid body formation, it is preferable that at least the inner surface of the well is composed of the low cell adhesion material, the coating composition described above, or the coating layer described above.

If cells tend to adhere to the equipment used to culture the cells, the recovery rate of the cultured cells will decrease, the cells will not be recovered in their proliferated form, or the properties of the cells will change. I'll be relaxed. 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 is the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned In the case of the coating layer, cells are difficult to adhere, and cells obtained by culturing can be recovered at a high recovery rate and in a preferable state of cell shape and properties.

Examples of the cells include hematopoietic stem cells, neural stem cells, mesenchymal hepatocytes, mesodermal stem cells, hepatic stem cells, pancreatic stem cells, embryonic stem cells, and the like, cells obtained by differentiating stem cells into target cells, or the immune system Cells, blood cells, neurons, vascular endothelial cells, fibroblasts, epithelial cells, keratinocytes, corneal cells, osteoblasts, chondrocytes, adipocytes, epidermal cells, hepatocytes, pancreatic β cells, cardiomyocytes, Cells derived from living bodies such as bone marrow cells, amniotic cells, umbilical cord blood cells, NIH3T3 cells, 3T3-L1 cells, 3T3-E1 cells, Hela ( HeLa) cells, PC-12 (PC Zelb) cells, P19 (Pineteen) cells, CHO (Chinese hamster oocytes) cells, C S (COS) cells, HEK (Heek-Ek) cells, Hep-G2 (Hepsey two) cells, L929 (El Nine to Nine) cells, C2C12 (C2C Zerub) cells, Daudi (Daudi) cells, Jurkat ( Jurkat) cells, KG-1a cells, CTLL-2 cells, NS-1 cells, MOLT-4 cells, HUT78 Eight) cells, established cell lines such as MT-4 (MT4) cells, various hybridoma cell lines that are antibody-producing cells, or cells obtained by genetically modifying these cells.

Even when culturing adhesive cells, which are cells that are particularly likely to adhere to a polymer material, the low cell adhesion article of the present invention can suppress adhesion of cultured cells.

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

Embryoid body formation is a widely adopted technique because it is effective in inducing differentiation of pluripotent stem cells including ES cells in vitro. In embryoid body formation, it is important to culture ES cells and iPS cells in a floating state where they are not allowed to adhere to the culture vessel, and it is difficult to form embryoid bodies in adhesion culture using a normal culture vessel. When the embryoid body-forming incubator is composed of the above-mentioned low cell adhesion material, the above-mentioned coating composition, or the above-mentioned coating layer, it is uniform, efficient, and high-quality embryoid body 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 examples was measured by the following method.

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

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

[Molecular weight and molecular weight distribution]
The average molecular weight was calculated from 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 for the detector, and a polystyrene standard sample was used for the calibration curve sample, and the measurement was performed at a flow rate of 1 ml / min and a sample injection amount of 200 μL.

[Measurement of degree of saponification by IR analysis]
Measurements were made at room temperature with a Fourier transform infrared spectrophotometer.

[Melting point (Tm)]
Using DSC (differential scanning calorimeter), the temperature corresponding to the maximum value in the heat of fusion curve when the temperature was raised (second run) at 10 ° C./min was defined as Tm (° C.).

[Decomposition temperature]
The decomposition temperature was the temperature at the inflection point that showed a significant weight loss in the thermal decomposition curve in the TGA (calorimeter) measurement. Specifically, in the TGA curve, the decomposition temperature was obtained by a method (intersection method) in which an auxiliary line was drawn before and after a large weight loss to obtain an intersection point.

[Quartz crystal microbalance method (QCM-D)]
The amount of protein adsorbed on the coating films of P-1 to P-4 was determined using QCM-D (QCM-D300 manufactured by Meiwa Forsys). A QCM chip manufactured by qsense (QSX 301, Au) was used. This QCM chip was first coated with PET and then coated with P-1 to 4 for measurement. The details of the PET coating method on the QCM chip are shown in Comparative Example 1.

The amount of protein adsorbed on the coating films of P-1 to P-4 is determined by calculating the frequency after an adsorption time of 30 minutes obtained from the experiment using the real mass [ng / cm ² from the Sauerbrey formula introduced in the analysis software QTools. ] Was calculated. Details of the experiment are 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 comprising 13% by mass of polymer P-2 and 87% by mass of methanol was prepared, spread 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 mass% sodium hypochlorite and 0.4 mass% sodium hydroxide, and kept at room temperature for 24 hours. As a result of measuring the rate of weight change, only about 1% was reduced, and sufficient alkali resistance was confirmed. Further, there was no change in the visual appearance.

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

The obtained coating film was incorporated into QCM-D, and a protein adsorption test (derived from bovine serum albumin and fibrinogen bovine plasma) on the coating film 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 over the PET-coated QCM chip by the above method is attached to the QCM-D, and stabilized in phosphate buffered saline (PBS) at 23.4 ° C. It was.
After confirming that the frequency baselines are parallel, 0.5 mL of a phosphate buffered saline solution containing protein (protein concentration 0.05 mg / mL) is injected, and the amount of change in frequency over an adsorption time of 30 minutes is measured. It was measured. As 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 Sauerbrey equation introduced in the analysis software QTools, based on the frequency after 30 minutes of adsorption time obtained from the experiment.

Examples 2-4
In the same manner as in Example 1, coating films of polymers P-2 to P-4 were obtained. The obtained coating film was subjected to a protein adsorption test in the same manner as in Example 1.

Comparative Example 1 (PET)
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 Spin coating was applied to one side reaction surface of a chip (disk shape: diameter 1.4 cm). Specifically, 30 μL of the above solution was dropped on a QCM chip and rotated at 2000 rpm for 60 seconds. Then, it dried under reduced pressure with the rotary vacuum pump at 50 degreeC for 3 hours, and obtained the coating film.
The obtained coating film was subjected to the protein adsorption test in the same manner as in Example 1.

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

[Cell adhesion test]
A coating film obtained by spin-coating 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 silicon adhesive. After washing with ultrapure water 3 times, it was immersed in PBS at 37 ° C. for 12 hours.

Mouse fibroblasts (NIH3T3) were cultured in Dulbecco's modified Eagle medium (FCS-containing DMEM) containing 10% fetal calf 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 detached from the cell culture plate. The cell suspension was centrifuged to collect NIH3T3 cells, and then resuspended in FCS-containing DMEM 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 three times with PBS, and adherent cells on the coating membrane were immobilized using 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 at a concentration of 1% by mass, and the number of adherent cells was counted by arbitrarily observing three visual fields using an optical microscope.

Examples 6-8
In the same manner as in Example 5, coating films of polymers P-2 to P-4 were obtained. The resulting coating film was subjected to a cell adhesion test in the same manner as in Example 5.

Comparative Example 2 (PET)
A cell adhesion test was performed 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 performed 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 (21)

A low protein adsorptive material comprising a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit. The low protein adsorptive material according to claim 1, wherein the content of fluorine-containing olefin units in the fluorine-containing copolymer is 60 to 10 mol%. The low protein adsorptive material according to claim 1 or 2, wherein the fluorine-containing copolymer has an alternating ratio of fluorine-containing olefin units and vinyl alcohol units of 1 to 75%. The low protein adsorptive material according to claim 1, 2 or 3, wherein the fluorinated olefin is at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene. The low protein adsorptive material according to claim 1, wherein the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit. 6. The low protein adsorptive material according to claim 1, which is a coating film. A coating composition comprising the low protein adsorptive material according to claim 1, 2, 3, 4 or 5. A coating layer formed from the low protein adsorptive material according to claim 1, 2, 3, 4 or 5. A bag, sheet, film, vial comprising the low protein adsorptive material according to claim 1, the coating composition according to claim 7, or the coating layer according to claim 8. A low protein adsorptive article characterized by being a petri dish or a flask. A low protein-adsorbing material according to claim 1, 2, 3, 4, 5 or 6, a coating composition according to claim 7, or a coating layer according to claim 8, comprising a biopharmaceutical bag and a biopharmaceutical. A low protein adsorptive article characterized by being a sheet, a biopharmaceutical film, a biopharmaceutical vial, a biopharmaceutical petri dish, or a biopharmaceutical flask. A low cell adhesion material comprising a fluorine-containing copolymer having a fluorine-containing olefin unit and a vinyl alcohol unit. The low cell adhesion material according to claim 11, wherein the fluorine-containing olefin unit content in the fluorine-containing copolymer is 60 to 10 mol%. The low cell adhesion material according to claim 11 or 12, wherein an alternating rate of the fluorine-containing olefin unit and the vinyl alcohol unit in the fluorine-containing copolymer is 1 to 75%. The low cell adhesion material according to claim 11, 12 or 13, wherein the fluorinated olefin is at least one selected from the group consisting of tetrafluoroethylene, chlorotrifluoroethylene and hexafluoropropylene. The low cell adhesion material according to claim 11, 12, 13, or 14, wherein the fluorine-containing copolymer has a fluorine-containing olefin unit, a vinyl alcohol unit, and a vinyl ester monomer unit. The low cell adhesion material according to claim 11, 12, 13, 14, or 15, which is a coating film. A coating composition comprising the low cell adhesion material according to claim 11, 12, 13, 14 or 15. A coating layer formed from the low cell adhesion material according to claim 11, 12, 13, 14 or 15. A bag, sheet, film, vial comprising the low cell adhesion material according to claim 11, 12, 13, 14, 15, or 16, the coating composition according to claim 17, or the coating layer according to claim 18. A low cell adhesion article characterized by being a bottle, a petri dish or a flask. A cell culture bag, a cell culture comprising the low cell adhesion material according to claim 11, 12, 13, 14, 15 or 16, the coating composition according to claim 17, or the coating layer according to claim 18. A low cell-adhesive article characterized by being a sheet for cell culture, a film for cell culture, a vial for cell culture, a petri dish for cell culture, or a flask for cell culture. A culture container for embryoid body formation comprising the low cell adhesion material according to claim 11, 12, 13, 14, 15 or 16, the coating composition according to claim 17, or the coating layer according to claim 18. A low cell-adhesive article characterized by