Cell culture member and method for modifying surface thereof

Abstract

To provide cell culture members with an excellent cell culture performance and long-term stability thereof, and to provide methods for modifying a surface thereof.SOLUTION: In the cell culture member according to the present invention, at least a holding region of adhesive cells is consisting of a polymer compound. At least a part of the holding region is a surface modification region in which a fluorine atom is directly chemically bonded to a part of carbon atoms and / or silicon atoms constituting the polymer compound. The cell culture member improves adhesiveness of the adhesive cells with respect to the surface modification region, suppresses deterioration of adhesiveness over time, and has excellent cell culture performance and long-term stability.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture component and a method for modifying its surface, and more particularly to a cell culture component and a method for modifying its surface that have excellent cell culture performance and long-term stability. [Background technology]

[0002] Cell culture technology is used in research in various fields, including elucidating biochemical phenomena, producing useful substances, regenerative medicine, and drug evaluation. Regarding cell culture vessels, due to increasing awareness of infection and contamination, as well as considerations of moldability and manufacturing cost, it is common to use polymer compounds made of thermoplastic resins as the material. The shape and physical properties of cell culture vessels significantly affect the efficiency of research and analytical accuracy in various applications.

[0003] In particular, in research using adherent cells (all mammalian cells except for certain cells such as cancer cells and hematopoietic cells), the adhesion between the adherent cells and the cell culture vessel is important for cell proliferation efficiency and maintenance of physiological activity. However, in most cell culture vessels using the aforementioned thermoplastic resin, the surface is hydrophobic, resulting in poor adhesion of adherent cells and a significant inhibition of their cellular activity.

[0004] To address such problems, for example, Patent Document 1 describes surface modification of a polypropylene substrate by plasma treatment in order to deposit or immobilize cells or biomolecules on the polypropylene substrate. According to Patent Document 1, surface modification of the polypropylene substrate is performed by plasma treatment, thereby optimizing the interaction between the surface of the polypropylene substrate and cells or biomolecules.

[0005] However, when performing surface modification by discharge treatment typified by plasma treatment, it is preferable that the surface of the object to be treated is a smooth surface without irregularities. Therefore, there is a problem that it is difficult to apply to cell culture containers having a complex shape, such as those used in the cultivation of three-dimensional cultures, which have been increasing in demand in recent years. In addition, the surface subjected to the discharge treatment exhibits sufficient cell culture performance immediately after the discharge treatment, but the cell culture performance deteriorates over time, so there is a problem that its long-term stability is inferior.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention has been made in view of the above problems, and an object thereof is to provide a cell culture member having excellent cell culture performance and its long-term stability, and a method for surface modification thereof.

Means for Solving the Problems

[0008] The cell culture member according to the present invention is a cell culture member in which at least a holding region for adherent cells is made of a polymer compound, and at least a part of the holding region is a surface modification region in which a fluorine atom is directly chemically bonded to a part of a carbon atom and / or a silicon atom constituting the polymer compound.

[0009] In the above configuration, it is preferable that the peak value of the binding energy measured by X-ray photoelectron spectroscopy of the fluorine atom is in the range of 680 eV to 690 eV.

[0010] In the above configuration, a surface modification group is directly chemically bonded to some of the other carbon atoms and / or other silicon atoms constituting the polymer compound in the surface modification region, and the surface modification group is -OR 1 base;-COOR 2 base;-COR 3 A group; hydrocarbon group; silyl group; hydrocarbon group having at least one heteroatom, halogen atom and unsaturated bond; silyl group having at least one heteroatom, halogen atom and unsaturated bond; cyano group; nitro group; nitroso group; phosphate group; sulfonyl group; thiol group; thionyl group; and at least one selected from the group consisting of halogen atoms excluding fluorine atoms, and the R 1 and R 2 Each is independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a heteroatom, a halogen atom, and an unsaturated bond; or a silyl group having at least one of a heteroatom, a halogen atom, and an unsaturated bond, and the R 3 Preferably, this is a hydrocarbon group; a hydrocarbon group having at least one heteroatom and / or an unsaturated bond; or a silyl group having at least one heteroatom and / or an unsaturated bond.

[0011] In the above configuration, it is preferable that the polymer compound is at least one polymer selected from the group consisting of polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polyurethane, cyclic polyolefin, polyetheretherketone, polyimide, polyamideimide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile butadiene styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and silicon-containing polymer compounds.

[0012] The surface modification method of the cell culture member of the present invention is a method for modifying the surface of a cell culture member in which a holding region for adherent cells is made of a polymer compound, and at least a part of the holding region is brought into contact with a first treatment gas containing a gas containing a fluorine atom and an inert gas as an optional component, thereby forming a surface modification region in which the fluorine atom is directly chemically bonded to a part of the carbon atom and / or silicon atom constituting the polymer compound.

[0013] In the above configuration, it is preferable that the peak value of the binding energy measured by X-ray photoelectron spectroscopy of the fluorine atom in the surface modification region is in the range of 680 eV to 690 eV.

[0014] In the above configuration, in the step, by using a first treatment gas further containing a gas containing an oxygen atom, a surface modification group is directly chemically bonded to a part of the other carbon atom and / or the other silicon atom constituting the polymer compound, and the surface modification group is an -OR 1 group; -COOR 2 group; -COR 3 group; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a hetero atom, a halogen atom, and an unsaturated bond; a silyl group having at least one of a hetero atom, a halogen atom, and an unsaturated bond; a nitro group; a nitroso group; a phosphate group; a sulfonyl group; a thionyl group; and at least one selected from the group consisting of halogen atoms other than a fluorine atom, and the R 1 and the R 2 each independently is a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a hetero atom, a halogen atom, and an unsaturated bond; or a silyl group having at least one of a hetero atom, a halogen atom, and an unsaturated bond, and the R 3 may be any of a hydrocarbon group; a hydrocarbon group having at least one of a hetero atom and an unsaturated bond; or a silyl group having at least one of a hetero atom and an unsaturated bond.

[0015] In the above configuration, the process further includes contacting a second processing gas containing other oxygen atoms with at least a portion of the holding region that has been contacted with the first processing gas, thereby directly chemically bonding surface modification groups to some of the other carbon atoms and / or other silicon atoms constituting the polymer compound, and R 1 and R 2 Each is independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a heteroatom, a halogen atom, and an unsaturated bond; or a silyl group having at least one of a heteroatom, a halogen atom, and an unsaturated bond, and the R 3 This may be a hydrocarbon group; a hydrocarbon group having at least one heteroatom and / or an unsaturated bond; or a silyl group having at least one heteroatom and / or an unsaturated bond.

[0016] In the above configuration, it is preferable to further include a step of replacing the fluorine atom and / or surface modifying group with the surface modifying group derived from the treatment compound by contacting the fluorine atom and / or surface modifying group with the surface modifying group derived from the treatment compound with the fluorine atom and / or surface modifying group which is directly chemically bonded to a portion of the carbon atom and / or silicon atom, and / or the surface modifying group which is directly chemically bonded to a portion of the other carbon atom and / or other silicon atom.

[0017] In the above configuration, it is preferable that the polymer compound is at least one polymer selected from the group consisting of polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polyurethane, cyclic polyolefin, polyetheretherketone, polyimide, polyamideimide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile butadiene styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and silicon-containing polymer compounds. [Effects of the Invention]

[0018] According to the cell culture material of the present invention, in the cell-holding region of the adhesive cell composed of a polymer compound, a surface-modified region is provided by directly chemically bonding fluorine atoms to some of the carbon atoms and / or silicon atoms constituting the polymer compound. This improves the adhesion of adhesive cells to the surface-modified region compared to regions without such surface modification. As a result, a cell culture material with excellent cell culture performance can be provided. Furthermore, since the fluorine atoms are directly chemically bonded and immobilized to some of the carbon atoms constituting the polymer compound, the cell culture material of the present invention can suppress the deterioration of adhesion of adhesive cells over time compared to conventionally surface-modified materials such as those treated with plasma. This makes it possible to provide a cell culture material with excellent long-term stability of cell culture performance.

[0019] Furthermore, according to the surface modification method for cell culture members of the present invention, by simply contacting at least a portion of the holding region of the cell culture member with a first treatment gas containing at least a gas containing fluorine atoms, it is possible to directly chemically bond fluorine atoms to some of the carbon atoms in the polymer compound constituting the holding region, thereby forming a surface-modified region. As a result, a cell culture member with excellent cell culture performance and long-term stability can be manufactured by an extremely simple method. [Brief explanation of the drawing]

[0020] [Figure 1] This is a conceptual diagram illustrating the surface modification region in a cell culture member according to an embodiment of the present invention. [Figure 2] This is a conceptual diagram illustrating the surface modification method for cell culture members according to the above embodiment. [Figure 3] This graph shows the XPS measurement results of the microplate according to Example 1. [Figure 4] This figure shows a bright-field image obtained by inverted microscopy after culturing mouse astrocyte cells using the microplate from Example 1. [Figure 5]This figure shows a bright-field image obtained by inverted microscopy after culturing mouse astrocyte cells using the microplate of Comparative Example 1. [Figure 6] This figure shows a bright-field image obtained by inverted microscopy after culturing mouse astrocyte cells using the microplate of Comparative Example 2. [Modes for carrying out the invention]

[0021] (Cell culture parts) First, the cell culture member of this embodiment will be described based on Figure 1. Figure 1 is a conceptual diagram illustrating the surface modification region in the cell culture member 10 according to this embodiment.

[0022] The cell culture member 10 of this embodiment includes at least a holding region (cell culture surface) capable of holding adherent cells. The cell culture member 10 of this embodiment allows adherent cells to adhere to this holding region, enabling the culture of adherent cells in said holding region.

[0023] In this specification, "cell culture component" means a component having at least a solid surface that can serve as a scaffold for adherent cells in order for them to proliferate, differentiate, and survive. Therefore, the cell culture component 10 can be, for example, a film (membrane), a sheet, a microplate, a flask, a dish, a tube, a hollow fiber membrane, or a substantially spherical shape. In the case where the cell culture component 10 is a film (membrane) or a sheet, this includes, for example, when it is a component of a finished product such as a cell culture vessel.

[0024] Furthermore, in this specification, "adherent cells" means cells that require a scaffold on a solid surface for proliferation, differentiation, and survival, and that adhere to a solid surface. Examples of adherent cells include epithelial cells such as human embryonic kidney cells (HEK293T cells), Syrian hamster kidney cells (BHK-21(C-13) cells), and mouse cerebral cortical neurons, as well as tumor cells, endothelial cells, fibroblasts, muscle cells, nerve / endocrine gland cells, primary cells, and glial cells (glia) such as mouse astrocytes. However, adherent cells are not limited to these exemplified cells.

[0025] The cell culture member 10 only needs to have a holding region (cell culture surface) made of a polymer compound. Furthermore, the holding region may be a surface-modified surface made of a polymer compound and further modified by a known surface treatment such as plasma treatment.

[0026] The polymer compound is at least one polymer selected from the group consisting of polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polyurethane, cyclic polyolefin, polyether ether ketone, polyimide, polyamide imide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile-butadiene-styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and silicon-containing polymer compounds. The polymer includes not only thermoplastic resins but also elastomers. Of these polymer compounds, polystyrene, polyethylene terephthalate, and polypropylene are preferred from the viewpoint of moldability and manufacturing cost.

[0027] At least a portion of the retention region is a surface-modified region in which fluorine atoms are directly chemically bonded to some of the carbon atoms and / or silicon atoms (hereinafter referred to as "carbon atoms, etc.") that constitute the polymer compound (see Figure 1). As a result, in the surface-modified region where fluorine atoms are directly chemically bonded, the adhesion of adherent cells is increased compared to the region where the fluorine atoms are not directly chemically bonded, thereby improving cell culture performance. Furthermore, since the fluorine atoms are directly chemically bonded to some of the carbon atoms, etc. of the polymer compound that forms the retention region, for example, the surface-modified region can suppress the deterioration of adherent cell adhesion over time compared to the region that has undergone other surface treatments such as plasma treatment. As a result, the long-term stability of cell culture performance can also be improved.

[0028] In this invention, the surface modification region only needs to be formed in at least a part of the retaining region. Therefore, in this invention, the entire retaining region may be the surface modification region.

[0029] The peak binding energy of fluorine atoms in the surface-modified region, measured by X-ray photoelectron spectroscopy (XPS), is preferably in the range of 680 eV to 690 eV, more preferably in the range of 682 eV to 690 eV, and particularly preferably in the range of 683 eV to 690 eV. By setting the peak binding energy to 680 eV or higher, the surface-modified region becomes hydrophilic, increasing adhesion performance. By setting the peak binding energy to 690 eV or lower, excessive hydrophobicity due to surface modification is prevented, and appropriate adhesion between the surface-modified region and adhesive cells can be maintained. Furthermore, the peak value of the fluorine atom's binding energy can be measured, for example, using an X-ray photoelectron spectrometer (model: PHI VersaProbeIII, manufactured by ULVAC-PHI, Inc.), with monochromatized AlKα rays used as the irradiated X-rays, an X-ray output of 50W, an acceleration voltage of 15kV, a measurement area of ​​approximately 200μm in diameter, and a binding energy measurement interval of 0.05eV.

[0030] Some of the other carbon atoms and / or other silicon atoms (hereinafter referred to as "other carbon atoms, etc.") constituting the polymer compound in the surface modification region may also have surface modification groups directly chemically bonded to them.

[0031] The surface modification group is -OR 1 base;-COOR 2 base;-COR 3 A group; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one heteroatom, halogen atom, and unsaturated bond (hereinafter referred to as "hydrocarbon group having a heteroatom, etc."); a silyl group having at least one heteroatom, halogen atom, and unsaturated bond (hereinafter referred to as "silyl group having a heteroatom, etc."); a cyano group; a nitro group; a nitroso group; a phosphate group; a sulfonyl group; a thiol group; a thionyl group; and at least one selected from the group consisting of halogen atoms excluding fluorine atoms.

[0032] The -OR in the surface modifying group 1 Base R 1 , and the -COOR 2 Base R 2 Each of these is independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a heteroatom, a halogen atom, and an unsaturated bond; or a silyl group having at least one of a heteroatom, a halogen atom, and an unsaturated bond.

[0033] The aforementioned R 1 and R 2 The metal atoms in this context are not particularly limited and include, for example, alkali metals such as lithium, sodium, and potassium; alkaline earth metals such as beryllium, magnesium, and calcium; Group 13 elements of the periodic table such as aluminum, gallium, and indium; and lanthanides such as lanthanum.

[0034] The aforementioned R 1 and R 2The hydrocarbon group in this specification is not particularly limited and includes, for example, a linear hydrocarbon group having 1 to 100 carbon atoms, preferably 1 to 50, more preferably 1 to 20; and a cyclic hydrocarbon group having 3 to 30 carbon atoms, preferably 3 to 20, more preferably 3 to 12. More specifically, the linear hydrocarbon group includes, for example, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, and an n-octyl group; and a branched hydrocarbon group includes, for example, an isopropyl group, an isobutyl group, a tert-butyl group, and an isopentyl group. More specifically, the cyclic hydrocarbon group includes, for example, a cyclopentyl group and a cyclohexyl group. In this specification, when a range of carbon atoms is expressed, that range means that all integer carbon atoms included in that range are included. Therefore, for example, a hydrocarbon group with "1 to 3 carbon atoms" means all hydrocarbon groups with 1, 2, and 3 carbon atoms.

[0035] The aforementioned R 1 and R 2 The silyl group in this context is not particularly limited and includes, for example, trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, triisopropylsilyl group, and tert-butyldiphenylsilyl (TBDPS) group.

[0036] The aforementioned R 1 and R 2 In hydrocarbon groups having heteroatoms, a heteroatom means an oxygen atom, nitrogen atom, or sulfur atom, and a halogen atom means a fluorine atom, chlorine atom, bromine atom, or iodine atom. A hydrocarbon group having heteroatoms means that some or all of the hydrogen and carbon in the hydrocarbon group are substituted with one of these heteroatoms. A hydrocarbon group having halogen atoms means that some or all of the hydrogen and carbon in the hydrocarbon group are substituted with one of these halogen atoms.

[0037] The aforementioned R 1 and R 2The number of carbon atoms in the hydrocarbon group having heteroatoms, etc., is 1 to 100, preferably 1 to 50, more preferably 1 to 20. Also, the R 1 and R 2 The number of unsaturated bonds in the hydrocarbon group having heteroatoms, etc., can be set as appropriate and as needed.

[0038] The aforementioned R 1 and R 2 More specifically, examples of hydrocarbon groups having heteroatoms, etc., in this context include, for example, a 2-methoxyethyl group.

[0039] The aforementioned R 1 and R 2 In a silyl group having a heteroatom, the heteroatom and halogen atom are R 1 and R 2 This is similar to the heteroatoms and halogen atoms in hydrocarbon groups having heteroatoms, etc. Therefore, a detailed explanation is omitted.

[0040] The aforementioned R 1 and R 2 The silyl group having a heteroatom, etc., in is not particularly limited, and examples include the 2-methoxysilyl group. 1 and R 2 The number of unsaturated bonds in the silyl group having heteroatoms, etc., can be set as appropriate and as needed.

[0041] The -COR in the surface modifying group 3 Base R 3 This is a hydrocarbon group; a hydrocarbon group having at least one heteroatom and / or an unsaturated bond (hereinafter referred to as "hydrocarbon group having heteroatoms, etc."); or a silyl group having at least one heteroatom and / or an unsaturated bond (hereinafter referred to as "silyl group having heteroatoms, etc.").

[0042] The aforementioned R 3The hydrocarbon group in this context is not particularly limited and includes, for example, a linear hydrocarbon group having 1 to 100 carbon atoms, preferably 1 to 50, more preferably 1 to 20; and a cyclic hydrocarbon group having 3 to 30 carbon atoms, preferably 3 to 20, more preferably 3 to 12. More specifically, the linear hydrocarbon group includes, for example, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, and an n-octyl group; and a branched hydrocarbon group includes, for example, an isopropyl group, an isobutyl group, a tert-butyl group, and an isopentyl group. More specifically, the cyclic hydrocarbon group includes, for example, a cyclopentyl group and a cyclohexyl group.

[0043] The aforementioned R 3 A hydrocarbon group having a heteroatom, etc., and the R 3 In a silyl group having a heteroatom, the heteroatom is R 1 and R 2 This is similar to the heteroatoms in hydrocarbon groups that have heteroatoms. Therefore, a detailed explanation is omitted.

[0044] The aforementioned R 3 The hydrocarbon group having a heteroatom, etc., in is not particularly limited, and examples include the 2-methoxyethyl group. 3 The number of unsaturated bonds in the hydrocarbon group having heteroatoms, etc., can be set as appropriate and as needed.

[0045] The aforementioned R 3 The silyl group having a heteroatom, etc., in is not particularly limited, and examples include a 2-methoxysilyl group. 3 The number of unsaturated bonds in the silyl group having heteroatoms, etc., can be set as appropriate and as needed.

[0046] The hydrocarbon group in the surface modification group is not particularly limited, and examples include a linear hydrocarbon group having 1 to 100 carbon atoms, preferably 1 to 50, more preferably 1 to 20; and a cyclic hydrocarbon group having 3 to 30 carbon atoms, preferably 3 to 20, more preferably 3 to 12. More specifically, the linear hydrocarbon group includes, for example, a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an n-heptyl group, and an n-octyl group; and a branched hydrocarbon group includes, for example, an isopropyl group, an isobutyl group, a tert-butyl group, and an isopentyl group. More specifically, the cyclic hydrocarbon group includes, for example, a cyclopentyl group and a cyclohexyl group.

[0047] The silyl group in the surface modification group is not particularly limited, and examples include trimethylsilyl group, triethylsilyl group, tert-butyldimethylsilyl group, triisopropylsilyl group, and tert-butyldiphenylsilyl (TBDPS) group.

[0048] The number of carbon atoms in the hydrocarbon group having heteroatoms, etc., in the surface modification group is 1 to 100, preferably 1 to 50, and more preferably 1 to 20. Furthermore, the number of unsaturated bonds in the hydrocarbon group having heteroatoms, etc., in the surface modification group can be set as appropriate and as needed.

[0049] More specifically, examples of hydrocarbon groups having heteroatoms in the surface modification group include, for example, a 2-methoxyethyl group.

[0050] In the hydrocarbon group having a heteroatom in the surface modification group, the heteroatom and halogen atom are R 1 and R 2 This is similar to the heteroatoms and halogen atoms in hydrocarbon groups having heteroatoms, etc. Therefore, a detailed explanation is omitted.

[0051] In the silyl group having a heteroatom in the surface modification group, the heteroatom and halogen atom are R1 and R 2 This is similar to the heteroatoms and halogen atoms in hydrocarbon groups having heteroatoms, etc. Therefore, a detailed explanation is omitted.

[0052] The silyl group having a heteroatom in the surface modification group is not particularly limited, and examples include a 2-methoxysilyl group. The number of unsaturated bonds in the silyl group having a heteroatom in the surface modification group can be set as appropriate and as needed.

[0053] The sulfonyl group in the surface modification group is not particularly limited, and examples include a mesyl group, tosyl group, nosyl group, trifluoromethanesulfonyl group, etc.

[0054] The thionyl group in the surface modification group is not particularly limited, and examples include thionyl chloride group, thionyl fluoride group, and the like.

[0055] As described above, the cell culture member 10 of this embodiment has a structure in which fluorine atoms and surface modifying groups such as -OH groups and -COOH groups are directly chemically bonded in the adherent cell retention region. Therefore, the cell culture member 10 of this embodiment can sufficiently enhance adhesion to adherent cells and has excellent cell culture performance. Furthermore, the cell culture member 10 of this embodiment also exhibits excellent long-term stability of its superior culture performance.

[0056] (Method for surface modification of cell culture materials) Next, the surface modification method for the cell culture member according to this embodiment will be described with reference to Figure 2. The surface modification method for the cell culture member of this embodiment includes at least the step of applying a first treatment gas to at least a portion of the holding area of ​​the cell culture member 10 to perform a surface modification treatment and form a surface modification area.

[0057] The first treatment gas contains a gas containing fluorine atoms and an inert gas as an optional component. By bringing the first treatment gas into contact with at least a portion of the holding area, fluorine atoms can be directly chemically bonded to some of the carbon atoms and other elements constituting the polymer compound in the holding area, thereby performing a fluorination treatment. As a result, a fluorinated surface modification area can be formed in the area of ​​the holding area that has come into contact with the first treatment gas. Since the surface modification treatment in this embodiment directly chemically bonds fluorine atoms to some of the carbon atoms and other elements constituting the polymer compound, it differs from, for example, plasma treatment which uses plasma to activate the surface and impart hydrophilicity, and thus enables a surface treatment with excellent long-term stability.

[0058] One method for bringing the first processing gas into contact with the holding area is, for example, a method performed in the gas phase.

[0059] The concentration of the fluorine-containing gas in the first processing gas is preferably in the range of 0.01 to 60 vol%, more preferably in the range of 0.05 to 30 vol%, and particularly preferably in the range of 0.1 to 20 vol% relative to the total volume of the first processing gas. By setting the concentration of the fluorine-containing gas to 0.01 vol% or higher, it is possible to prevent insufficient fluorination of the retaining region. Furthermore, by setting the concentration of the fluorine-containing gas to 60 vol% or lower, it is possible to prevent the polymer compound in the retaining region and the fluorine atoms from reacting violently and burning during processing.

[0060] The gas containing fluorine atoms is not particularly limited as long as it contains fluorine atoms. Examples of such fluorine-containing gases include hydrogen fluoride (HF), fluorine (F2), chlorine trifluoride (ClF3), sulfur tetrafluoride (SF4), boron trifluoride (BF3), nitrogen trifluoride (NF3), carbonyl fluoride (COF2), and phosphorus pentafluoride (PF5). These may be used individually or in mixtures of two or more.

[0061] The first processing gas may contain an inert gas. The inert gas is not particularly limited, but gases that react with fluorine atoms and adversely affect the surface modification treatment of the retaining area, gases that react with polymer compounds and adversely affect them, and gases that contain impurities that adversely affect them are undesirable. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These can be used individually or in mixtures of two or more. The purity of the inert gas is not particularly limited, but the amount of the adversely affecting impurities is preferably 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less.

[0062] Furthermore, the first treatment gas may contain a gas containing oxygen atoms. This makes it possible to introduce surface modifying groups in addition to fluorination in the surface modification treatment of this embodiment. That is, by including a gas containing oxygen atoms in the first treatment gas, the surface modifying groups can be directly chemically bonded to some of the other carbon atoms, etc., that constitute the polymer compound in the retention region.

[0063] The gas containing oxygen atoms is not particularly limited, but gases that react with oxygen atoms and adversely affect the surface modification treatment (fluorination treatment) of the cell culture member 10, gases that react with the materials constituting the cell culture member 10 and adversely affect them, and gases that contain impurities that adversely affect them are undesirable. Specific examples of oxygen-containing gases include oxygen, ozone, water vapor, carbon monoxide, carbon dioxide, phosgene, and sulfur dioxide. These can be used individually or in mixtures of two or more.

[0064] The processing temperature during the surface modification treatment is not particularly limited as long as it is below the glass transition temperature of the polymer compound constituting the cell culture member 10, but is preferably -20°C to 150°C, more preferably -10°C to 120°C, and even more preferably 0°C to 100°C. By setting the processing temperature to -20°C or higher, the surface modification treatment, particularly the fluorination treatment, can be accelerated. On the other hand, by setting the processing temperature to 150°C or lower, it is possible to suppress an excessive increase in defects in the carbon skeleton and / or silicon skeleton that occur with the introduction of fluorine atoms (fluorine groups) to the surface of the cell culture member 10, and to prevent excessive destruction of the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture member 10. Furthermore, it is possible to prevent thermal deformation of the cell culture member 10 and suppress a decrease in yield.

[0065] The processing time (reaction time) for the surface modification treatment is preferably in the range of 1 second to 24 hours, more preferably in the range of 1 minute to 12 hours, and particularly preferably in the range of 3 minutes to 1 hour. By making the processing time 1 second or longer, sufficient surface modification, especially fluorination, of the surface of the cell culture member 10 can be achieved. On the other hand, by making the processing time 24 hours or less, a decrease in processing efficiency due to prolonged processing time can be prevented.

[0066] The pressure conditions for surface modification treatment are not particularly limited and can be carried out under normal pressure, increased pressure, or reduced pressure. From an economic and safety standpoint, it is preferable to carry out the treatment under normal pressure. Note that "normal pressure" means standard atmospheric pressure (101.3 kPa), but in this invention, it may also include pressure conditions of ±10% of standard atmospheric pressure.

[0067] The reaction vessel for surface modification treatment is not particularly limited, and conventionally known vessels such as fixed beds and fluidized beds can be used.

[0068] The method of contacting the cell culture member 10 with the first treatment gas is not particularly limited. For example, contact can be made in a sealed state under a flow of the first treatment gas or in an atmosphere containing at least the first treatment gas. Furthermore, the surface modification treatment may be performed multiple times. This allows for the introduction of more fluorine atoms and surface modification groups onto the surface of the cell culture member 10, further improving the long-term stability of the cell culture member 10 surface.

[0069] The surface modification treatment of the cell culture member 10 only needs to be applied to at least any portion of the area where the adherent cells are held. Therefore, the surface modification treatment may be applied to the entire area where the cells are held. In addition, the area where the cells are held may have been subjected to other known surface treatments beforehand. Examples of known surface treatments include discharge treatments such as plasma treatment. It should be noted that the present invention may also include cases where known surface treatments have been applied to areas other than the area where the cells are held.

[0070] When performing surface modification treatment on any portion of a surface (partial surface modification treatment), this can be done by masking the areas other than the area to be treated. The masking material used for masking is not particularly limited, except that it has heat resistance to the treatment temperature during the surface modification treatment. Specific examples of masking materials include fluororesins such as polytetrafluoroethylene, polytetrafluorochloroethylene, polyvinyl fluoride, polyvinylidene fluoride, polydichlorodifluoroethylene, and polytrifluorochloroethylene, as well as ceramics, polyimide, polyetheretherketone (PEEK), and metals.

[0071] Furthermore, a post-treatment step may be performed immediately after the surface modification treatment. The post-treatment step involves replacing the first treatment gas with an inert gas to create an inert atmosphere and cooling the cell culture member 10 to room temperature. Cooling to room temperature may be done by air cooling. Alternatively, the system may be evacuated to replace the gas with an inert gas, and then the pressure may be reduced to atmospheric pressure using the inert gas. This prevents fluorine gas from physically adsorbing and remaining on the surface of the cell culture member 10 that has undergone surface modification treatment. As a result, the generation of hydrogen fluoride as a by-product due to hydrolysis of fluorine gas is prevented, and problems such as the destruction of cultured cells by hydrogen fluoride do not occur. The inert gas is not particularly limited and, for example, nitrogen gas can be used.

[0072] Furthermore, after the surface modification treatment, a second treatment gas containing other oxygen atoms may be brought into contact with the surface-modified region to introduce the aforementioned surface-modifying groups (first surface-modifying group introduction treatment).

[0073] Other gases containing oxygen atoms include, specifically, oxygen, ozone, water vapor, carbon monoxide, carbon dioxide, phosgene, and sulfur dioxide. These can be used individually or in combination of two or more.

[0074] The second processing gas may contain an inert gas. The inert gas is not particularly limited, but gases that react with oxygen atoms and adversely affect the surface modification treatment of the retaining area, gases that react with polymer compounds and adversely affect them, and gases that contain impurities that adversely affect them are undesirable. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These can be used individually or in mixtures of two or more. The purity of the inert gas is not particularly limited, but the amount of the adversely affecting impurities is preferably 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less.

[0075] The processing temperature during the first surface modification group introduction treatment is not particularly limited as long as it is below the glass transition temperature of the polymer compound constituting the cell culture member 10, but is preferably -20°C to 150°C, more preferably -10°C to 120°C, and even more preferably 0°C to 100°C. By setting the processing temperature to -20°C or higher, the introduction of surface modification groups can be promoted. On the other hand, by setting the processing temperature to 150°C or lower, it is possible to suppress an excessive increase in defects in the carbon skeleton and / or silicon skeleton that occur as a result of treatment with the second processing gas on the surface of the cell culture member 10, and to prevent excessive destruction of the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture member 10. Furthermore, it is possible to prevent thermal deformation of the cell culture member 10 and suppress a decrease in yield.

[0076] The processing time (reaction time) for the first surface modification group introduction treatment is preferably in the range of 1 second to 24 hours, more preferably in the range of 1 minute to 12 hours, and particularly preferably in the range of 3 minutes to 1 hour. By making the processing time 1 second or longer, sufficient introduction of surface modification groups can be achieved. On the other hand, by making the processing time 24 hours or less, a decrease in processing efficiency due to prolonged processing time can be prevented.

[0077] The pressure conditions for the first surface modification group introduction treatment are not particularly limited and can be carried out under normal pressure, under increased pressure, or under reduced pressure. From an economic and safety standpoint, it is preferable to carry out the treatment under normal pressure.

[0078] The reaction vessel for the first surface modification group introduction treatment is not particularly limited, and conventionally known vessels such as fixed beds and fluidized beds can be used.

[0079] The method of contacting the cell culture member 10 with the second treatment gas is not particularly limited. For example, contact can be made in a sealed state under a flow of the second treatment gas or in an atmosphere containing at least the second treatment gas. Furthermore, the first surface modification group introduction treatment may be performed multiple times. This allows for the introduction of hydrophilic groups such as -OH groups and -COOH groups onto the surface of the cell culture member 10, and further improves the long-term stability of the surface of the cell culture member 10.

[0080] The first surface modification group introduction treatment may be applied to at least any portion of the retention region of the adherent cell. Therefore, the first surface modification group introduction treatment may be performed on the entire retention region. When the first surface modification group introduction treatment is applied to any portion of the region (partial surface modification group introduction treatment), this can be done by masking the regions other than the region to which the first surface modification group introduction treatment is to be performed. The masking material used for masking is not particularly limited, except that it has heat resistance to the treatment temperature during the first surface modification group introduction treatment. Specific examples of masking materials include the aforementioned masking materials used when performing partial surface modification treatment.

[0081] Furthermore, immediately after the first surface modification group introduction treatment, other surface modification groups may be introduced to the surface-modified region (second surface modification group introduction treatment). The second surface modification group introduction treatment is carried out by contacting a treatment compound that reacts with a fluorine atom directly chemically bonded to a portion of a carbon atom, etc., and / or a surface modification group directly chemically bonded to a portion of another carbon atom, etc. This allows the fluorine atom and / or surface modification group to be replaced with a surface modification group derived from the compound in the second surface modification group introduction treatment.

[0082] The surface modification groups derived from the aforementioned treated compound are the same as those in the first surface modification group introduction treatment. Therefore, a detailed explanation is omitted.

[0083] The aforementioned treatment compound can be any compound that reacts with fluorine atoms and / or surface-modifying groups, and can be used in gaseous, liquid, or solid form without any particular limitations.

[0084] The gaseous treatment compound is not particularly limited and can be, for example, water vapor. The gaseous treatment compound may also contain an inert gas. The inert gas is not particularly limited, but it is undesirable if it reacts with the treatment compound and adversely affects the surface modification treatment of the retaining area, reacts with the polymer compound and adversely affects it, or contains impurities that cause such adverse effects. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These can be used individually or in mixtures of two or more. The purity of the inert gas is not particularly limited, but it is preferable that the amount of such adversely affecting impurities be 100 ppm or less, more preferably 10 ppm or less, and particularly preferably 1 ppm or less.

[0085] The liquid treatment compound is not particularly limited and can be, for example, water. If the treatment compound is water, it can also be used as a washing treatment for the cell culture component 10 described later. Details of the washing treatment will be described later.

[0086] The processing temperature during the second surface modification group introduction treatment is not particularly limited as long as it is below the glass transition temperature of the polymer compound constituting the cell culture member 10, but is preferably -20°C to 150°C, more preferably -10°C to 120°C, and even more preferably 0°C to 100°C. By setting the processing temperature to -20°C or higher, the introduction of surface modification groups derived from the processing compound can be promoted. On the other hand, by setting the processing temperature to 150°C or lower, it is possible to suppress an excessive increase in defects in the carbon skeleton and / or silicon skeleton that occur as a result of processing the surface of the cell culture member 10 with the processing compound, thereby preventing excessive destruction of the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture member 10. Furthermore, it is possible to prevent thermal deformation of the cell culture member 10 and suppress a decrease in yield.

[0087] The processing time (reaction time) for the second surface modification group introduction treatment is preferably in the range of 1 second to 24 hours, more preferably in the range of 1 minute to 12 hours, and particularly preferably in the range of 3 minutes to 1 hour. By making the processing time 1 second or longer, sufficient introduction of surface modification groups derived from the processed compound can be achieved. On the other hand, by making the processing time 24 hours or less, a decrease in processing efficiency due to prolonged processing time can be prevented.

[0088] The pressure conditions for introducing the second surface modification group are not particularly limited and can be carried out under normal pressure, under increased pressure, or under reduced pressure. From an economic and safety standpoint, it is preferable to carry out the process under normal pressure.

[0089] The reaction vessel for the second surface modification group introduction treatment is not particularly limited, and conventionally known vessels such as fixed beds and fluidized beds can be used.

[0090] When the treatment compound is in gaseous form, the method of contacting the cell culture member 10 with the treatment compound is not particularly limited. For example, contact can be made under a flow of the treatment compound or in an atmosphere containing at least the treatment compound, while in a sealed state. Furthermore, the second surface modification group introduction treatment may be performed multiple times.

[0091] The second surface modification group introduction treatment only needs to be applied to at least any portion of the retention area of ​​the adherent cell. Therefore, the second surface modification group introduction treatment may be performed on the entire retention area. When the second surface modification group introduction treatment is applied to any portion of the area (partial surface modification group introduction treatment), this can be done by masking the areas other than the area to which the second surface modification group introduction treatment is to be performed. The masking material used for masking is not particularly limited, except that it has heat resistance to the treatment temperature during the second surface modification group introduction treatment. Specific examples of masking materials include the aforementioned masking materials used when performing the first surface modification group introduction treatment.

[0092] In this embodiment, post-treatment may be performed immediately after the first surface modification group introduction treatment and the second surface modification group introduction treatment. The post-treatment is a step of replacing the second treatment gas or gaseous treatment compound with an inert gas to create an inert atmosphere and cooling the cell culture member 10 to room temperature. Cooling to room temperature may be done by air cooling. Alternatively, the area may be evacuated to replace with an inert gas, and then the pressure may be reduced to atmospheric pressure with the inert gas. This prevents the second treatment gas from adsorbing and remaining on the surface of the cell culture member 10 that has undergone the first surface modification group introduction treatment if the post-treatment step is performed immediately after the first surface modification group introduction treatment. Similarly, if the post-treatment step is performed immediately after the second surface modification group introduction treatment, it prevents the treatment compound from adsorbing and remaining on the surface of the cell culture member 10 that has undergone the second surface modification group introduction treatment. This prevents the generation of by-products due to the decomposition of the second treatment gas or gaseous treatment compound, and prevents problems such as the destruction of cultured cells by by-products. The inert gas is not particularly limited and can be nitrogen gas, for example.

[0093] Furthermore, in this embodiment, a washing treatment may be performed after the surface modification treatment, the first surface modification group introduction treatment, the second surface modification group introduction treatment, and post-treatment. For example, if the washing agent is water, washing can react some of the fluorine atoms (fluorine groups) that are directly chemically bonded to carbon atoms, etc., with water molecules, and replace the fluorine groups with -OH groups, -COOH groups, etc., that can be directly chemically bonded. As a result, the surface of the cell culture member 10 can be made even more hydrophilic. In addition, the first treatment gas, second treatment gas, treatment compound, and by-products that were not immobilized on the surface of the cell culture member 10 can be removed. The washing agent used is not particularly limited, and examples include ethanol, isopropyl alcohol, water (e.g., ultrapure water), toluene, acetone, etc. The washing conditions are not particularly limited, but are usually carried out within the range of washing temperature (temperature of the washing agent) 0°C to 100°C and washing time 1 second to 60 minutes.

[0094] It is preferable to perform a drying treatment after the washing treatment. The drying method is not particularly limited and can be, for example, natural drying or drying by spraying nitrogen gas. The drying conditions are also not particularly limited, but are usually carried out within the range of a drying temperature (or the temperature of the nitrogen gas if sprayed) of 0°C to 100°C and a drying time of 1 second to 24 hours.

[0095] As described above, the surface modification method for the cell culture member 10 according to this embodiment allows for surface modification that improves adhesion to adherent cells in the adherent cell retention area simply by bringing a first treatment gas containing a gas with fluorine atoms into contact with the adherent cell retention area. As a result, it becomes possible to provide a cell culture member 10 that has excellent cell culture performance and excellent long-term stability. [Examples]

[0096] Preferred embodiments of this invention are described in detail below. However, unless otherwise specified, the materials and proportions described in these embodiments do not limit the scope of this invention to those described.

[0097] (Example 1) First, a polystyrene microplate (manufactured by AGC Techno Glass Co., Ltd., product name: IWAKI suspension culture microplate 24WELL, hereinafter referred to as "microplate") was prepared and placed inside a SUS316L chamber (capacity 18L).

[0098] Next, the chamber was purged with nitrogen gas under vacuum, and the ambient temperature inside the chamber was raised at a rate of 4°C / min under a nitrogen gas flow (4 L / min) until it reached 40°C. After that, the microplate was subjected to a 1-hour constant temperature treatment.

[0099] Next, a first treatment gas was introduced into the chamber to perform surface modification (fluorination) on the microplate. The first treatment gas was introduced into the chamber by vacuum displacement until the pressure inside the chamber reached atmospheric pressure. The first treatment gas was a mixed gas consisting of fluorine gas at a concentration of 0.25 vol% of the total volume of the first treatment gas and nitrogen gas. The surface modification treatment was performed with the chamber sealed, the ambient temperature inside the chamber (treatment temperature) set to 40°C, and the treatment time for 17 minutes. After that, the inside of the chamber was vacuum-purged with nitrogen gas, and the microplate was allowed to cool to room temperature under a nitrogen gas flow (4 L / min).

[0100] Next, the microplates after surface modification were thoroughly washed with ultrapure water, and then dried by blowing nitrogen gas at 25°C. This prepared microplates with surface modification treatment for use as cell culture materials in this embodiment. The ultrapure water temperature during washing was 25°C, and the washing time was 5 minutes. The nitrogen gas temperature during drying was also 25°C, and the drying time was 8 hours.

[0101] (Comparative Example 1) In this comparative example, a microplate that had not undergone the surface modification treatment used in Example 1 (manufactured by AGC Technoglass Co., Ltd., product name: IWAKI Suspension Culture Microplate 24WELL) was used.

[0102] (Comparative Example 2) In this comparative example, instead of the surface modification treatment in Example 1, a microplate that had undergone electrical discharge treatment (manufactured by AGC Technoglass Co., Ltd., product name: IWAKI Adhesive Culture Microplate 24WELL) was used.

[0103] (Elemental analysis) Elemental analysis was performed on each microplate related to Example 1, Comparative Example 1, and Comparative Example 2. Elemental analysis was carried out by X-ray photoelectron spectroscopy using a PHI5000 VersaProbe III (trade name, manufactured by ULVAC-PHI, Inc.). The results are shown in Table 1.

[0104] (contact angle) The water contact angle was measured for each microplate in Example 1, Comparative Example 1, and Comparative Example 2. The water contact angle was measured using a contact angle meter (Kyowa Interface Science Co., Ltd., model number: DM-300). The results are shown in Table 1.

[0105] (Measurement of the bond energy of fluorine atoms) The surface of the microplate according to Example 1 (the surface that has been treated with fluorination) was analyzed by X-ray photoelectron spectroscopy (XPS) to measure the binding energy of the fluorine atoms.

[0106] The XPS measurement conditions were as follows: An X-ray photoelectron spectrometer (model: PHIVersaProbeIII, manufactured by ULVAC-PHI, Inc.) was used, and monochromatized AlKα rays were used as the irradiating X-rays. The X-ray output was set to 50W, the acceleration voltage to 15kV, the measurement area to be approximately 200μm in diameter, and the measurement interval for the binding energy to 0.05eV. The intensity in the binding energy range of 679 to 699eV was measured. As a result of the measurement, as shown in Figure 3, the binding energy of the fluorine atom was detected as a waveform with a peak value at 686.5eV. Figure 3 is a graph showing the XPS measurement results of the microplate according to Example 1.

[0107] (Cell culture evaluation) Cell culture evaluations were performed on each microplate related to Example 1, Comparative Example 1, and Comparative Example 2 using human embryonic kidney cells (HEK293T cells) and Syrian hamster kidney cells (BHK-21(C-13) cells), respectively.

[0108] Cell culture evaluation begins with the number of cells being 1 × 10⁶ 4 The culture medium was seeded into each microplate to achieve a cell / WELL ratio. Fetal bovine serum (FBS) in the culture medium was set to 10%.

[0109] Next, human embryonic kidney cells and Syrian hamster kidney cells were cultured in this culture medium for 4 days at 37°C and 5% CO2. After that, the culture medium was removed, the cells were detached, and the number of viable cells in each cell type was counted. The results are shown in Table 1.

[0110] (Evaluation of cell culture under nutrient-low conditions) Cell culture evaluations were performed on each microplate related to Example 1, Comparative Example 1, and Comparative Example 2 using Syrian hamster kidney cells (BHK cells) under nutrient-low conditions.

[0111] Specifically, a culture medium containing 5% fetal bovine serum (FBS) was used, and Syrian hamster kidney cells were cultured using the same method as described above for cell culture evaluation, and the number of viable cells was counted. The results are shown in Table 1.

[0112] (Evaluation of long-term stability of cell culture performance) Next, the long-term stability of cell culture performance was evaluated for each microplate related to Example 1, Comparative Examples 1 and 2, using human embryonic kidney cells (HEK293T cells).

[0113] Long-term stability evaluation begins with the number of cells being 1 × 10⁶. 4 The culture medium was seeded into each microplate to achieve a cell / WELL ratio. Fetal bovine serum (FBS) in the culture medium was set to 10%.

[0114] Next, this culture medium was stored in air at 25°C for 30 days. After that, human embryonic kidney cells were cultured for 4 days at 37°C in a 5% CO2 environment. After culturing, the culture medium was removed, the cells were detached, and the number of viable human embryonic kidney cells was counted. The results are shown in Table 1.

[0115] [Table 1]

[0116] (Evaluation of cell culture using primary cells) Next, cell culture evaluations were performed using mouse astrocyte cells in each of the microplates for Example 1, Comparative Example 1, and Comparative Example 2.

[0117] For cell culture evaluation, the cerebral cortex was first extracted from a mouse (ICR strain) fetus, enzymatically degraded, and then seeded onto a microplate coated with laminin and polylysine. After culturing for 15 days to confirm the presence of mouse astrocyte cells, the microplate was detached.

[0118] Next, each microplate contains 1 × 10 cells. 5 The cells were reseeded to achieve a cells / well ratio. Three days later, the mouse astrocyte cells in each microplate were fluorescently stained with calcein, and the condition of the mouse astrocyte cells was observed using an inverted microscope. The results are shown in Figures 4 to 6. Figures 4 to 6 show the bright-field images observed under an inverted microscope after culturing mouse astrocyte cells using microplates in Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0119] (result) Elemental analysis by XPS revealed that 14.0 at% fluorine atoms were detected in the fluorinated microplate of Example 1. Furthermore, an increase in the amount of oxygen atoms was observed compared to the untreated microplate of Comparative Example 1. XPS peak positions also confirmed the formation of -OH and -COOH groups as surface modification groups in the microplate of Example 1. On the other hand, no fluorine atoms were detected in the microplates of Comparative Examples 1 and 2.

[0120] Furthermore, measurements of the water contact angle revealed that the microplate of Example 1 had a smaller contact angle compared to Comparative Example 1, which used an untreated microplate. This clearly demonstrated that the microplate of Example 1 had improved wettability with water.

[0121] Furthermore, cell culture evaluation results showed that the number of viable cells in the microplate of Example 1 was 31.7 × 10⁶. 4 The number of microplates was 1.6 × 10⁶, while the untreated microplate in Comparative Example 1 had 1.6 × 10⁶ microplates. 4 This confirmed that the microplate of Example 1, which underwent surface modification treatment, had superior cell culture performance compared to the untreated microplate of Comparative Example 1.

[0122] In particular, in the evaluation of cell culture under nutrient-low conditions, the number of viable cells in the fluorinated microplate of Example 1 was 3.2 × 10⁶. 4 The number of cells was small, and it was confirmed that the cell culture performance was superior to that of the microplates used in Comparative Examples 1 and 2.

[0123] Furthermore, regarding the long-term stability of cell culture performance, the number of viable cells in the microplate of Example 1, which was treated with fluorination, was 31.5 × 10⁶. 4 The number of cells was 23.4 × 10⁶, and it was confirmed that the cell culture performance was maintained at the same level as before storage in the atmosphere for 30 days. On the other hand, in the microplate of Example 1 that was plasma-treated, the number of viable cells was 23.4 × 10⁶. 4 The number of viable cells before 30 days of storage in the atmosphere was 31.2 × 10⁶. 4 The number of cells decreased significantly, confirming that cell culture performance declined over time.

[0124] Furthermore, in cell culture evaluations using primary cells, as shown in Figure 4, mouse astrocyte cells adhered to the microplate surface of Example 1, which had been fluorinated, and their neurites were observed to be extended. On the other hand, in the microplate of Comparative Example 1, which had not undergone surface modification, mouse astrocyte cells did not adhere to the microplate surface, as shown in Figure 5. In the microplate of Comparative Example 2, which had been plasma-treated, mouse astrocyte cells did adhere to the microplate surface, as shown in Figure 6, but the extension of their neurites was not as sufficient compared to Example 1. From these results, it became clear that the microplate of Example 1 had superior cell culture performance compared to the microplates of Comparative Examples 1 and 2, even in cell culture evaluations using primary cells.

[0125] From the above results, it was found that the microplate after fluorination treatment in Example 1 had better cell culture performance compared to the untreated microplate in Comparative Example 1. Furthermore, it was found that the microplate after surface modification treatment in Example 1 maintained superior cell culture performance even after 30 days of storage in air, compared to the microplate after plasma treatment in Comparative Example 2. [Explanation of Symbols]

[0126] 10 Cell culture materials

Claims

1. A cell culture member in which at least the holding region for adherent cells is made of a polymer compound, wherein at least a part of the holding region is a surface-modified region in which fluorine atoms are directly chemically bonded to some of the carbon atoms and / or silicon atoms constituting the polymer compound by bringing a first treatment gas containing a fluorine atom-containing gas and an inert gas as an optional component into contact with the polymer compound without performing plasma treatment, and the polymer compound is at least one polymer selected from the group consisting of polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polyurethane, cyclic polyolefin, polyetheretherketone, polyimide, polyamideimide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile-butadiene-styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and a silicon-containing polymer compound.

2. The cell culture member according to claim 1, wherein the peak value of the binding energy measured by X-ray photoelectron spectroscopy of the fluorine atoms is in the range of 680 eV to 690 eV.

3. Surface modification groups are directly chemically bonded to some of the other carbon atoms and / or other silicon atoms constituting the polymer compound in the surface-modified region, The surface modification group is -OR １ group; -COOR ２ group; -COR ３ group; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a heteroatom, a halogen atom, and an unsaturated bond; a silyl group having at least one of a heteroatom, a halogen atom, and an unsaturated bond; a cyano group; a nitro group; a nitroso group; a phosphoric acid group; a sulfonyl group; a thiol group; a thionyl group; and at least one selected from the group consisting of halogen atoms excluding a fluorine atom, Said R １ and said R ２ are each independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least any one of a heteroatom, a halogen atom, and an unsaturated bond; or a silyl group having at least any one of a heteroatom, a halogen atom, and an unsaturated bond, Said R ３ is any one of a hydrocarbon group; a hydrocarbon group having at least one of a hetero atom and an unsaturated bond; or a silyl group having at least one of a hetero atom and an unsaturated bond, according to claim 1 or 2 of the cell culture member.

4. A method for surface modification of a cell culture member in which a holding region for adherent cells is made of a polymer compound, the method including a step of forming a surface-modified region in which fluorine atoms are directly chemically bonded to some of the carbon atoms and / or silicon atoms constituting the polymer compound by bringing a first treatment gas containing a fluorine atom-containing gas and an inert gas as an optional component into contact with at least a part of the holding region without performing plasma treatment, and the polymer compound is at least one polymer selected from the group consisting of polyvinyl chloride, polystyrene, polyethylene, polypropylene, polyvinyl acetate, polyurethane, cyclic polyolefin, polyetheretherketone, polyimide, polyamideimide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile-butadiene-styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and a silicon-containing polymer compound.

5. The method for surface modification of a cell culture member according to claim 4, wherein the peak value of the binding energy measured by X-ray photoelectron spectroscopy of the fluorine atoms in the surface modification region is in the range of 680 eV to 690 eV.

6. By using, as the first treatment gas, a gas containing a gas further containing oxygen atoms, a surface modification group is directly chemically bonded to a part of other carbon atoms and / or other silicon atoms constituting the polymer compound. The surface modification group is -OR １ group; -COOR ２ group; -COR ３ group; a hydrocarbon group; a silyl group; a hydrocarbon group having at least any one of a hetero atom, a halogen atom and an unsaturated bond; a silyl group having at least any one of a hetero atom, a halogen atom and an unsaturated bond; a nitro group; a nitroso group; a phosphoric acid group; a sulfonyl group; a thionyl group; and at least one selected from the group consisting of halogen atoms excluding a fluorine atom, Said R １ and said R ２ are each independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least any one of a hetero atom, a halogen atom and an unsaturated bond; or a silyl group having at least any one of a hetero atom, a halogen atom and an unsaturated bond. Said R ３ is any one of a hydrocarbon group; a hydrocarbon group having at least one of a hetero atom and an unsaturated bond; or a silyl group having at least one of a hetero atom and an unsaturated bond, according to claim 4 or 5, the method for surface modification of a cell culture member.

7. By bringing a second treatment gas containing other oxygen atoms into contact with at least a part of the holding region in which the first treatment gas has been brought into contact, without performing plasma treatment, a step of directly chemically bonding a surface modification group to a part of other carbon atoms and / or other silicon atoms constituting the polymer compound is further included. The surface modification group is -OR １ group; -COOR ２ group; -COR ３ group; a hydrocarbon group; a silyl group; a hydrocarbon group having at least one of a hetero atom, a halogen atom, and an unsaturated bond; a silyl group having at least one of a hetero atom, a halogen atom, and an unsaturated bond; a nitro group; a nitroso group; a phosphoric acid group; a sulfonyl group; a thionyl group; and at least one selected from the group consisting of halogen atoms excluding a fluorine atom, Said R １ and said R ２ are each independently a hydrogen atom; a metal atom; a hydrocarbon group; a silyl group; a hydrocarbon group having at least any one of a hetero atom, a halogen atom, and an unsaturated bond; or a silyl group having at least any one of a hetero atom, a halogen atom, and an unsaturated bond, Said R ３ is any one of a hydrocarbon group; a hydrocarbon group having at least one of a heteroatom and an unsaturated bond; or a silyl group having at least one of a heteroatom and an unsaturated bond, according to claim 4 or 5, the method for surface modification of a cell culture member.

8. By bringing a treatment compound that reacts with the fluorine atoms and / or the surface modification group into contact with the fluorine atoms directly chemically bonded to a part of the carbon atoms and / or silicon atoms, and / or the surface modification group directly chemically bonded to a part of the other carbon atoms and / or other silicon atoms. The method for surface modification of a cell culture member according to claim 6 or 7, further including a step of substituting the fluorine atoms and / or the surface modification group with the surface modification group derived from the treatment compound.

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