WO2016121994A1 - Cell culture apparatus - Google Patents

Abstract

The present invention addresses the problem of providing a cell culture apparatus wherein at least a portion of the surface is capable of sufficiently inhibiting the adhesion of anchorage-dependent cells. As a means of solving said problem, a cell culture apparatus comprising a support body (A) and a surface layer (B), which includes a fluorine-containing compound, is provided, wherein the surface layer (B) is disposed directly or with one or more other layers therebetween on at least a portion of the surface of the support body (A), and the water contact angle of the surface of the surface layer (B) at 37°C is 80° or greater.

Description

Cell culture equipment

The present invention relates to cell culture equipment and uses thereof.

Cell culture equipment having at least part of a surface on which anchorage-dependent cells are difficult to adhere is used. Such a cell culture device is used for, for example, suspension culture (three-dimensional culture) of anchorage-dependent cells or selective culture of anchorage-independent cells.

Conventionally, it has been proposed that a surface having a high contact angle in water with respect to water (approximately 90 ° or more) may be used as a surface on which anchorage-dependent cells are difficult to adhere (Non-Patent Document 1).

The inventors of the present invention have found that even a surface having a high contact angle in water with respect to water (approximately 90 ° or more) may not actually sufficiently suppress adhesion of anchorage-dependent cells. Therefore, an object of the present invention is to provide a cell culture device having at least a surface capable of sufficiently suppressing the adhesion of anchorage-dependent cells.

The inventors of the present invention have intensively studied to solve the above-mentioned problems, and unlike the conventionally proposed theory, if the surface has a high contact angle in water with respect to water, the anchorage-dependent cell adhesion can be sufficiently achieved. It was found that can be suppressed.

The present invention has been completed by further trial and error based on such findings, and includes the following embodiments.
Item 1.
(A) a support; and (B) a cell culture device containing a surface layer containing a fluorine-containing compound,
The surface layer (B) is disposed on at least a part of the surface of the support (A) directly or via one or more other layers,
The cell culture equipment whose water contact angle in 37 degreeC of the surface of the said surface layer (B) is 80 degrees or more.
Item 2.
Item 2. The cell culture device according to Item 1, wherein the surface layer (B) is a layer that has been surface-modified using a fluorine-containing compound.
Item 3.
Item 3. The cell culture device according to Item 2, wherein the surface layer (B) is a layer subjected to plasma treatment using a fluorine-containing gas.
Item 4.
Item 2. The cell culture device according to Item 1, wherein the surface layer (B) is a layer containing a fluorine-containing polymer.
Item 5.
The fluorine-containing polymer is at least one fluorine-containing polymer having a softening point of 30 ° C. or higher selected from the group consisting of a fluoro (meth) acrylate-based polymer, a fluoro (meth) acrylamide-based polymer, and an olefin-based fluorine-containing polymer. Item 5. The cell culture equipment according to Item 4.
Item 6.
Item 6. The cell culture device according to any one of Items 1, 4, and 5, wherein the surface layer (B) is bonded to a surface to be a base via a chemical bond.
Item 7.
Item 7. A composition capable of forming the surface layer (B), which is used for producing the cell culture device according to any one of Items 1 and 4 to 6.
Item 8.
Item 8. The composition according to Item 7, comprising a fluorine-containing polymer and / or a monomer capable of forming a fluorine-containing polymer.

The cell culture device of the present invention has an effect that adhesion of anchorage-dependent cells is sufficiently suppressed on at least a part of the surface thereof.

1. Cell culture equipment The cell culture equipment of the present invention is:
(A) a support; and (B) a cell culture device containing a surface layer containing a fluorine-containing compound,
The surface layer (B) is disposed on at least a part of the surface of the support (A) directly or via one or more other layers,
It is a cell culture equipment whose water contact angle in 37 degreeC of the surface of the said surface layer (B) is 80 degrees or more.

In the boundary between two layers in the present invention, the composition of both may change critically or may change in an inclined manner.

The cell culture device of the present invention contains a fluorine-containing compound and has a surface layer having an underwater contact angle of 80 ° or more at 37 ° C., so that anchorage-dependent cells are difficult to adhere. Using this characteristic, it can be used for various applications. Although it does not specifically limit, For example, the cell culture equipment for carrying out suspension culture (three-dimensional culture) of an anchorage dependent cell, the cell culture equipment for selectively culture | cultivating an anchorage independent cell, etc. are mentioned.

Although not particularly limited, the purpose of suspension culture (three-dimensional culture) includes, for example, ES cell embryoid body (EB) formation and spheroid formation, as well as production of useful substances and drug screening.

Although not particularly limited, examples of anchorage-independent cells include cancer cells and blood cells.

In addition to those listed above, these cell culture equipments are used for a wide range of purposes such as cell medicine production, tissue engineering, stem cell differentiation and cell culture experiments, protein preparations and / or cell preparation storage bags, etc. it can.

The cell culture device of the present invention does not include those exhibiting temperature responsiveness below 37 ° C., more specifically those having an underwater contact angle below 37 ° C. smaller than the underwater contact angle at 37 ° C. .

(A) Support The support can be appropriately selected depending on the purpose of use of the cell culture equipment, and is not particularly limited. The material of the support is selected from resin, glass, ceramic, metal and the like. Specific examples thereof include polyolefins such as polystyrene, polyethylene terephthalate, polyethylene and polypropylene, EVA (Ethylene-vinyl acetate), cycloolefin, polytetrafluoroethylene, and stainless steel.

The shape of the support is not particularly limited as long as it is usually used, and examples thereof include a dish, a plate, and a bag. The surface shape of the support may be smooth or uneven, and may have a smooth region and an uneven region. The well shape of the plate may be a flat bottom or a round bottom, and the well diameter is not particularly limited, and may be, for example, 34 mm (6 wells), 9 mm (96 wells), or the like. Alternatively, it may be a micro space plate partitioned into a micrometer order space, for example, a space having a width of 200 μm and a depth of 100 μm.

The support is preferable in that the surface layer (B) can be formed on the surface by surface graft polymerization if at least the surface thereof contains a material capable of forming a polymerization initiation point by radiation irradiation. In this case, the support may contain only a material capable of forming a polymerization starting point on the surface, or may contain such a material as a whole.

Examples of materials that can form the polymerization starting point include resins, and specifically, acrylics such as polystyrene, polyethylene terephthalate, low density polyethylene, medium density polyethylene, high density polyethylene, polyurethane, urethane acrylate, and polymethyl methacrylate. Examples thereof include resin, polyamide (nylon), polycarbonate, natural rubber having conjugated bond, synthetic rubber having conjugated bond, and silicone rubber. The material may be a blend polymer or polymer alloy containing two or more of these.

The support may be surface-treated as necessary as long as the effect of the present invention is not hindered, and may further have another layer on the surface.

(B) Surface layer The surface layer (B) contains a fluorine-containing compound, and the contact angle in water at 37 ° C is 80 ° or more. By having a surface having such characteristics, the cell culture device of the present invention can sufficiently suppress adhesion of anchorage-dependent cells. In this respect, the surface layer (B) preferably has an underwater contact angle at 37 ° C. of 80 ° or more, more preferably 90 ° or more. The underwater contact angle is usually 120 ° or less.

The surface layer (B) is disposed on at least a part of the surface of the support (A) directly or via one or more other layers. There is no particular limitation, and for example, the surface layer (B) may be disposed directly or via one or more other layers in an area of at least 50% of the surface of the support (A). It may be arranged directly or via one or more other layers in an area of at least 90% of the surface of the support (A).

In the present invention, the underwater contact angle is specifically measured as follows.

¡Fix the measurement sample in water and let it stand on a hot plate whose temperature is adjusted to 37 ° C for 30 minutes to achieve an equilibrium state. A few drops of bubbles (about 10 μL) were attached to the surface of the measurement sample using a syringe equipped with a three-phase reverse needle, and the contact angle of the bubbles was determined using DropMaster 701 (Kyowa Interface Science) or equivalent underwater contact angle. Measure.

The surface layer (B) may be a surface-modified layer using a fluorine-containing compound. In this case, the surface layer (B) is the surface portion of the support that is surface-modified, if the other layer is interposed between the support and the surface layer (B), the outermost layer thereof. Point to.

Examples of the fluorine-containing compound used in the surface modification include at least one fluorine-containing compound selected from the group consisting of fluorine gas, fluorine-containing gas, and fluorine-containing polymer. The fluorinated gas is not particularly limited, tetrafluoromethane _(CF 4), hexafluoroethane _{(C 2 F} 6), perfluoro ethylene _{(C 3 F} 6), hexafluoropropene oxide _(C 3 _OF 6), In addition, fluorine groups can be introduced into the surface of the polymer substrate by plasma treatment using perfluoropropane (C ₃ F ₈ ) or the like. The conditions for the plasma treatment can be set as appropriate according to the type of polymer substrate and gaseous fluorine-containing compound. Usually, so-called low-temperature plasma treatment is performed in the presence of a gaseous fluorine-containing compound.

The surface layer (B) may be a layer containing a fluorine-containing compound as an aspect different from the above-described surface-modified one. That is, in this case, the surface layer (B) is not a part of the modified lower layer but is originally a different layer from the lower layer.

Thus, although it does not specifically limit as a fluorine-containing compound used in the aspect which provides a surface layer (B) as a layer different from a lower layer originally, For example, an acrylic acid type compound, an acrylate type compound, an acrylamide type compound, an olefin And compounds in which at least one hydrogen atom is substituted with a fluorine atom in a benzene compound, a styrene compound, an acrylonitrile compound, a vinyl pyrrolidone compound, a vinyl ether compound, a pyrrole compound, and the like.

In the above, the acrylic acid-based compound is not particularly limited, and examples thereof include acrylic acid, methacrylic acid, itaconic acid, maleic anhydride, maleic acid, fumaric acid, and crotonic acid.

In the above, the acrylate compound is not particularly limited. For example, methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, n-propyl acrylate, n-propyl methacrylate, isopropyl acrylate, isopropyl methacrylate, lauryl acrylate, stearyl acrylate, stearyl Examples thereof include α, β-ethylenically unsaturated carboxylic acid esters such as methacrylate, behenyl acrylate, and behenyl methacrylate.

In the above, the acrylamide compound is not particularly limited, and examples thereof include octyl acrylamide and stearyl acrylamide.

As an acrylate compound or an acrylamide compound, for example, it has a fluoroalkyl group that is ester-bonded or amide-bonded directly or via a divalent organic group to the carboxyl group, and may have a substituent at the α-position, Examples thereof include acrylic acid esters (hereinafter sometimes abbreviated as “fluoroalkyl group-containing acrylic acid esters”) or acrylamides (hereinafter sometimes abbreviated as “fluoroalkyl group-containing acrylamides”).

Preferable specific examples of the fluoroalkyl group-containing acrylate ester or the fluoroalkyl group-containing acrylamide include the following general formula (1):
CH ₂ = C (-X) -C (= O) -YZ-Rf (1)
[Wherein, X represents a hydrogen atom, a linear or branched alkyl group having 1 to 21 carbon atoms, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a CFX ¹ X ² group (where X ¹ and X ² is a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom), a cyano group, a linear or branched fluoroalkyl group having 1 to 21 carbon atoms, a substituted or unsubstituted benzyl group or A substituted or unsubstituted phenyl group;
Y is —O— or —NH—;
Z is an aliphatic group having 1 to 10 carbon atoms, an aromatic group having 6 to 10 carbon atoms or a cyclic aliphatic group,
—CH ₂ CH ₂ N (R ¹ ) SO ₂ — group (where R ¹ is an alkyl group having 1 to 4 carbon atoms).
), —CH ₂ CH (OZ ¹ ) CH ₂ — group (wherein Z ¹ is a hydrogen atom or an acetyl group.
), — (CH ₂ ) _m —SO ₂ — (CH ₂ ) _n — group, — (CH ₂ ) _m —S— (CH ₂ ) _n — group (m is 1 to 10, and n is 0 to Or a — (CH ₂ ) _m —COO— group, where m is 1 to 10;
Rf is a linear or branched fluoroalkyl group having 1 to 20 carbon atoms which may have a hetero atom. An acrylate ester or acrylamide represented by the formula:

In the general formula (1), the fluoroalkyl group represented by Rf is an alkyl group which may have a hetero atom, in which at least one hydrogen atom is substituted with a fluorine atom, and all the hydrogen atoms are fluorine. A perfluoroalkyl group which may be substituted with an atom and may have a hetero atom is also included. Examples of the fluoroalkyl group having a heteroatom represented by Rf include a perfluoropolyether group.

In the acrylic ester or acrylamide represented by the general formula (1), Rf is preferably a linear or branched fluoroalkyl group having 1 to 6 carbon atoms, and particularly having 1 to 3 carbon atoms. It is preferably a linear or branched perfluoroalkyl group. In recent years, it has been pointed out by the EPA (US Environmental Protection Agency) that a compound having a fluoroalkyl group having 8 or more carbon atoms is a high environmental load that may decompose and accumulate in the environment and living organisms. However, in the acrylic ester or acrylamide represented by the general formula (1), when Rf is a linear or branched fluoroalkyl group having 1 to 6 carbon atoms, such environmental problems are pointed out. Because it is not.

In the above formula (1), examples of the Rf group include —CF ₃ , —CF ₂ CF ₃ , —CF ₂ CF ₂ H, —CF ₂ CF ₂ CF ₃ , —CF ₂ CFHCF ₃ , —CF (CF ₃ ). ₂ , -CF ₂ CF ₂ CF ₂ CF ₃ , -CF ₂ CF (CF ₃ ) ₂ , -C (CF ₃ ) ₃ ,-(CF ₂ ) ₄ CF ₃ ,-(CF ₂ ) ₂ CF (CF ₃ ) ₂ , -CF ₂ C (CF ₃ ) ₃ , -CF (CF ₃ ) CF ₂ CF ₂ CF ₃ ,-(CF ₂ ) ₅ CF ₃ ,-(CF ₂ ) ₃ CF (CF ₃ ) ₂ etc. .

Further, the fluorine-containing compound is preferably a non-telomer. In this respect, the Rf group may be a fluoroalkyl group having 1 to 2 carbon atoms, or two or more carbon atoms having 1 to 3 carbon atoms interposed by a hetero atom. A fluoroalkyl group is preferred. Specific examples include C ₃ F ₇ OCF (CF ₃ ) CF ₂ OCF (CF ₃ )-, (CF ₃ ) ₂ NC _n F _2n- (n = 1 to 6), and the like.

Specific examples of the acrylic acid ester or acrylamide represented by the general formula (1) are as follows.

CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ -Rf
CH ₂ = C (-H) -C (= O) -O-C ₆ H ₄ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₂ -Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ N (-CH ₃ ) SO ₂ -Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ N (-C ₂ H ₅ ) SO ₂ -Rf
CH ₂ = C (-H) -C (= O) -O-CH ₂ CH (-OH) CH ₂ -Rf
CH ₂ = C (-H) -C (= O) -O-CH ₂ CH (-OCOCH ₃ ) CH ₂ -Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-H) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-H) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O-C ₆ H ₄ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ N (-CH ₃ ) SO ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ N (-C ₂ H ₅ ) SO ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O-CH ₂ CH (-OH) CH ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O-CH ₂ CH (-OCOCH ₃ ) CH ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CH ₃ ) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CN) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S-Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -NH- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-F) -C (= O) -O- (CH ₂ ) ₃ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-F) -C (= O) -NH- (CH ₂ ) ₃ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-Cl) -C (= O) -O- (CH ₂ ) ₃ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-CF ₂ H) -C (= O) -O- (CH ₂ ) ₃ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-CN) -C (= O) -O- (CH ₂ ) ₃ -SO _2- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -S-Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -S- (CH ₂ ) ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₃ -SO ₂ -Rf
CH ₂ = C (-CF ₂ CF ₃ ) -C (= O) -O- (CH ₂ ) ₂ -SO _2- (CH ₂ ) ₂ -Rf
[In the above formula, Rf is a fluoroalkyl group having 1 to 6, preferably 1 to 3 carbon atoms. ]
C ₃ F ₇ OCF (CF ₃ ) CF ₂ O-CF (CF ₃ ) CH ₂ -MAc
C ₃ F ₇ OCF (CF ₃ ) CF ₂ O-CF (CF ₃ ) CH ₂ -Ac
(CF ₃ ) ₂ CH-Ac
C ₂ F ₅ CH ₂ -MAc
C ₂ F ₅ CH ₂ -Ac
[In the above formula, Ac represents acrylate, and MAc represents methacrylate, respectively. ]
The above-mentioned fluoroalkyl group-containing acrylic acid ester or acrylamide can be used alone or in combination of two or more.

The olefin compound is not particularly limited, and examples thereof include alkenes and alkyl vinyl ethers. The alkene is not particularly limited, and examples thereof include ethylene and propylene. Alkenes may have at least a part of hydrogen atoms substituted with fluorine atoms, and are not particularly limited. Specific examples include tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride. The alkyl vinyl ether is not particularly limited, and examples thereof include alkyl vinyl ethers having an alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. The alkyl vinyl ether may be a perfluoroalkyl vinyl ether. Specific examples of the perfluoroalkyl vinyl ether are not particularly limited, and examples thereof include perfluoropropyl vinyl ether.

The above olefinic compounds can be used alone or in combination of two or more.

In the above, the styrene compound is not particularly limited, and examples thereof include styrene and alkylstyrene. The above styrenic compounds can be used singly or in combination of two or more.

In the above, the acrylonitrile-based compound is not particularly limited, and examples thereof include acrylonitrile. The above acrylonitrile compounds can be used singly or in combination of two or more.

In the above, the vinyl pyrrolidone compound is not particularly limited, and examples thereof include vinyl pyrrolidone. The above-mentioned vinylpyrrolidone compounds can be used singly or in combination of two or more.

In the above, the vinyl ether compound is not particularly limited, and examples thereof include alkyl vinyl ethers such as ethyl vinyl ether and butyl vinyl ether. The above vinyl ether compounds can be used singly or in combination of two or more.

In the above, the pyrrole compound is not particularly limited, and examples thereof include pyrrole. The above pyrrole compounds can be used singly or in combination of two or more.

Examples of the fluorine-containing compound used in a mode in which the surface layer (B) is originally provided as a layer different from the lower layer include a fluorine-containing polymer. In this case, the surface layer (B) may further contain a fluorine-containing compound different from that in addition to the fluorine-containing polymer.

The fluorine-containing polymer is not particularly limited, but for example, at least one of acrylate compounds, acrylamide compounds, olefin compounds, styrene compounds, acrylonitrile compounds, vinyl pyrrolidone compounds, vinyl ether compounds and pyrrole compounds. And those having a structural unit based on one in which two hydrogen atoms are substituted with fluorine atoms. Moreover, the surface layer (B) may contain 1 type, or 2 or more types of fluorine-containing polymers. As the various compounds serving as the basis of these structural units, those described above as the fluorine-containing compounds that can be contained in the surface layer (B) can be used.

In the above, the fluorine-containing polymer is preferably a fluoro (meth) acrylate-based polymer, a fluoro (meth) acrylamide-based polymer, or an olefin-based fluorine-containing polymer. Furthermore, these polymers are more preferable if the softening point is 30 ° C. or higher.

The fluoro (meth) acrylate polymer, fluoro (meth) acrylamide polymer or olefinic fluorine-containing polymer used as the fluorine-containing polymer is more preferable as the softening point is higher in terms of the effect of suppressing the anchorage-dependent cell adhesion. Show the trend. In this respect, the numerical range of the softening point is preferably in the order of 40 ° C. or higher, 50 ° C. or higher, 60 ° C. or higher, and 70 ° C. or higher.

The fluoro (meth) acrylate polymer and the fluoro (meth) acrylamide polymer are polymers containing structural units based on at least fluoro (meth) acrylate and fluoro (meth) acrylamide, respectively. Although not particularly bound by theory, the polymer having a softening point of 30 ° C. or higher is easy to maintain hydrophobicity because reorientation of Rf group (hydrophobic) and carbonyl group (hydrophilicity) hardly occurs in water at 37 ° C. it is conceivable that. In this case, preferred specific examples of the fluoro (meth) acrylate polymer and the fluoro (meth) acrylamide polymer are configurations based on the fluoroalkyl group-containing (meth) acrylic acid ester and / or the fluoroalkyl group-containing (meth) acrylamide. What has a unit etc. are mentioned.

For example, even if it has a structural unit based on the fluoroalkyl group-containing (meth) acrylic acid ester and / or fluoroalkyl group-containing (meth) acrylamide, the one in which Rf is a perfluoropolyether group has a softening point. It becomes less than 30 ° C.

The olefinic fluorine-containing polymer means a fluorine-containing polymer which may contain a structural unit based on a polymerized olefin monomer and a structural unit based on at least one other comonomer. In the above, the olefinic fluorine-containing polymer preferably contains a structural unit based on a polymerized olefin monomer corresponding to a majority quantity based on the weight of the polymer.

In the above, the fluorine atom may be contained only in the structural unit based on the polymerized olefin monomer, may be contained only in the structural unit based on the other comonomer, or may be contained in both of them. Good. At least one of the structural units may be one in which all of the hydrogen atoms are substituted with fluorine atoms, or both of the structural units may be one in which all of the hydrogen atoms are substituted with fluorine atoms. . In the above, in other structural units, a part of hydrogen atoms may be substituted with fluorine atoms, or the hydrogen atoms may not be substituted at all with fluorine atoms.

In the above, the polymerized olefin monomer is not particularly limited, and examples thereof include alkenes and alkyl vinyl ethers. The alkene is not particularly limited, and examples thereof include ethylene and propylene. Alkenes may have at least a part of hydrogen atoms substituted with fluorine atoms, and are not particularly limited. Specific examples include tetrafluoroethylene, hexafluoropropylene, and vinylidene fluoride. The alkyl vinyl ether is not particularly limited, and examples thereof include alkyl vinyl ethers having an alkyl group having 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms. The alkyl vinyl ether may be a perfluoroalkyl vinyl ether. Specific examples of the perfluoroalkyl vinyl ether are not particularly limited, and examples thereof include perfluoropropyl vinyl ether.

Although not particularly limited, specific examples of the olefinic fluoropolymer include, for example, polytetrafluoroethylene (PTFE), tetrafluoroethylene-hexafluoropropylene copolymer (FEP), and tetrafluoroethylene-perfluoroalkyl vinyl ether copolymer. (PFA), tetrafluoroethylene-ethylene copolymer (ETFE), polyvinylidene fluoride (PVDF), and vinylidene fluoride-tetrafluoroethylene copolymer. These may be used alone or in combination.

The surface layer (B) may further contain a non-fluorine-containing compound. It does not specifically limit, An acrylamide type compound, an olefin type compound, a styrene type compound, etc. are mentioned. Among these, a single species may be used, or a plurality of species may be used in combination.

The acrylamide compound is not particularly limited, and examples thereof include acrylamide and methylol methacrylamide. These may be used alone or in combination.

In the above, the styrene compound is not particularly limited, and examples thereof include styrene and alkylstyrene. These may be used alone or in combination.

The surface layer (B) may contain a silicone compound and is not particularly limited, and examples thereof include a polymer of dimethylpolysiloxane and a silicone macromonomer.

The surface layer (B) may have a functional group capable of chemically bonding such as an epoxy group, an isocyanate group, a succinimide group, an amino group, a carboxyl group, or a hydroxyl group. The chemical bonds of the present invention refer to covalent bonds, ionic bonds, metal bonds, hydrogen bonds, and intermolecular forces.

In the present invention, the softening point of a polymer means a melting point in the case of a crystalline polymer and a glass transition temperature in the case of an amorphous polymer. These are measured as follows. The polymer softening point is the melting point (Tm) or glass transition point (Tg) measured by differential scanning calorimetry. From the thermogram of the 2nd cycle when 10 mg of polymer powder was measured with a differential scanning calorimeter (DSC) under the conditions of a temperature range of −50 to 150 ° C. and a heating rate of 10 ° C./min. The melting point is read from the top of the melting peak, and in the case of an amorphous polymer, the extrapolated glass transition end temperature (JIS K7121-1987) is read.

The surface layer (B) may be disposed on the underlying surface by physical adsorption or may be bonded via a chemical bond. The surface layer (B) is preferably bonded to the underlying surface via a chemical bond in that the effect of suppressing cell adhesion can be maintained over a long period of time.

Although not particularly limited, for example, by performing electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, etc., the surface layer (B) is fixed to the underlying surface via chemical bonding. It may be.

The thickness of the surface layer (B) is 0.2 μm or more, preferably 1 μm or more, more preferably 5 μm or more. If thickness is 0.2 micrometer or more, it will become easy to exhibit sufficient cell adhesion suppression ability.

(C) Other layers The other layers are not particularly limited, and examples thereof include a support reinforcing layer, a primer layer, and a radiation modification auxiliary layer.

The support reinforcing layer is a layer that mechanically or chemically reinforces the support. Specifically, it is used when the mechanical strength of the support is low, or when chemical corrosion occurs on the support due to the influence of the solvent and / or monomer used in forming the surface layer. For example, the mechanical strength can be improved by laminating a polyethylene terephthalate film having a thickness of 250 μm on a polyethylene film having a thickness of 10 μm. Moreover, the chemical corrosion with respect to a solvent and / or a monomer can be prevented by sticking a FEP film on a polystyrene base material.

The primer layer is a layer for improving the adhesion between the support (A) and the surface layer (B), and is not particularly limited. For example, both surfaces for attaching an FEP film on the above-mentioned polystyrene base material. Tape etc. are mentioned.

The radiation modification auxiliary layer is used when the support (A) is made of a material that hardly generates radicals when irradiated with radiation, such as polystyrene. For example, by attaching a polyethylene film as a radiation modification auxiliary layer on the surface of polystyrene equipment, a large amount of radicals are generated when irradiated with radiation, and graft polymerization can be easily performed.

2. Although the manufacturing method of a cell culture equipment is not specifically limited, the cell culture equipment of this invention is obtained by applying the surface treatment agent which can make the water contact angle in 37 degreeC of the process surface 80 degree or more to the surface used as a foundation | substrate. be able to.

Hereinafter, a specific method will be described by taking as an example the case of using a surface treatment agent containing a fluorine-containing compound. In addition, also when using the surface treating agent containing a compound different from a fluorine-containing compound, it can carry out similarly by referring to the following examples and changing conditions suitably as needed.

The surface treatment agent can be applied to the underlying surface by a coating method such as a solvent casting method, a dipping method, a spray method, a spin coating method, a bar coating method, or a brush coating method. For example, in the solvent casting method, a solution in which components such as the above-mentioned fluorine-containing compounds are dissolved in a solvent is uniformly applied to the base surface, and then the solvent is evaporated to form a film on the base surface. Let The solvent used to make the above-mentioned fluorine-containing compound or the like into a solution by various coating methods is not particularly limited, but, for example, those having a boiling point of 120 ° C. or less, particularly 50 to 110 ° C. under normal pressure are preferable. As specific examples, any fluorine-containing compound, alcohol, ether, etc. may be used as long as the solvent has a fluorine atom in the molecule and the solubility of the fluorine-containing polymer is good. Any of the family may be used. For example, perfluoroaliphatic hydrocarbon, polyfluoroaromatic hydrocarbon, polyfluoroaliphatic hydrocarbon, hydrofluorocarbon (HFC), hydrochlorofluorocarbon (HCFC), hydrofluoroether (HFE), hydrofluoroolefin (HFO) and Examples thereof include alkyl perfluoroalkyl ethers.

The perfluoroaliphatic hydrocarbon is not particularly limited, but preferably has 5 to 12 carbon atoms. Specific examples include perfluorohexane, perfluoromethylcyclohexane, perfluoro-1,3-dimethylcyclohexane, perfluorodihydropropanol (pentafluoropropanol), and the like.

The polyfluoroaromatic hydrocarbon is not particularly limited, and examples thereof include bis (trifluoromethyl) benzene and hexafluoro-m-xylene.

The polyfluoroaliphatic hydrocarbon is not particularly limited. For example, C ₆ F ₁₃ CH ₂ CH ₃ [for example, Asahi Clin (registered trademark) AC-6000 manufactured by Asahi Glass Co., Ltd.], 1, 1, 2, 2 , 3,3,4-heptafluorocyclopentane [for example, ZEOLOR (registered trademark) H manufactured by Nippon Zeon Co., Ltd.].

Hydrofluorocarbon (HFC) is not particularly limited, and examples thereof include 1,1,1,3,3-pentafluorobutane (HFC-365mfc).

Hydrochlorofluorocarbon (HCFC) is not particularly limited, but preferably has 2 to 5 carbon atoms. Specific examples include, but are not limited to, HCFC-225 [dichloropentafluoropropane: Asahiklin (registered trademark) AK225], HCFC141b (dichlorofluoroethane), CFC316 (2,2,3,3-tetrachlorohexafluorobutane) )) And C ₅ H ₂ F ₁₀ (for example, Vertrel (registered trademark) XF manufactured by DuPont).

The hydrofluoroether (HFE), is not particularly limited, for example, perfluoropropyl methyl ether _{(C 3 F 7 OCH 3)} [ for example, Sumitomo 3M Limited of Novec (TM) 7000] and _CF 3 CH ₂ OCF ₂ CHF ₂ [for example, Asahi Clin (registered trademark) AE-3000 manufactured by Asahi Glass Co., Ltd.] and the like.

The hydrofluoroolefin (HFO) is not particularly limited. For example, 1,2-dichloro-1,3,3,3-tetrafluoro-1-propene [for example, Vertrel (registered by Mitsui DuPont Fluorochemical) (registered) Trademark) Scion] and the like.

In the alkyl perfluoroalkyl ether, the perfluoroalkyl group and the alkyl group may be either linear or branched. Specific examples include, but are not limited to, perfluorobutyl methyl ether (C ₄ F ₉ OCH ₃ ) [for example, Novec (trade name) 7100 manufactured by Sumitomo 3M Limited], perfluorobutyl ethyl ether (C ₄ F _9). OC ₂ H ₅ ) [for example, Novec (trade name) 7200 manufactured by Sumitomo 3M Limited] and perfluorohexyl methyl ether (C ₂ F ₅ CF (OCH ₃ ) C ₃ F ₇ ) [for example, manufactured by Sumitomo 3M Limited. Novec (trade name) 7300] and the like.

Other solvents used to make the above-mentioned fluorine-containing compounds and the like into solutions include alcohol solvents such as methanol, ethanol and isopropyl alcohol, ketone solvents such as acetone and methyl isobutyl ketone, ethyl acetate and butyl acetate. An ester solvent such as tetrahydrofuran and an ether solvent such as tetrahydrofuran can be used.

As the solvent for dissolving the fluorine-containing compound, only one kind of solvent may be used, or two or more kinds of solvents may be mixed and used. If necessary, an additive may be added to the solvent.

When the fluorine-containing compound used for the surface layer (B) has a functional group capable of chemically bonding such as an epoxy group, an isocyanate group, a succinimide group, an amino group, a carboxyl group, or a hydroxyl group, In order to chemically react with a carboxyl group or a hydroxyl group (previously introduced by a technique such as plasma treatment or corona treatment), a solution of a fluorine-containing compound or the like is applied to the surface of the container and chemically bonded. A cell culture vessel can also be obtained by a method of removing unreacted polymer after evaporation to form a coating layer.

In the present invention, the fluorine compound or the like may be fixed to the base surface by chemical bonding also by the following method.
(1) a step of dissolving components such as the above-mentioned fluorine-containing compound in a solvent; and (2) a step of applying the solution obtained in (1) above to the underlying surface and further polymerizing them as necessary. The equipment of the present invention can be obtained by the method of including.

In step (1), the solvent for dissolving the fluorine-containing compound is the same as in various coating methods.

In the polymerization step (2), the surface of the base may be coated with the fluorine-containing compound dissolved in the solvent and then polymerized to form a polymer on the surface. After the formation, the base surface may be covered with the obtained polymer.

The polymerization method is not particularly limited, but may be, for example, radical polymerization, and more specifically, electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, and organic polymerization reaction. Etc.

The method of coating the surface of the equipment with a polymer formed in advance is not particularly limited, and may be performed by simple physical adsorption such as coating and kneading, and further includes electron beam irradiation (EB), γ-ray irradiation, By performing ultraviolet irradiation, plasma treatment, corona treatment, or the like, the polymer may be fixed to the surface of the base via a chemical bond.

3. Surface Treatment Agent The surface treatment agent of the present invention is a composition capable of forming the surface layer (B) used for producing the cell culture device of the present invention.

The surface treatment agent of the present invention is a surface treatment agent that can make the contact angle of the treated surface at 37 ° C. in water of 80 ° or more. The surface treatment agent of the present invention is a surface treatment agent capable of setting the contact angle of the treated surface at 37 ° C. in water to preferably 80 ° or more, more preferably 90 ° or more.

The surface treatment agent of the present invention is a surface treatment agent applied to a surface having a contact angle in water at 37 ° C. of preferably 50 ° or less, more preferably 40 ° or less.

Although it does not specifically limit as an application object, For example, the cell culture equipment for carrying out suspension culture (three-dimensional culture | cultivation) of an anchorage-dependent cell, the cell culture equipment for selectively culture | cultivating an anchorage-independent cell, etc. Can be mentioned.

Although not particularly limited, the purpose of suspension culture (three-dimensional culture) includes, for example, ES cell embryoid body (EB) formation and spheroid formation, as well as production of useful substances and drug screening.

Although not particularly limited, examples of anchorage-independent cells include cancer cells and blood cells.

In addition to those listed above, these cell culture equipments can be used for a wide range of purposes, such as cell medicine production, tissue engineering, stem cell differentiation and cell culture experiments, and protein preparations and / or cell preparation storage bags. Can be used.

The surface treatment agent of the present invention is not particularly limited as an active ingredient, but includes, for example, the above-mentioned fluorine-containing compound.

The surface treatment agent of the present invention contains a solvent that dissolves the active ingredient. For example, when the fluorine-containing compound is contained as an active ingredient, the solvent for dissolving it is not particularly limited, but those described in the above “2. Method for producing cell culture equipment” may be used.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

1. Preparation Method of (Meth) acrylate Polymer by Solution Polymerization (Meth) acrylate polymer shown in Table 1 was prepared.

As the polymerization solvent, HCFC-225 was used in the case of fluorine-containing (meth) acrylate, and isooctane was used in the case of non-fluorinated (meth) acrylate. The monomer concentration was 20% by weight, and 1 mol% (based on monomer) of azobisisobutylnitrile was used as a polymerization initiator, and polymerization was carried out at 50 ° C. for 12 hours. The polymer was purified by dropping the obtained polymer solution into a poor solvent and precipitating, isolating and drying.

2. Surface treatment method of cell culture dish Surface treatment was performed using the polymer obtained in (1).

2.1 As a cast film solvent, 0.5 mL of a 1 wt% solution prepared using HCFC-225 in the case of a fluorine-containing (meth) acrylate polymer and isooctane in the case of a non-fluorine-containing (meth) acrylate polymer, A film was formed on a Petri dish (diameter: 3.5 cm) [Falcon 1008 manufactured by Corning International, Inc. (formerly Becton Dickinson Labware), USA] by a solvent casting method.

2.2 Radiation-modified film (1) Graft polymerization of monomer A suitable amount of a 10 wt% monomer solution substituted with nitrogen was injected into a Petri dish (diameter 3.5 cm), and then packed in a pack in a nitrogen atmosphere. Next, graft polymerization was performed on the surface of the PS device by irradiating an electron beam or gamma rays at a dose of 100 kGy. A radiation-modified film was prepared by washing with a good monomer solvent and drying.

(2) Crosslinking of cast film The cast film was irradiated with an electron beam or gamma ray to crosslink the polymer to PS equipment, and then washed with a good monomer solvent and dried to prepare a radiation modified film.

2.3 Fluororesin film A flat-bottomed 6-well plate (# 92006) (diameter: 3.4 cm) made by TPP, a double-sided tape was applied to the well, and an FEP film (film thickness of 50 μm) was attached thereon.

2.4 A double-sided tape was applied in the same well as the polyethylene (PE) film 2.3, and a PE film (film thickness 100 μm) was stuck thereon.

2.5 A double-sided tape was affixed in the same well as the PE film 2.3 plasma treated with CF4 gas, and a PE film (film thickness 100 μm) vacuum-treated with CF4 gas was produced thereon.

3. Measurement of polymer softening point The polymer softening point was the melting point (Tm) or glass transition point (Tg) measured by differential scanning calorimetry. In the case of a crystalline polymer, a 10 mg polymer powder was measured with DSC822e (manufactured by Mettler Toledo, USA) under the conditions of a temperature range of −50 to 150 ° C. and a heating rate of 10 ° C./min. Read the melting point from the top of the melting peak and, in the case of an amorphous polymer, the extrapolated glass transition end temperature (JIS K7121-1987).

4). Contact angle (in air, underwater)
For the contact angle, the bottom surface of the cell culture dish was cut out, measured with a Drop Master 701 manufactured by Kyowa Interface Science Co., Ltd., and an average value of five measurements was adopted.

4.1 In air at a contact angle of 25 ° C., 2 μl of water or n-hexadecane (hereinafter abbreviated as HD) was dropped, and the contact angle one second after the dropping was measured.

4.2 An underwater contact angle glass cell (60 × 60 × 40 mm, thickness 2 mm) was filled with water, and the bottom of the cut cell culture dish was fixed so that it was completely immersed in water with the treated surface facing downward. Next, after heating at 37 ° C. for 30 minutes, 10 μl of bubbles were attached to the treated surface fixed in water using a microsyringe, and the contact angle of the bubbles was measured. The underwater contact angle was calculated from “180- (bubble contact angle)”.

5). Cell adhesion test A cell adhesion test was carried out using C3H / 10T1 / 2clone 8 (CL8) cells (cultured cells like mesenchymal stem cells derived from mouse embryos). Prior to this, a 6-well plate (TPP) was prepared as a sample to be subjected to the cell adhesion test, in which each well bottom surface was treated with various polymers.

(1) Culture conditions Medium: DMEM (Dulbecco's modified Eagle's medium) -high glucose (Nacalai Tesque) 2 ml containing 10% FBS (fetal bovine serum)
(2) Cell preparation method for evaluation Once the cells are cultured in a cell culture dish (Falcon 3001), the cells are detached and collected, the number of cells is counted, and the medium is adjusted to 1 × 10 ⁶ cells / ml. A cell suspension was prepared using

100 μl of the cell suspension was seeded in a 6-well plate in which 2 ml of medium was filled in each well in advance.

For the observation of cells, a microscope “Nikon TE300” (magnification 40 to 100 culture) and camera “Leica MC120HD” were used.

(3) Judgment criteria After culturing for 3 days, the sample was observed under a microscope and judged according to the following criteria.

A: Completely non-adhering B: Extension of 1 or more and less than 5 cells per field is observed C: Extension of 5 or more and less than 10 cells per field is observed D: 10 per field As described above, the extension of less than 20 cells is observed. E: The extension of 20 or more cells per field of view is observed. X: Results equivalent to the petri dish for culture (6-well plate manufactured by TPP) The results are shown in Table 2. Show. It was found that the surface where the contact angle in water at 37 ° C. is 80 ° or more is difficult for cells to adhere. Furthermore, it was found that when the contact angle in water at 37 ° C. is 80 ° or more and the softening point is 30 ° C. or more, the cells are more difficult to adhere.

 

Claims (8)

1. (A) a support; and (B) a cell culture device containing a surface layer containing a fluorine-containing compound,
   The surface layer (B) is disposed on at least a part of the surface of the support (A) directly or via one or more other layers,
   The cell culture equipment whose water contact angle in 37 degreeC of the surface of the said surface layer (B) is 80 degrees or more.
2. The cell culture device according to claim 1, wherein the surface layer (B) is a layer whose surface has been modified using a fluorine-containing compound.
3. The cell culture device according to claim 2, wherein the surface layer (B) is a layer subjected to plasma treatment using a fluorine-containing gas.
4. The cell culture device according to claim 1, wherein the surface layer (B) is a layer containing a fluorine-containing polymer.
5. The fluorine-containing polymer is at least one fluorine-containing polymer having a softening point of 30 ° C. or higher selected from the group consisting of a fluoro (meth) acrylate-based polymer, a fluoro (meth) acrylamide-based polymer, and an olefin-based fluorine-containing polymer. The cell culture equipment according to claim 4.
6. The cell culture device according to any one of claims 1, 4, and 5, wherein the surface layer (B) is bonded to a base surface via a chemical bond.
7. A composition capable of forming the surface layer (B), which is used for producing the cell culture device according to any one of claims 1 and 4 to 6.
8. The composition of Claim 7 containing the monomer which can form a fluoropolymer and / or a fluoropolymer.