Cell culture component and surface modification method 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 substrate and a method for modifying its surface, and more particularly to a cell culture substrate having excellent cell culture performance and long-term stability, and a method for modifying its surface. [Background technology]

[0002] Cell culture technology is used in research in a variety of fields, including elucidating biochemical phenomena, producing useful substances, regenerative medicine, and drug evaluation. Among cell culture technologies, polymer compounds made from thermoplastic resins are generally used as the material for cell culture vessels, due to growing concerns about infection and contamination, as well as from the perspectives of moldability and manufacturing costs. The shape and physical properties of cell culture vessels have a significant impact on the efficiency of research and analytical accuracy in various applications.

[0003] In particular, in research using adherent cells (all mammalian cells except for some cells such as cancer cells and hematopoietic cells), the adhesiveness between the adherent cells and the cell culture vessel is important for cell growth efficiency and maintaining physiological activity. However, most cell culture vessels made of the above-mentioned thermoplastic resins have hydrophobic surfaces, which result in poor adhesion of adherent cells and a significant inhibition of their cellular activity.

[0004] To address this problem, for example, Patent Document 1 describes a method of modifying the surface of a polypropylene substrate by plasma treatment in order to deposit or immobilize cells or biomolecules on the polypropylene substrate. Patent Document 1 describes that the surface of the polypropylene substrate is modified by plasma treatment, thereby optimizing the interaction between the surface of the polypropylene substrate and the cells or biomolecules.

[0005] However, when surface modification is performed using discharge treatment, such as plasma treatment, it is preferable that the surface of the object to be treated be smooth and free of irregularities. Therefore, there is a problem that it is difficult to apply this method to cell culture vessels with complex shapes, such as those used in three-dimensional culture, which has seen increasing demand in recent years. Furthermore, although discharge-treated surfaces exhibit sufficient cell culture performance immediately after the discharge treatment, the cell culture performance deteriorates over time, resulting in poor long-term stability. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 2014-515692 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of the above problems, and an object of the present invention is to provide a cell culture component having excellent cell culture performance and long-term stability, and a method for modifying the surface thereof. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, the cell culture device of the present invention is a cell culture device in which at least the holding area for adhesive cells is made of a polymer compound, and at least a part of the holding area is a surface-modified area in which fluorine atoms are directly chemically bonded to some of the carbon atoms and / or silicon atoms that constitute the polymer compound.

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

[0010] In the above-mentioned structure, a surface modifying group is directly chemically bonded to a part of other carbon atoms and / or other silicon atoms constituting the polymer compound in the surface modified region, and the surface modifying group is -OR 1 base;-COOR 2 base;-COR 3 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 cyano group; a nitro group; a nitroso group; a phosphate group; a sulfonyl group; a thiol group; a thionyl group; and a halogen atom other than a fluorine atom, 1 and the above R 2 are each independently any one of 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, 3 is preferably 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.

[0011] In the above-mentioned 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, polyether ether ketone, polyimide, polyamide imide, polycarbonate, polymethyl methacrylate, polyethylene terephthalate, acrylonitrile-butadiene-styrene, polyacrylonitrile, polyamide, polyvinyl alcohol, polyolefin, and silicon-containing polymer compounds.

[0012] In order to solve the above-mentioned problems, the surface modification method of the cell culture member of the present invention is a surface modification method of a cell culture member in which a holding area for adhesive cells is made of a polymer compound, and is characterized by including a step of contacting at least a portion of the holding area with a first treatment gas containing a gas containing fluorine atoms and an inert gas as an optional component, thereby forming a surface modified area in which the fluorine atoms are directly chemically bonded to a portion of the carbon and / or silicon atoms constituting the polymer compound.

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

[0014] In the above-described configuration, the step uses a gas further containing an oxygen atom as the first processing gas, thereby directly chemically bonding a surface modifying group to a part of other carbon atoms and / or other silicon atoms constituting the polymer compound, and the surface modifying group is -OR 1 base;-COOR 2 base;-COR 3 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 a halogen atom other than a fluorine atom, 1 and the above R 2 are each independently any one of 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, 3 may be any 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.

[0015] In the above-described configuration, the method further includes a step of contacting a second processing gas containing a gas containing other oxygen atoms with at least a part of the holding region that has been contacted with the first processing gas, thereby directly chemically bonding a surface modifying group to a part of other carbon atoms and / or other silicon atoms that constitute the polymer compound, and 1 and the above R 2 are each independently any one of 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, 3 may be any 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.

[0016] In the above-described configuration, it is preferable to further include a step of bringing the fluorine atoms directly chemically bonded to a portion of the carbon atoms and / or silicon atoms and / or the surface modifying groups directly chemically bonded to a portion of the other carbon atoms and / or other silicon atoms into contact with a treating compound that reacts with the fluorine atoms and / or surface modifying groups, thereby substituting the fluorine atoms and / or surface modifying groups with the surface modifying groups derived from the treating compound.

[0017] In the above-mentioned 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, polyether ether ketone, polyimide, polyamide imide, 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 device of the present invention, a surface-modified region is formed in an adherent cell-holding region made of a polymer compound by directly chemically bonding fluorine atoms to a portion of the carbon and / or silicon atoms constituting the polymer compound. This improves the adhesiveness of adherent cells to the surface-modified region compared to regions that have not undergone such surface modification. As a result, a cell culture device with excellent cell culture performance can be provided. Furthermore, because the fluorine atoms are directly chemically bonded and fixed to a portion of the carbon atoms, etc. constituting the polymer compound, the cell culture device of the present invention can suppress deterioration of adhesive cell adhesion over time compared to conventional surface modifications such as those achieved by plasma treatment. This allows for the provision of a cell culture device with excellent long-term stability of cell culture performance.

[0019] Furthermore, according to the surface modification method of the cell culture component of the present invention, by simply contacting at least a portion of the holding area of ​​the cell culture component with a first treatment gas containing at least a gas containing fluorine atoms, fluorine atoms can be directly chemically bonded to some of the carbon atoms, etc., in the polymer compound that constitutes the holding area, thereby forming a surface-modified area. As a result, a cell culture component with excellent cell culture performance and long-term stability can be produced by an extremely simple method. [Brief explanation of the drawings]

[0020] [Figure 1] 1 is a conceptual diagram for explaining a surface-modified region in a cell culture member according to an embodiment of the present invention. [Figure 2] 3A to 3C are conceptual diagrams illustrating a method for modifying the surface of a cell culture component according to the embodiment. [Figure 3] 1 is a graph showing the results of XPS measurement of the microplate according to Example 1. [Figure 4] FIG. 1 shows a bright-field image observed with an inverted microscope after mouse astrocyte cells were cultured using the microplate of Example 1. [Figure 5]FIG. 10 is a bright-field image observed with an inverted microscope after mouse astrocyte cells were cultured using the microplate of Comparative Example 1. [Figure 6] FIG. 10 is a bright-field image observed with an inverted microscope after mouse astrocyte cells were cultured using the microplate of Comparative Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0021] (Cell culture parts) First, the cell culture component of this embodiment will be described with reference to Fig. 1. Fig. 1 is a conceptual diagram illustrating a surface-modified region in a cell culture component 10 according to this embodiment.

[0022] The cell culture component 10 of this embodiment includes at least a holding area (cell culture surface) capable of holding adhesive cells. The cell culture component 10 of this embodiment allows adhesive cells to adhere to this holding area, enabling the adhesive cells to be cultured in the holding area.

[0023] As used herein, the term "cell culture component" refers to a component having at least a solid surface that can serve as a scaffold for adherent cells to grow, differentiate, survive, and the like. Therefore, examples of the cell culture component 10 that can be used include films (membranes), sheets, microplates, flasks, dishes, tubes, hollow fiber membranes, and generally spherical objects. When the cell culture component 10 is a film (membrane) or sheet, it also includes components of finished products such as cell culture vessels.

[0024] Furthermore, as used herein, "adherent cells" refers to cells that require a scaffold on a solid surface for proliferation, differentiation, survival, etc., and that adhere to the 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, tumor cells, endothelial cells, fibroblasts, muscle cells, neural / endocrine cells, primary cells, and glial cells (neuronal glial cells) such as mouse astrocyte cells. However, adhesive cells are not limited to these exemplified cells.

[0025] The cell culture member 10 may be any member as long as at least the holding area (cell culture surface) is made of a polymer compound. The holding area may also be made of a polymer compound and have a surface 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. Polymers include thermoplastic resins and elastomers. Among these polymer compounds, polystyrene, polyethylene terephthalate, and polypropylene are preferred from the viewpoints of moldability and production costs.

[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). This allows the surface-modified region to have increased adhesiveness of adherent cells compared to regions in which the fluorine atoms are not directly chemically bonded, thereby improving cell culture performance. Furthermore, because the fluorine atoms are directly chemically bonded to some of the carbon atoms, etc., of the polymer compound that forms the retention region, the surface-modified region can suppress deterioration of adhesiveness of adherent cells over time compared to regions that have been subjected to other surface treatments, such as plasma treatment. As a result, the long-term stability of cell culture performance can also be improved.

[0028] In the present invention, the surface-modified region may be formed in at least a part of the holding region, and therefore the entire holding region may be a surface-modified region.

[0029] The peak value of the 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 value of the binding energy to 680 eV or higher, the surface-modified region becomes hydrophilic, thereby improving adhesive performance. By setting the peak value of the binding energy to 690 eV or lower, hydrophobicity due to excessive surface modification can be prevented, and appropriate adhesiveness between the surface-modified region and adhesive cells can be maintained. The peak value of the binding energy of fluorine atoms can be measured, for example, using an X-ray photoelectron spectrometer (model number: PHI VersaProbeIII, manufactured by ULVAC-PHI, Inc.) with AlKα rays monochromated by a monochromator as the irradiated X-rays, an X-ray output of 50 W, an acceleration voltage of 15 kV, a measurement area of ​​approximately 200 μm in diameter, and a binding energy measurement interval of 0.05 eV.

[0030] A surface modifying group may be further directly chemically bonded to some of the other carbon atoms and / or other silicon atoms (hereinafter referred to as "other carbon atoms, etc.") that constitute the polymer compound in the surface-modified region.

[0031] The surface modifying group is —OR 1 base;-COOR 2 base;-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 (hereinafter referred to as a "hydrocarbon group having a heteroatom, etc."); a silyl group having at least one of a heteroatom, a halogen atom, and an unsaturated bond (hereinafter referred to as a "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 a halogen atom other than a fluorine atom.

[0032] The -OR in the surface modifying group 1 Group R 1 , and -COOR 2 Group R 2 are each independently any one of 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] R 1 and R 2 The metal atom in is not particularly limited, and examples thereof include alkali metals such as lithium, sodium, and potassium; alkaline earth metals such as beryllium, magnesium, and calcium; elements of Group 13 of the periodic table such as aluminum, gallium, and indium; and lanthanoids such as lanthanum.

[0034] R 1 and R 2The hydrocarbon group in the formula (I) is not particularly limited, and examples thereof include linear hydrocarbon groups having 1 to 100 carbon atoms, preferably 1 to 50 carbon atoms, and more preferably 1 to 20 carbon atoms; and cyclic hydrocarbon groups having 3 to 30 carbon atoms, preferably 3 to 20 carbon atoms, and more preferably 3 to 12 carbon atoms. More specific examples of the linear hydrocarbon groups include linear hydrocarbon groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl; and branched hydrocarbon groups such as isopropyl, isobutyl, tert-butyl, and isopentyl. More specific examples of the cyclic hydrocarbon groups include cyclopentyl and cyclohexyl groups. When a range of carbon atoms is expressed herein, the range includes all integer carbon numbers within that range. Therefore, for example, a hydrocarbon group having "1 to 3 carbon atoms" refers to all hydrocarbon groups having 1, 2, and 3 carbon atoms.

[0035] R 1 and R 2 The silyl group in the formula (I) is not particularly limited, and examples thereof include a trimethylsilyl group, a triethylsilyl group, a tert-butyldimethylsilyl group, a triisopropylsilyl group, and a tert-butyldiphenylsilyl (TBDPS) group.

[0036] R 1 and R 2 In the hydrocarbon group having a heteroatom or the like, the heteroatom means an oxygen atom, a nitrogen atom, a sulfur atom, or the like, and the halogen atom means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. The hydrocarbon group having a heteroatom means a hydrocarbon group in which some or all of the hydrogen and carbon atoms in the hydrocarbon group have been substituted with any of these heteroatoms. Furthermore, the hydrocarbon group having a halogen atom means a hydrocarbon group in which some or all of the hydrogen and carbon atoms in the hydrocarbon group have been substituted with any of these halogen atoms.

[0037] R 1 and R 2The number of carbon atoms in the hydrocarbon group having a heteroatom or the like is 1 to 100, preferably 1 to 50, and more preferably 1 to 20. 1 and R 2 The number of unsaturated bonds in the hydrocarbon group having a hetero atom or the like can be appropriately set as required.

[0038] R 1 and R 2 More specifically, the hydrocarbon group having a hetero atom or the like in the above formula includes, for example, a 2-methoxyethyl group.

[0039] R 1 and R 2 In the silyl group having a hetero atom or the like, the hetero atom and the halogen atom are 1 and R 2 The heteroatoms and halogen atoms in the hydrocarbon group having heteroatoms etc. in the above are the same as those in the above, and therefore a detailed description thereof will be omitted.

[0040] R 1 and R 2 The silyl group having a hetero atom or the like in the formula (I) is not particularly limited, and examples thereof include a 2-methoxysilyl group. 1 and R 2 The number of unsaturated bonds in the silyl group having a heteroatom or the like can be appropriately set as needed.

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

[0042] R 3The hydrocarbon group in is not particularly limited, and examples thereof include chain hydrocarbon groups having 1 to 100, preferably 1 to 50, and more preferably 1 to 20 carbon atoms; and cyclic hydrocarbon groups having 3 to 30, preferably 3 to 20, and more preferably 3 to 12 carbon atoms. Specific examples of the chain hydrocarbon groups include linear hydrocarbon groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl; and branched hydrocarbon groups such as isopropyl, isobutyl, tert-butyl, and isopentyl. Specific examples of the cyclic hydrocarbon groups include cyclopentyl and cyclohexyl.

[0043] R 3 and the hydrocarbon group having a heteroatom or the like in the above R 3 In the silyl group having a heteroatom or the like in the above, the heteroatom is 1 and R 2 The heteroatom in the hydrocarbon group having a heteroatom in the above is the same as the heteroatom in the above. Therefore, a detailed description thereof will be omitted.

[0044] R 3 The hydrocarbon group having a heteroatom or the like in the formula (I) is not particularly limited, and examples thereof include a 2-methoxyethyl group. 3 The number of unsaturated bonds in the hydrocarbon group having a hetero atom or the like can be appropriately set as required.

[0045] R 3 The silyl group having a hetero atom or the like in the formula (I) is not particularly limited, and examples thereof include a 2-methoxysilyl group. 3 The number of unsaturated bonds in the silyl group having a heteroatom or the like can be appropriately set as needed.

[0046] The hydrocarbon group in the surface modifying group is not particularly limited, and examples thereof include chain hydrocarbon groups having 1 to 100, preferably 1 to 50, and more preferably 1 to 20 carbon atoms; and cyclic hydrocarbon groups having 3 to 30, preferably 3 to 20, and more preferably 3 to 12 carbon atoms. Specific examples of the chain hydrocarbon groups include linear hydrocarbon groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl; and branched hydrocarbon groups such as isopropyl, isobutyl, tert-butyl, and isopentyl. Specific examples of the cyclic hydrocarbon groups include cyclopentyl and cyclohexyl.

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

[0048] The number of carbon atoms in the hydrocarbon group having a heteroatom or the like in the surface modifying group is 1 to 100, preferably 1 to 50, and more preferably 1 to 20. The number of unsaturated bonds in the hydrocarbon group having a heteroatom or the like in the surface modifying group can be set appropriately as needed.

[0049] More specifically, examples of the hydrocarbon group having a heteroatom or the like in the surface modifying group include a 2-methoxyethyl group.

[0050] In the hydrocarbon group having a heteroatom or the like in the surface modifying group, the heteroatom and the halogen atom are 1 and R 2 The heteroatoms and halogen atoms in the hydrocarbon group having heteroatoms etc. in the above are the same as those in the above, and therefore a detailed description thereof will be omitted.

[0051] In the silyl group having a heteroatom or the like in the surface modifying group, the heteroatom and the halogen atom are1 and R 2 The heteroatoms and halogen atoms in the hydrocarbon group having heteroatoms etc. in the above are the same as those in the above, and therefore a detailed description thereof will be omitted.

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

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

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

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

[0056] (Surface modification method for cell culture substrate) Next, the surface modification method of the cell culture component of this embodiment will be described with reference to FIG. The surface modification method of the cell culture member of this embodiment includes at least the step of subjecting at least a part of the holding region of the cell culture member 10 to a surface modification treatment by bringing a first processing gas into contact with the part, thereby forming a surface-modified region.

[0057] The first processing gas contains a gas containing fluorine atoms and an optional inert gas. By contacting at least a portion of the holding region with the first processing gas, fluorine atoms are directly chemically bonded to some of the carbon atoms and other components of the polymer compound in the holding region, thereby performing a fluorination treatment. As a result, a fluorinated surface-modified region can be formed in the region of the holding region that comes into contact with the first processing gas. The surface modification treatment of this embodiment chemically bonds fluorine atoms directly to some of the carbon atoms and other components of the polymer compound, and therefore enables surface treatment with excellent long-term stability, unlike, for example, plasma treatments that use plasma to activate the surface and impart hydrophilicity.

[0058] The first processing gas may be brought into contact with the holding region in a gas phase, for example.

[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 0.05 to 30 vol%, and particularly preferably 0.1 to 20 vol%, based on the total volume of the first processing gas. By setting the concentration of the fluorine-containing gas to 0.01 vol% or more, insufficient fluorination of the holding area can be prevented. Furthermore, by setting the concentration of the fluorine-containing gas to 60 vol% or less, it is possible to prevent the polymer compound in the holding area from violently reacting with the fluorine atoms and burning during processing.

[0060] The gas containing fluorine atoms is not particularly limited as long as it is a gas containing fluorine atoms. Examples of such gases containing fluorine atoms 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 alone or in combination of two or more.

[0061] The first processing gas may contain an inert gas. While the inert gas is not particularly limited, it is preferable to use an inert gas that reacts with a gas containing fluorine atoms to adversely affect the surface modification treatment of the holding region, a gas that reacts with a polymer compound to adversely affect the surface modification treatment, or a gas containing impurities that adversely affect the surface modification treatment. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These gases may be used alone or in combination of two or more. Furthermore, the purity of the inert gas is not particularly limited, but the content 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] The first processing gas may also contain a gas containing oxygen atoms. This allows the surface modification treatment of this embodiment to introduce surface modifying groups in addition to the fluorination treatment. That is, by including a gas containing oxygen atoms in the first processing gas, the surface modifying groups can be further chemically bonded directly to some of the other carbon atoms constituting the polymer compound in the retention region.

[0063] The gas containing oxygen atoms is not particularly limited, but is preferably one that reacts with the gas containing oxygen atoms to adversely affect the surface modification treatment (fluorination treatment) of the cell culture member 10, one that reacts with the materials constituting the cell culture member 10 to adversely affect the cell culture member 10, or one that contains impurities that have such adverse effects. Specific examples of gases containing oxygen atoms include oxygen, ozone, water vapor, carbon monoxide, carbon dioxide, phosgene, and sulfur dioxide. These gases can be used alone or in combination of two or more.

[0064] The treatment temperature during the surface modification treatment is not particularly limited as long as it is equal to or lower than the glass transition point of the polymer compound constituting the cell culture component 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. Setting the treatment temperature at −20°C or higher can promote the surface modification treatment, particularly the fluorination treatment. On the other hand, setting the treatment temperature at 150°C or lower can suppress excessive increases in defects in the carbon skeleton and / or silicon skeleton that occur with the introduction of fluorine atoms (fluorine groups) into the surface of the cell culture component 10, thereby preventing excessive damage to the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture component 10. Furthermore, thermal deformation of the cell culture component 10 can be prevented, suppressing a decrease in yield.

[0065] The treatment time (reaction time) of 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 setting the treatment time to 1 second or longer, the surface modification, particularly fluorination, of the surface of the cell culture component 10 can be made sufficient. On the other hand, by setting the treatment time to 24 hours or shorter, a decrease in treatment efficiency due to a long treatment time can be prevented.

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

[0067] The reaction vessel for carrying out the surface modification treatment is not particularly limited, and a conventionally known vessel such as a fixed bed or a fluidized bed can be used.

[0068] The method for contacting the cell culture component 10 with the first processing gas is not particularly limited, and examples thereof include contacting the cell culture component 10 in a sealed state under a flow of the first processing gas or in an atmosphere containing at least the first processing gas. The surface modification treatment may be performed multiple times. This allows additional fluorine atoms or surface modifying groups to be introduced into the surface of the cell culture component 10, further improving the long-term stability of the surface of the cell culture component 10.

[0069] The surface modification treatment of the cell culture component 10 may be performed on at least a portion of the adhesive cell retention area. Therefore, the surface modification treatment may be performed on the entire retention area. The retention area may also be subjected to other known surface treatments before the surface modification treatment. Examples of known surface treatments include discharge treatments such as plasma treatment. The present invention also includes cases where known surface treatments are performed on areas other than the retention area.

[0070] When performing surface modification treatment on an arbitrary portion of a region (partial surface modification treatment), the surface modification treatment can be performed by masking the region other than the region to be subjected to the surface modification treatment. The masking material used for masking is not particularly limited, as long as 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, ceramics, polyimide, polyether ether ketone (PEEK), and metals.

[0071] Alternatively, a post-treatment process may be performed immediately after the surface modification treatment. The post-treatment process is a process in which the first treatment gas is replaced with an inert gas to create an inert atmosphere, and the cell culture component 10 is cooled to room temperature. Cooling to room temperature may be performed by standing to cool. Alternatively, the air may be evacuated to a vacuum to replace the gas with an inert gas, and then the pressure may be increased to atmospheric pressure with the inert gas. This prevents fluorine gas from being physically adsorbed and remaining on the surface of the cell culture component 10 that has been subjected to the surface modification treatment. As a result, hydrogen fluoride is prevented from being produced as a by-product by hydrolysis of fluorine gas, and problems such as destruction of cultured cells by hydrogen fluoride are prevented. The inert gas is not particularly limited, and examples thereof include nitrogen gas.

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

[0073] Specific examples of other gases containing oxygen atoms include oxygen, ozone, water vapor, carbon monoxide, carbon dioxide, phosgene, sulfur dioxide, etc. These can be used alone or in combination of two or more.

[0074] The second processing gas may contain an inert gas. While the inert gas is not particularly limited, it is preferable to use an inert gas that reacts with a gas containing oxygen atoms to adversely affect the surface modification treatment of the holding region, a gas that reacts with a polymer compound to adversely affect the surface modification treatment, or a gas containing impurities that adversely affect the surface modification treatment. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These gases may be used alone or in combination of two or more. Furthermore, the purity of the inert gas is not particularly limited, but the content 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 treatment temperature for the first surface-modifying group introduction treatment is not particularly limited as long as it is equal to or lower than the glass transition point 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. Setting the treatment temperature to −20°C or higher can promote the introduction of the surface-modifying group. On the other hand, setting the treatment temperature to 150°C or lower can suppress excessive increases in defects in the carbon skeleton and / or silicon skeleton that occur during treatment of the surface of the cell culture member 10 with the second treatment gas, thereby preventing excessive damage to the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture member 10. Furthermore, thermal deformation of the cell culture member 10 can be prevented, suppressing a decrease in yield.

[0076] The treatment time (reaction time) for the first surface modifying 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 setting the treatment time to 1 second or longer, the introduction of the surface modifying group can be ensured. On the other hand, by setting the treatment time to 24 hours or shorter, a decrease in treatment efficiency due to a long treatment time can be prevented.

[0077] The pressure conditions for carrying out the first surface modifying group introduction treatment are not particularly limited, and the treatment can be carried out under normal pressure, elevated pressure, or reduced pressure. From the standpoints of economy and safety, it is preferable to carry out the treatment under normal pressure.

[0078] The reaction vessel for carrying out the first surface modifying group introduction treatment is not particularly limited, and a conventionally known vessel such as a fixed bed or a fluidized bed can be used.

[0079] The method for contacting the cell culture component 10 with the second processing gas is not particularly limited, and examples include a method in which the cell culture component 10 is contacted in a sealed state under a flow of the second processing gas or in an atmosphere containing at least the second processing gas. The first surface-modifying group introduction treatment may be performed multiple times. This allows additional hydrophilic groups, such as -OH groups and -COOH groups, to be introduced onto the surface of the cell culture component 10, and further improves the long-term stability of the surface of the cell culture component 10.

[0080] The first surface modifying group introduction treatment may be performed on at least a portion of any desired region of the adhesive cell retention region. Therefore, the first surface modifying group introduction treatment may be performed on the entire surface of the retention region. When the first surface modifying group introduction treatment is performed on any desired region (partial surface modifying group introduction treatment), this can be performed by masking regions other than the desired region for the first surface modifying group introduction treatment. The masking material used for masking is not particularly limited, as long as it is heat resistant to the treatment temperature during the first surface modifying group introduction treatment. Specific examples of the masking material include the aforementioned masking materials used when performing partial surface modification treatment.

[0081] Immediately after the first surface-modifying group introduction treatment, another surface-modifying group may be introduced into the surface-modified region (second surface-modifying group introduction treatment). The second surface-modifying group introduction treatment is carried out by contacting the surface with a treatment compound that reacts with fluorine atoms directly chemically bonded to a portion of the carbon atoms, etc., and / or surface-modifying groups directly chemically bonded to a portion of other carbon atoms, etc. In this way, the second surface-modifying group introduction treatment can replace the fluorine atoms and / or surface-modifying groups with surface-modifying groups derived from the compound.

[0082] The surface modifying groups derived from the treatment compound are the same as those in the first surface modifying group introduction treatment, and therefore, a detailed description thereof will be omitted.

[0083] The treating compound may be in gaseous, liquid or solid form and is not particularly limited as long as it reacts with fluorine atoms and / or surface modifying groups.

[0084] The gaseous treatment compound is not particularly limited, and examples thereof include water vapor. The gaseous treatment compound may also contain an inert gas. The inert gas is not particularly limited, but it is not preferable to use an inert gas that reacts with the treatment compound to adversely affect the surface modification treatment of the holding area, an inert gas that reacts with the polymer compound to adversely affect the surface modification treatment, or an inert gas that contains impurities that have such adverse effects. Specific examples of inert gases include nitrogen, argon, helium, neon, krypton, and xenon. These may be used alone or in combination of two or more. The purity of the inert gas is not particularly limited, but it is preferable that the content of the impurities that have such adverse effects 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 examples thereof include water. When the treatment compound is water, it can be used as a cleaning treatment for the cell culture device 10, which will be described later. Details of the cleaning treatment will be described later.

[0086] The treatment temperature for the second surface-modifying group introduction treatment is not particularly limited as long as it is equal to or lower than the glass transition point 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. Setting the treatment temperature at −20°C or higher can promote the introduction of the surface-modifying group derived from the treatment compound. On the other hand, setting the treatment temperature at 150°C or lower can suppress excessive increases in defects in the carbon skeleton and / or silicon skeleton that occur during treatment of the surface of the cell culture member 10 with the treatment compound, thereby preventing excessive damage to the carbon skeleton and / or silicon skeleton and a decrease in the mechanical strength of the cell culture member 10. Furthermore, thermal deformation of the cell culture member 10 can be prevented, suppressing a decrease in yield.

[0087] The treatment time (reaction time) for the second surface modifying 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 setting the treatment time to 1 second or longer, the introduction of the surface modifying group derived from the treatment compound can be ensured. On the other hand, by setting the treatment time to 24 hours or shorter, a decrease in treatment efficiency due to a prolonged treatment time can be prevented.

[0088] The pressure conditions for carrying out the second surface-modifying group introduction treatment are not particularly limited, and the treatment can be carried out under normal pressure, elevated pressure, or reduced pressure. From the standpoints of economy and safety, it is preferable to carry out the treatment under normal pressure.

[0089] The reaction vessel for carrying out the second surface modifying group introduction treatment is not particularly limited, and a conventionally known vessel such as a fixed bed or a fluidized bed can be used.

[0090] When the treatment compound is gaseous, the method for contacting the treatment compound with the cell culture member 10 is not particularly limited, and examples thereof include a method in which the treatment compound is contacted in a sealed state under a flow of the treatment compound or in an atmosphere containing at least the treatment compound. The second surface-modifying group introduction treatment may be performed multiple times.

[0091] The second surface modifying group introduction treatment may be performed on at least a portion of any desired region of the adhesive cell retention region. Therefore, the second surface modifying group introduction treatment may be performed on the entire surface of the retention region. When the second surface modifying group introduction treatment is performed on a portion of any desired region (partial surface modifying group introduction treatment), the treatment can be performed by masking regions other than the desired region for the second surface modifying group introduction treatment. The masking material used for masking is not particularly limited, as long as it is heat-resistant to the treatment temperature during the second surface modifying group introduction treatment. Specific examples of the masking material include the aforementioned masking materials used when performing the first surface modifying group introduction treatment.

[0092] In this embodiment, a post-treatment may be performed immediately after the first surface-modifying group introduction treatment and the second surface-modifying group introduction treatment. The post-treatment is a process 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 performed by standing to cool. Alternatively, the cell culture member 10 may be evacuated to a vacuum to replace the gas with an inert gas, and then the pressure may be increased to atmospheric pressure with the inert gas. When the post-treatment is performed immediately after the first surface-modifying group introduction treatment, the second treatment gas can be prevented from adsorbing and remaining on the surface of the cell culture member 10 that has been subjected to the first surface-modifying group introduction treatment. When the post-treatment is performed immediately after the second surface-modifying group introduction treatment, the treatment compound can be prevented from adsorbing and remaining on the surface of the cell culture member 10 that has been subjected to the second surface-modifying group introduction treatment. This prevents the generation of by-products due to decomposition of the second treatment gas or gaseous treatment compound, and prevents problems such as destruction of cultured cells due to the by-products. The inert gas is not particularly limited, and examples thereof include nitrogen gas.

[0093] In this embodiment, a cleaning treatment may be performed after the surface modification treatment, the first surface-modifying group introduction treatment, the second surface-modifying group introduction treatment, and the post-treatment. For example, when the cleaning agent is water, some of the fluorine atoms (fluorine groups) directly chemically bonded to carbon atoms or the like can be reacted with water molecules by cleaning, thereby converting the fluorine groups into -OH groups, -COOH groups, or the like that are directly chemically bonded. As a result, the surface of the cell culture substrate 10 can be further hydrophilized. Furthermore, the first processing gas, the second processing gas, processing compounds, and by-products that are not immobilized on the surface of the cell culture substrate 10 can be removed. The cleaning agent used is not particularly limited, and examples thereof include ethanol, isopropyl alcohol, water (e.g., ultrapure water), toluene, and acetone. The cleaning conditions are not particularly limited, but the cleaning temperature (temperature of the cleaning agent) is usually 0°C to 100°C, and the cleaning time is usually 1 second to 60 minutes.

[0094] It is preferable to carry out a drying treatment after the washing treatment. The drying method is not particularly limited, and examples thereof include natural drying and drying by blowing nitrogen gas or the like. The drying conditions are also not particularly limited, but the drying temperature (when blowing nitrogen gas or the like, the temperature of the nitrogen gas) is usually 0°C to 100°C, and the drying time is within the range of 1 second to 24 hours.

[0095] As described above, the surface modification method of the cell culture component 10 according to this embodiment enables surface modification that improves the adhesiveness of the adherent cells in the holding area by simply contacting the adherent cell holding area with a first treatment gas containing a gas containing fluorine atoms. As a result, it is possible to provide a cell culture component 10 that has excellent cell culture performance and long-term stability. [Example]

[0096] Preferred examples of the present invention are described in detail below. However, the materials and blending amounts described in these examples are not intended to limit the scope of the present invention unless otherwise specified.

[0097] Example 1 First, a polystyrene microplate (manufactured by AGC Technoglass Co., Ltd., trade name: IWAKI Suspension Culture Microplate 24WELL, hereinafter referred to as "microplate") was prepared, and the microplate was placed in a SUS316L chamber (volume 18 L).

[0098] Next, the chamber was evacuated and filled with nitrogen gas, and the temperature was raised at 4°C / min under a nitrogen gas flow (4 L / min) until the ambient temperature in the chamber reached 40°C. The microplate was then subjected to constant temperature treatment for 1 hour.

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

[0100] Next, the microplate after the surface modification treatment was thoroughly washed with ultrapure water, and then dried by spraying nitrogen gas at 25°C. In this way, a microplate subjected to surface modification treatment was produced as the cell culture device according to this example. The temperature of the ultrapure water in the washing treatment was 25°C, and the washing time was 5 minutes. The temperature of the nitrogen gas in the drying treatment was 25°C, and the drying time was 8 hours.

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

[0102] (Comparative Example 2) In this comparative example, a microplate (manufactured by AGC Technoglass Co., Ltd., trade name: IWAKI Microplate for Adherent Culture 24WELL) that had been subjected to a discharge treatment instead of the surface modification treatment of Example 1 was used.

[0103] (Elemental analysis) Elemental analysis was performed on each of the microplates according to Example 1, Comparative Example 1, and Comparative Example 2. The elemental analysis was performed 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 of the microplates according to Example 1, Comparative Example 1, and Comparative Example 2. The water contact angle was measured using a contact angle meter (manufactured by Kyowa Interface Science Co., Ltd., model number: DM-300). The results are shown in Table 1.

[0105] (Measurement of the binding energy of fluorine atoms) The surface of the microplate according to Example 1 (the surface subjected to the fluorination treatment) 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 number: PHIVersaProbeIII, manufactured by ULVAC-PHI, Inc.) was used, and AlKα rays monochromated by a monochromator were used as the irradiated X-rays. The X-ray output was 50 W, the acceleration voltage was 15 kV, the diameter of a single measurement area was approximately 200 μm, and the measurement interval for binding energy was 0.05 eV, and the intensity was measured in the binding energy range of 679 to 699 eV. As a result of the measurement, as shown in FIG. 3, the binding energy of fluorine atoms was detected as a waveform having a peak value at 686.5 eV. FIG. 3 is a graph showing the XPS measurement results for the microplate according to Example 1.

[0107] (Cell culture evaluation) For each of the microplates according to Example 1, Comparative Example 1, and Comparative Example 2, cell culture evaluation was carried out using human embryonic kidney cells (HEK293T cells) and Syrian hamster kidney cells (BHK-21 (C-13) cells), respectively.

[0108] Cell culture evaluation was performed by first culturing 1×10 cells. 4 The culture medium was seeded into each microplate so that cells would be seeded in the well. The culture medium contained 10% fetal bovine serum (FBS).

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

[0110] (Cell culture evaluation in low-nutrient medium) For each of the microplates according to Example 1, Comparative Example 1, and Comparative Example 2, cell culture evaluation was carried out in a low-nutrient medium using Syrian hamster kidney cells (BHK cells).

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

[0112] (Long-term stability evaluation of cell culture performance) Next, for each of the microplates according to Example 1 and Comparative Examples 1 and 2, an evaluation of the long-term stability of cell culture performance was carried out using human embryonic kidney cells (HEK293T cells).

[0113] The long-term stability evaluation was performed by first using a cell count of 1 × 10 4 The culture medium was seeded into each microplate so that cells would be seeded in the well. The culture medium contained 10% fetal bovine serum (FBS).

[0114] The culture medium was then stored at 25°C under atmospheric conditions for 30 days. Human fetal kidney cells were then cultured for 4 days at 37°C in a 5% CO2 atmosphere. After culturing, the medium was removed, the cells were detached, and the viable human fetal kidney cells were counted. The results are shown in Table 1.

[0115] [Table 1]

[0116] (Cell culture evaluation using primary cells) Next, for each of the microplates of Example 1, Comparative Example 1, and Comparative Example 2, cell culture evaluation was carried out using mouse astrocyte cells.

[0117] For cell culture evaluation, the cerebral cortex was first removed from mouse (ICR strain) fetuses, digested with enzymes, and then seeded onto microplates coated with laminin and polylysine. After culturing for 15 days, mouse astrocyte cells were confirmed to have been cultured, and then detached.

[0118] Then, each microplate was filled with 1 × 10 cells. 5 The cells were replated to give 1000 cells / well. After three days, the mouse astrocytes in each microplate were fluorescently stained with calcein, and the state of the mouse astrocytes was observed using an inverted microscope. The results are shown in Figures 4 to 6. Figures 4 to 6 show bright-field images observed under an inverted microscope after culturing mouse astrocytes using the microplates of Example 1, Comparative Example 1, and Comparative Example 2, respectively.

[0119] (result) As a result of elemental analysis by XPS, 14.0 at% of fluorine atoms were detected in the microplate after fluorination treatment in Example 1. It was also revealed that the amount of oxygen atoms was increased compared to the untreated microplate in Comparative Example 1. Furthermore, from the peak positions of XPS, it was also confirmed that -OH groups and -COOH groups were generated as surface modifying groups in the microplate in Example 1. On the other hand, no fluorine atoms were detected in the microplates in Comparative Examples 1 and 2.

[0120] Furthermore, as a result of measuring the water contact angle, the microplate of Example 1 had a smaller contact angle than the microplate of Comparative Example 1, which used an untreated microplate, which revealed that the microplate of Example 1 had improved wettability with water.

[0121] Furthermore, as a result of cell culture evaluation, the number of viable cells in the microplate of Example 1 was 31.7 × 10 4 pcs, and 1.6 × 10 in the untreated microplate of Comparative Example 1 4 This confirmed that the surface-modified microplate of Example 1 had superior cell culture performance compared to the untreated microplate of Comparative Example 1.

[0122] In particular, in the cell culture evaluation under a low-nutrient medium, the number of viable cells in the fluorinated microplate of Example 1 was 3.2 × 10 4 It was confirmed that the cell culture performance was superior to that of the microplates of Comparative Examples 1 and 2.

[0123] Furthermore, regarding the long-term stability of cell culture performance, the number of viable cells in the fluorinated microplate of Example 1 was 31.5 × 10 4 It was confirmed that the cell culture performance was maintained at the same level as before the 30-day storage in the atmosphere. On the other hand, the number of viable cells in the plasma-treated microplate of Example 1 was 23.4 × 10 4 The number of viable cells before 30 days of storage in air was 31.2 × 10 4 This significantly decreased the number of cells, confirming that the cell culture performance decreased over time.

[0124] Furthermore, in a cell culture evaluation using primary cells, it was confirmed that mouse astrocytes adhered to the surface of the fluorinated microplate of Example 1 and their neurites extended, as shown in Figure 4. On the other hand, in the microplate of Comparative Example 1, which was not subjected to surface modification, mouse astrocytes did not adhere to the surface, as shown in Figure 5. Furthermore, in the microplate of Comparative Example 2, which was subjected to plasma treatment, mouse astrocytes adhered to the surface, but their neurite extension was insufficient compared to Example 1, as shown in Figure 6. These results demonstrate that the microplate of Example 1 has superior cell culture performance compared to the microplates of Comparative Examples 1 and 2, even in a cell culture evaluation using primary cells.

[0125] From the above results, it was revealed that the fluorination-treated microplate of Example 1 had better cell culture performance than the untreated microplate of Comparative Example 1. It was also revealed that the surface-modified microplate of Example 1 maintained excellent cell culture performance even after 30 days of atmospheric storage, compared to the plasma-treated microplate of Comparative Example 2. [Explanation of symbols]

[0126] 10 Cell culture materials

Claims

1. A method for modifying the surface of a cell culture substrate, the retention area of ​​which is made of a polymer compound, comprising: a step of contacting at least a portion of the holding region with a first treatment gas containing a gas containing fluorine atoms, a gas containing oxygen atoms, and an inert gas as an optional component, without performing plasma treatment, thereby forming a surface-modified region in which the fluorine atoms are directly chemically bonded to a portion of the carbon atoms and / or silicon atoms constituting the polymer compound, and surface-modifying groups are directly chemically bonded to a portion of the other carbon atoms and / or other silicon atoms constituting the polymer compound; and bringing the fluorine atoms directly chemically bonded to a portion of the carbon atoms and / or silicon atoms and / or the surface modifying groups directly chemically bonded to a portion of the other carbon atoms and / or other silicon atoms into contact with a treating compound that reacts with the fluorine atoms and / or the surface modifying groups, thereby substituting the fluorine atoms and / or the surface modifying groups with the surface modifying groups derived from the treating compound, 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 surface modifying group is at least one selected from the group consisting of an —OR 1 group; a —COOR 2 group; a —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 nitro group; a nitroso group; a phosphate group; a sulfonyl group; a thionyl group; and a halogen atom other than a fluorine atom; R 1 and R 2 are each independently any one of 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; The method for modifying the surface of a cell culture device, wherein 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.

2. A method for modifying the surface of a cell culture substrate, the retention area of ​​which is made of a polymer compound, comprising: a step of contacting at least a portion of the holding region with a first treatment gas consisting only of a gas containing fluorine atoms and an inert gas as an optional component, without performing plasma treatment, to form a surface-modified region in which the fluorine atoms are directly chemically bonded to a portion of the carbon atoms and / or silicon atoms constituting the polymer compound; and contacting, without plasma treatment, at least a portion of the holding region that has been contacted with the first processing gas, with a second processing gas containing a gas containing other oxygen atoms, thereby directly chemically bonding surface modifying groups to a portion of other carbon atoms and / or other silicon atoms that constitute the polymer compound, 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 surface modifying group is —OR １ Group;-COOR ２ Group;-COR ３ a silyl 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 nitro group; a nitroso group; a phosphate group; a sulfonyl group; a thionyl group; and a halogen atom other than a fluorine atom, The R １ and the R ２ are each independently any one of 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, The R ３ is either 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.

3. 3. The surface modification method of claim 1, wherein the peak value of the binding energy of the fluorine atoms in the surface modification region measured by X-ray photoelectron spectroscopy is in the range of 680 eV to 690 eV.

4. by contacting the fluorine atoms directly chemically bonded to a portion of the carbon atoms and / or silicon atoms and / or the surface modifying groups directly chemically bonded to a portion of the other carbon atoms and / or other silicon atoms with a treatment compound that reacts with the fluorine atoms and / or the surface modifying groups, The method for modifying the surface of a cell culture device according to claim 2 , further comprising the step of substituting the fluorine atoms and / or the surface modifying groups with the surface modifying groups derived from the treating compound.

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