Cell culture vessel and method for producing the same

Abstract

To provide a cell culture vessel that can inhibit movement of cells toward a side wall.SOLUTION: A cell culture vessel comprising a container part for accommodating cells and a culture medium, the container part including a container body member and a bottom substrate, wherein a surface-modifying agent layer having cell adhesiveness is formed only on the bottom substrate, and the bottom substrate is joined to the container body member such that the surface of the surface-modifying agent layer faces a space for accommodating the cells and the culture medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture vessel and a method for manufacturing the cell culture vessel. [Background technology]

[0002] Cell culture vessels, such as well plates used in the field of life science, include a vessel portion for containing cells and culture medium, and cells are cultured in the internal space of the vessel portion. The bottom surface of the vessel portion of such cell culture vessels may be formed with a layer containing a surface modifier with cell adhesive properties (hereinafter, sometimes referred to as a "surface modifier layer"). By forming the surface modifier layer, cells can adhere well to the bottom surface of the vessel portion via the surface modifier layer, thereby improving the effects of cell proliferation and survival.

[0003] Conventionally, cell culture vessels with a surface modifier layer have been produced by dispensing a solution containing a surface modifier (surface modifier solution) into the vessel portion of the cell culture vessel using a dispenser or the like, leaving it to stand for a certain period of time to allow the surface modifier layer to adhere to the bottom surface of the vessel portion, and then removing any unimmobilized surface modifier residues by washing (Patent Document 1).

[0004] As a method for applying a liquid to the bottom surface of a cell culture vessel, in addition to the above-mentioned method using a dispenser, a method using an inkjet or the like can also be mentioned (Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-106531 [Patent Document 2] Japanese Patent Publication No. 2020-178612 Summary of the Invention [Problem to be solved by the invention]

[0006] However, even when the above-mentioned conventional method was used to apply a surface modifier solution and prepare a cell culture vessel, it was difficult to sufficiently suppress cell migration to the side wall because it was not possible to prevent the surface modifier solution from adhering to the side wall of the vessel and the formation of a surface modifier layer.

[0007] Therefore, an object of the present invention is to provide a cell culture vessel that can suppress cell migration to the side wall. Another object of the present invention is to provide a method for producing a cell culture vessel that can suppress cell migration to the side wall. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems, and have found that the use of a cell culture vessel in which a surface modifier layer having cell adhesive properties is formed only on the bottom substrate can suppress cell migration to the side wall, thereby completing the present invention.

[0009] That is, the present invention aims to advantageously solve the above-mentioned problems, and provides the following cell culture vessels [1] to [4] and the following methods for manufacturing cell culture vessels [5] to [8].

[0010] [1] A cell culture vessel having a vessel portion for containing cells and culture medium, the vessel portion comprising a vessel body member and a bottom substrate, a surface modifier layer having cell adhesive properties formed only on the bottom substrate, and the bottom substrate being joined to the vessel body member so that the surface of the surface modifier layer faces the space for containing the cells and culture medium. By using a cell culture vessel in which a surface modifier layer is formed only on the bottom substrate and the bottom substrate and the container body member are joined so that the surface of the surface modifier layer faces the space for containing cells and culture medium, migration of cells toward the side walls can be sufficiently suppressed.

[0011] [2] The cell culture vessel according to [1] above, wherein the bottom substrate is composed of one or more selected from the group consisting of cycloolefin polymer, polyester, polypropylene, and polystyrene. If the bottom base material of the vessel portion of the cell culture vessel is made of the above resin, it is possible to produce a cell culture vessel suitable for optical cell observation such as fluorescence observation with low autofluorescence.

[0012] [3] The cell culture vessel according to [1] or [2] above, wherein the bottom substrate has not been subjected to a surface treatment. If the bottom substrate is not surface treated, the surface modifier can be better fixed to the bottom substrate, improving the uniformity of the surface modifier layer.

[0013] [4] The cell culture vessel according to any one of [1] to [3] above, wherein the surface modifier layer contains an oligopeptide.

[0014] [5] A method for manufacturing a cell culture vessel having a vessel portion for containing cells and culture medium, the vessel portion having a vessel body member and a bottom substrate, the method including the steps of: applying a surface modifier solution onto the bottom substrate to obtain a bottom substrate with a surface modifier layer formed on the bottom substrate; and forming the vessel portion by insert injection molding using the bottom substrate with the surface modifier layer. By going through the two steps described above, a container portion having a surface modifier layer only on the bottom surface can be easily manufactured, and this container can be used to efficiently manufacture a cell culture container that can suppress cell migration to the side walls.

[0015] [6] A method for manufacturing a cell culture vessel according to [5] above, which does not include both a step of washing the bottom substrate with the surface modifier layer and a step of washing the vessel part. By not including the above steps, the manufacturing process of the cell culture vessel can be simplified and productivity can be improved.

[0016] [7] The method for producing a cell culture vessel according to [5] or [6] above, wherein the contact angle between the surface modifier solution and the bottom substrate is 0° or more and 40° or less. If the contact angle between the surface modifier solution and the bottom substrate is within the above range, the uniformity of the surface modifier layer formed can be improved. In the present invention, the "contact angle" between the surface modifier solution and the bottom substrate can be measured using the method described in the examples of this specification.

[0017] [8] The method for producing a cell culture vessel according to any one of [5] to [7] above, wherein the thickness of the coating film when the surface modifier solution is applied onto the bottom substrate is 150 μm or less. By setting the thickness of the applied film when the surface modifier solution is applied to the bottom substrate to the above value or less, the uniformity of the formed surface modifier layer can be improved even without including a cleaning step. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide a cell culture vessel capable of suppressing cell migration to the side wall. Furthermore, the present invention can provide a method for producing a cell culture vessel that can suppress cell migration to the side wall. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a diagram showing a schematic configuration of an example of a microwell plate, which is a cell culture vessel according to the present invention. [Figure 2] 1 is a diagram showing an example of a vessel part of a cell culture vessel according to the present invention. FIG. [Figure 3] FIG. 10 is a diagram showing another example of the vessel part of the cell culture vessel according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] Hereinafter, embodiments of the present invention will be described in detail. Here, the cell culture vessel of the present invention is not particularly limited as long as it is intended for culturing cells. Specific examples of the cell culture vessel of the present invention include well plates, petri dishes, dishes, flasks, etc. Furthermore, the cell culture vessel of the present invention can be manufactured, for example, using the manufacturing method of the cell culture vessel of the present invention.

[0021] (Cell culture container) The cell culture vessel of the present invention comprises a vessel portion capable of containing cells and culture medium. The cell culture vessel of the present invention may also comprise components other than the vessel portion. For example, when the cell culture vessel of the present invention is a microwell plate, the microwell plate may comprise a lid or the like in addition to wells as vessel portions, as shown in Figures 1 and 2. The cell culture vessel may also comprise only one vessel portion, or two or more vessel portions. Here, the cell culture vessel of the present invention is characterized in that a surface modifier layer is formed only on the bottom substrate of the vessel portion, and the bottom substrate is joined to the vessel body member so that the surface of the surface modifier layer faces the space for accommodating cells and culture medium.

[0022] An example of the structure of the cell culture vessel of the present invention will be further described below with reference to Figures 1 to 3. In Figures 1 to 3, the structure will be described using a microwell plate as an example of the cell culture vessel, but the cell culture vessel of the present invention is not limited to this.

[0023] FIG. 1 shows an example of a microwell plate as a cell culture vessel of the present invention. The microwell plate 1 shown in FIG. 1 includes multiple wells 10 as vessel portions and a lid 20. FIGS. 2 and 3 show enlarged views of the wells 10 of the microwell plate. The wells 10 have well sidewalls 11 as vessel body members and a well bottom surface 12 as a bottom substrate, and are capable of containing cells 30 and culture medium 40 in the space defined by the well sidewalls 11 and well bottom surface 12. A surface modifier layer 50 is formed on the well bottom surface 12, allowing cells 30 to adhere to the well bottom surface 12 via the surface modifier layer 50. The well bottom surface 12 is bonded to the well sidewalls 11 so that the surface of the surface modifier layer 50 faces the space for containing the cells 30 and culture medium 40.

[0024] The joining method between the well side wall 11 as the container body member and the well bottom surface 12 as the bottom substrate will now be described. In the embodiment shown in Fig. 2, the well side wall 11 and the well bottom surface 12 are joined so that the well side wall 11 is in direct contact with the well bottom surface 12. On the other hand, in the embodiment shown in Fig. 3, the well side wall 11 is joined to the well bottom surface 12 via a surface modifier layer 50 formed on the well bottom surface 12. In other words, in the embodiment of Fig. 3, there is no direct contact between the well side wall 11 and the well bottom surface 12 because the surface modifier layer 50 is interposed between them. Both of the above-described embodiments shown in FIG. 2 and FIG. 3 are included in the cell culture vessel of the present invention.

[0025] <Container part> The vessel portion of the cell culture vessel is a member having a space capable of accommodating cells and culture medium. The vessel portion includes at least a vessel body member and a bottom substrate. The vessel portion may further include members other than the vessel body member and the bottom substrate.

[0026] <<Bottom base material>> The bottom substrate is a member that constitutes the bottom of the container portion, and together with the container body member forms a space capable of accommodating cells and culture medium.

[0027] The shape of the bottom substrate is not particularly limited, and can be, for example, a circle, a triangle, a square, a rectangle, etc. Furthermore, when the cell culture vessel is viewed from the side, the bottom substrate may be flat or may be curved downward in a U-shape, but is preferably flat. In the cell culture vessel of the present invention, when the bottom substrate is curved downward in a U-shape, the bottom substrate refers to the portion located below the end point of the straight line that constitutes the side wall.

[0028] The material of the bottom substrate is not particularly limited, and for example, a thermoplastic resin that is solid at room temperature and normal pressure can be used. Examples of thermoplastic resins that are solid at room temperature and pressure include cycloolefin polymers; acrylic resins; silicone resins; fluororesins; polyethylene; polyester; polypropylene; ethylene-propylene copolymers; polymethylpentene; polyvinyl chloride; polyvinylidene chloride; polyvinyl acetate; ethylene-vinyl acetate copolymers; polyvinyl alcohol; polyacetal; polyethylene terephthalate; polybutylene terephthalate; polyethylene naphthalate; polystyrene; polyacrylonitrile; styrene-acrylonitrile copolymers; acrylonitrile-butadiene-styrene copolymers (ABS resins); styrene-butadiene block copolymers or hydrogenated products thereof; styrene-isoprene block copolymers or hydrogenated products thereof; polyphenylene ethers; modified polyphenylene ethers; aliphatic polyamides; aromatic polyamides; polyamideimides; polycarbonates; polyphenylene sulfides; polysulfones; polyethersulfones; polyethernitriles; polyetherketones; polyketones; polyurethanes; liquid crystal polymers; ionomers; and the like. These resins may be used alone or in combination of two or more in any ratio. In this specification, "normal temperature" refers to 23°C, and "normal pressure" refers to 1 atm (absolute pressure).

[0029] Of the above resins, from the viewpoint of producing a cell culture vessel suitable for optical cell observation such as fluorescence observation with low autofluorescence, it is preferable to use cycloolefin polymer, polyester, polystyrene, or polypropylene, it is more preferable to use cycloolefin polymer or polystyrene, and it is even more preferable to use cycloolefin polymer.

[0030] The cycloolefin polymer, which is a suitable material for the bottom substrate, will be described in detail below.

[0031] [Cycloolefin polymer] Here, the cycloolefin polymer is a polymer having an alicyclic structure in one or both of the main chain and the side chain. Examples thereof include norbornene-based polymers, monocyclic cyclic olefin-based polymers, cyclic conjugated diene-based polymers, vinyl alicyclic hydrocarbon-based polymers, and hydrogenated versions thereof. Among these, from the viewpoints of transparency and moldability, norbornene-based polymers are preferred, and norbornene-based polymers having no polar groups in the side chains are more preferred.

[0032] Examples of norbornene-based polymers include ring-opening polymers of monomers having a norbornene structure, ring-opening polymers of a monomer having a norbornene structure and an arbitrary monomer, or hydrogenated products thereof; addition polymers of a monomer having a norbornene structure, addition polymers of a monomer having a norbornene structure and an arbitrary monomer, or hydrogenated products thereof; and the like.

[0033] Here, the norbornene polymer is a polymer containing monomer units having a norbornene skeleton in an amount of 50% by mass or more, preferably 60% by mass or more, based on the total monomer units constituting the norbornene polymer. More specifically, norbornene polymers are obtained by polymerizing norbornene monomers, which are monomers having a norbornene skeleton, and are roughly classified into those obtained by ring-opening polymerization and those obtained by addition polymerization.

[0034] Examples of materials obtainable by ring-opening polymerization include ring-opening polymers of norbornene-based monomers, ring-opening polymers of norbornene-based monomers and other monomers that are ring-opening copolymerizable with the norbornene-based monomers, and hydrogenated products of these. Examples of the polymers obtained by addition polymerization include addition polymers of norbornene-based monomers and addition polymers of norbornene-based monomers and other monomers copolymerizable therewith. The norbornene polymers can be used either alone or in combination of two or more. Among these, a hydrogenated ring-opening polymer of a norbornene-based monomer (sometimes referred to as a "hydrogenated norbornene-based ring-opening polymer") is preferred because the effects of the present invention can be more easily obtained.

[0035] Norbornene monomers that can be used to synthesize norbornene polymers include bicyclo[2.2.1]hept-2-ene (common name: norbornene), 5-methyl-bicyclo[2.2.1]hept-2-ene, 5,5-dimethyl-bicyclo[2.2.1]hept-2-ene, 5-ethyl-bicyclo[2.2.1]hept-2-ene, 5-ethylidene-bicyclo[2.2.1]hept-2-ene, and 5-methyl-bicyclo[2.2.1]hept-2-ene. ]hept-2-ene, 5-vinyl-bicyclo[2.2.1]hept-2-ene, 5-propenylbicyclo[2.2.1]hept-2-ene, 5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, 5-cyanobicyclo[2.2.1]hept-2-ene, 5-methyl-5-methoxycarbonyl-bicyclo[2.2.1]hept-2-ene, and other bicyclic monomers; Tricyclo[4.3.0 1,6 .1 2,5 ] Tricyclic monomers such as deca-3,7-diene (trivial name: dicyclopentadiene), 2-methyldicyclopentadiene, 2,3-dimethyldicyclopentadiene, and 2,3-dihydroxydicyclopentadiene; Tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene (tetracyclododecene), tetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyltetracyclo[4.4.0.12,5 .1 7,10 ]-3-dodecene, 8-ethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethylidenetetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8,9-dimethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethyl-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-ethylidene-9-methyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 8-methyl-8-carboxymethyltetracyclo[4.4.0.1 2,5 .1 7,10 ]-3-dodecene, 7,8-benzotricyclo[4.3.0.1 2,5 ] tetracyclic monomers such as dec-3-ene (trivial name: methanotetrahydrofluorene; also known as 1,4-methano-1,4,4a,9a-tetrahydrofluorene), 1,4-methano-8-methyl-1,4,4a,9a-tetrahydrofluorene, 1,4-methano-8-chloro-1,4,4a,9a-tetrahydrofluorene, and 1,4-methano-8-bromo-1,4,4a,9a-tetrahydrofluorene; and the like. These norbornene-based monomers may have one or more substituents, such as alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups.

[0036] Other monomers that can be ring-opening copolymerized with norbornene-based monomers include monocyclic cycloolefin-based monomers such as cyclohexene, cycloheptene, cyclooctene, 1,4-cyclohexadiene, 1,5-cyclooctadiene, 1,5-cyclodecadiene, 1,5,9-cyclododecatriene, and 1,5,9,13-cyclohexadecatetraene.

[0037] Other monomers that can be addition copolymerized with norbornene monomers include α-olefin monomers having 2 to 20 carbon atoms, such as ethylene, propylene, 1-butene, 1-pentene, and 1-hexene; cyclobutene, cyclopentene, cyclohexene, cyclooctene, and tetracyclo[9.2.1.0]. 2,10 .0 3,8 ] Cycloolefin-based monomers such as tetradeca-3,5,7,12-tetraene (also known as 3a,5,6,7a-tetrahydro-4,7-methano-1H-indene); non-conjugated diene-based monomers such as 1,4-hexadiene, 4-methyl-1,4-hexadiene, 5-methyl-1,4-hexadiene, and 1,7-octadiene; and the like. Among these, as the other monomer capable of addition copolymerization with the norbornene-based monomer, an α-olefin-based monomer is preferred, and ethylene is more preferred. These other monomers may have one or more substituents, such as alkyl groups, alkylene groups, aryl groups, silyl groups, alkoxycarbonyl groups, and alkylidene groups.

[0038] A ring-opening polymer of a norbornene-based monomer, or a ring-opening polymer of a norbornene-based monomer and another monomer capable of ring-opening copolymerization therewith, can be obtained by polymerizing the monomer components in the presence of a known ring-opening polymerization catalyst. As the ring-opening polymerization catalyst, for example, a catalyst composed of a halide of a metal such as ruthenium or osmium, a nitrate or an acetylacetone compound, and a reducing agent, or a catalyst composed of a halide or an acetylacetone compound of a metal such as titanium, zirconium, tungsten, or molybdenum, and an organoaluminum compound can be used. The hydrogenated ring-opening polymer of a norbornene-based monomer can usually be obtained by adding a known hydrogenation catalyst containing a transition metal such as nickel or palladium to a polymerization solution of the ring-opening polymer and hydrogenating the carbon-carbon unsaturated bonds.

[0039] An addition polymer of a norbornene-based monomer or an addition polymer of a norbornene-based monomer and another monomer copolymerizable therewith can be obtained by polymerizing the monomer components in the presence of a known addition polymerization catalyst. As the addition polymerization catalyst, for example, a catalyst comprising a titanium, zirconium or vanadium compound and an organoaluminum compound can be used.

[0040] Although there are no particular limitations on the molecular weight of the norbornene polymer, the weight-average molecular weight (Mw) of the norbornene polymer, measured by gel permeation chromatography (GPC) of a cyclohexane solution (or a toluene solution if the polymer is not soluble) in terms of polyisoprene, is typically 1,000 or more, preferably 1,000 to 500,000, more preferably 8,000 to 350,000, and particularly preferably 10,000 to 200,000. A weight-average molecular weight within this range provides a high level of balance between mechanical strength and moldability, making it suitable. The number-average molecular weight (Mn), measured by gel permeation chromatography (GPC) of a cyclohexane solution (or a toluene solution if the polymer is not soluble) in terms of polyisoprene, is typically 5,000 to 100,000, preferably 6,000 to 70,000, and more preferably 7,000 to 60,000. The molecular weight distribution (Mw / Mn) is not particularly limited, but when it is usually 4.0 or less, preferably 3.0 or less, more preferably 2.5 or less, a good balance between mechanical strength and moldability is obtained.

[0041] The glass transition temperature of the norbornene polymer preferably used as the material constituting the bottom substrate may be selected appropriately depending on the intended use, but is preferably 150°C or higher, more preferably 155°C or higher, and even more preferably 160°C or higher. A glass transition temperature above the lower limit above allows for the production of a microwell plate with good flatness and enhances the heat resistance of the microwell plate. The upper limit of the glass transition temperature of the bottom substrate is not particularly limited, and can be, for example, 200°C or lower, or 180°C or lower. The glass transition temperature of the norbornene polymer is measured in accordance with JIS K 7121.

[0042] The norbornene polymers can be used either alone or in combination of two or more. In addition to the resin such as the norbornene-based polymer described above, compounding agents that are usually used in thermoplastic resin materials, such as soft polymers, antioxidants, ultraviolet absorbers, light stabilizers, near-infrared absorbers, release agents, colorants such as dyes and pigments, plasticizers, antistatic agents, and fluorescent brightening agents, may be added in their usual amounts.

[0043] The method of mixing the resin such as the norbornene polymer with the compounding agent is not particularly limited as long as the compounding agent is sufficiently dispersed in the resin. The order of compounding is also not particularly limited. Examples of the compounding method include a method of kneading the resin in a molten state using a mixer, a single-screw kneader, a twin-screw kneader, a roll, a Brabender, an extruder, or the like; a method of dissolving and dispersing the resin in a suitable solvent, and then removing the solvent by a coagulation method, a casting method, or a direct drying method; and the like. When a twin-screw kneader is used, after kneading, the material is usually extruded in a molten state into a rod shape, cut to an appropriate length with a strand cutter, and pelletized for use.

[0044] [Surface treatment] The bottom substrate may also be surface-treated. Surface treatment may be applied to both sides of the bottom substrate, or only one side of the bottom substrate. The type of surface treatment is not particularly limited and may be appropriately selected from known surface treatment methods. Examples of such known surface treatment methods include heat treatment, plasma treatment, vacuum ultraviolet treatment, corona treatment, ozone treatment, flame burning, and treatment with a surface treatment agent. Examples of surface treatment agents that can be used include polyester, (meth)acrylic acid ester, polyurethane, polyethyleneimine, silane coupling agent, and perfluorooctane sulfonic acid. From the perspective of favorably fixing the surface modifier to the bottom substrate and improving the uniformity of the surface modifier layer, it is preferable that at least the surface on which the surface modifier layer is formed is not surface-treated.

[0045] <<Container body parts>> The container body member is a member that, together with the bottom base material, forms a space capable of accommodating cells and a culture medium.

[0046] The material of the container body member is not particularly limited, and for example, the thermoplastic resin that is solid at room temperature and normal pressure described in the section on "bottom substrate" can be used. Among these, from the viewpoint of producing a cell culture vessel suitable for optical cell observation such as fluorescence observation with low autofluorescence, cycloolefin polymer, polyester, polystyrene, and polypropylene are preferred as the material of the container body member, with cycloolefin polymer and polystyrene being more preferred, and cycloolefin polymer being even more preferred.

[0047] The glass transition temperature of the norbornene-based polymer preferably used as the material constituting the container body member is not particularly limited, but is preferably 126°C or higher, more preferably 130°C or higher, and is preferably 150°C or lower, more preferably 140°C or lower. The glass transition temperature of the resin constituting the container body member is preferably lower than the glass transition temperature of the resin constituting the bottom substrate. Specifically, the glass transition temperature (Tg A) and the glass transition temperature (Tg B ) and (Tg B -Tg A ) is preferably 20°C or higher, more preferably 22°C or higher, even more preferably 24°C or higher, and particularly preferably 26°C or higher, and is preferably 50°C or lower, more preferably 40°C or lower, and even more preferably 30°C or lower. B -Tg A Within the above range, it is possible to produce a microwell plate with good flatness and to improve the heat resistance of the microwell plate.

[0048] <Surface modifier layer> The surface modifier layer is a layer having cell adhesive properties, and has the function of favorably adhering the cells being cultured to the bottom substrate of the cell culture vessel. Here, the surface modifier layer typically contains at least a surface modifier having cell adhesive properties. The surface modifier layer may optionally further contain a solvent. That is, the surface modifier layer may be a coating (liquid film) of a surface modifier solution, or a layer made of a dried product of the surface modifier solution. The surface modifier layer may optionally further contain components other than the surface modifier and the solvent.

[0049] The surface modifier is not particularly limited as long as it has cell adhesive properties, and examples thereof include biological constituents and stimuli-responsive polymers.

[0050] Specific examples of biological constituents include proteins such as collagen, fibronectin, and laminin; and oligopeptides. Examples of the stimuli-responsive polymer that can be used include temperature-responsive polymers, pH-responsive polymers, ion-responsive polymers, photoresponsive polymers, etc. Examples of the temperature-responsive polymer include (meth)acrylic polymers such as poly-Nn-isopropylacrylamide, poly-Nn-propylacrylamide, poly-Nn-propylmethacrylamide, and poly-N-ethoxyethylacrylamide.

[0051] Among the above, biological constituents are preferred as surface modifiers, and oligopeptides are more preferred. The number of amino acid residues in the oligopeptide is, for example, 2 to 50. The oligopeptide may have a first amino acid sequence that specifically binds to a biological substrate, such as a target protein or peptide, and a second amino acid sequence that specifically binds to a non-biological substrate, such as a cell culture vessel, thereby mediating the interaction between cells and the surface modifier layer. If necessary, the oligopeptide may further include a linker sequence between the first and second amino acid sequences. The surface modifier may be used alone or in combination of two or more kinds in any ratio.

[0052] <cell> The cells contained in the vessel portion of the cell culture vessel are not particularly limited, and examples of the cells include muscle cells, hepatocytes, spinal ganglion cells, embryonic lung cells, Schwann cells, epithelial cells (endothelial cells), kidney cells, and nerve cells. The cells may be used singly or in combination of two or more types in any ratio.

[0053] <Culture medium> The medium contained in the vessel portion of the cell culture vessel is not particularly limited, and can be appropriately selected from known cell culture media depending on the type of cells, etc. The medium can contain known additives such as antibiotics and amino acids, as needed.

[0054] The cell culture vessel of the present invention is not particularly limited, but can be suitably produced, for example, by the method for producing a cell culture vessel of the present invention.

[0055] (Method of manufacturing cell culture vessel) The method for producing a cell culture vessel of the present invention includes at least a step of applying a surface modifier solution to a bottom substrate to obtain a bottom substrate with a surface modifier layer formed on the bottom substrate (surface modifier layer forming step), and a step of forming a vessel part by insert injection molding using the bottom substrate with a surface modifier layer obtained in the surface modifier layer forming step (insert injection molding step). Note that the method for producing a cell culture vessel of the present invention may further include steps other than those described above.

[0056] Furthermore, the manufacturing method of the cell culture vessel of the present invention includes a surface modifier layer formation process and an insert injection molding process, so that the surface modifier layer can be formed only on the bottom substrate, thereby sufficiently suppressing cell migration toward the side walls.

[0057] Furthermore, the method for manufacturing a cell culture vessel of the present invention preferably does not include either a step of washing the bottom substrate with a surface modifier layer obtained in the surface modifier layer formation step or a step of washing the vessel part obtained in the insert injection molding step. According to the method for manufacturing a cell culture vessel of the present invention, all of the surface modifier in the surface modifier solution can be fixed on the bottom substrate, so no residue is generated and there is no need to remove the residue by washing. This simplifies the manufacturing process of the cell culture vessel and improves productivity.

[0058] <Surface modifier layer forming process> In the surface modifier layer forming step, a surface modifier solution is applied onto the bottom substrate to obtain a bottom substrate with a surface modifier layer formed on the bottom substrate.

[0059] <<Bottom base material>> Specific examples and preferred examples of the bottom substrate are the same as the specific examples and preferred examples of the bottom substrate in the cell culture vessel of the present invention described above.

[0060] <<Surface modifier solution>> The surface modifier solution contains a surface modifier and a solvent. The surface modifier solution may optionally contain further components other than the surface modifier and the solvent.

[0061] [Surface modifier] Specific examples and preferred examples of the surface modifier are the same as the specific examples and preferred examples of the surface modifier in the cell culture vessel of the present invention described above.

[0062] [solvent] The solvent contained in the surface modifier solution preferably contains at least one of water and alcohol, and more preferably contains both water and alcohol. The solvent contained in the surface modifier solution may be a mixed solvent containing water and an organic solvent other than alcohol (for example, acetonitrile).

[0063] Examples of alcohols that can be suitably used as a solvent include 1-propanol, 2-propanol (also called "isopropyl alcohol"), 2-methyl-2-propanol, 1-butanol, 2-butanol, 2-methyl-2-butanol, etc. Among these, 1-propanol is preferred.

[0064] When the solvent contained in the surface modifier solution is a mixed solvent containing both water and alcohol, the mass ratio of water to alcohol (water / alcohol) can be appropriately set depending on the type of alcohol used, etc. From the viewpoint of improving the uniformity of the surface modifier layer to be formed, the mass ratio of water to alcohol (water / alcohol) is preferably 10 / 90 or more, more preferably 20 / 80 or more, and is preferably 90 / 10 or less, and more preferably 80 / 20 or less.

[0065] The SP value of the solvent contained in the surface modifier solution is preferably 12 or more, more preferably 14 or more, and preferably 23 or less, more preferably 20 or less, and even more preferably 18 or less. If the SP value of the solvent is above the lower limit, the surface modifier can be sufficiently dissolved, and the uniformity of the surface modifier layer formed can be improved. On the other hand, if the SP value of the solvent is below the upper limit, the uniformity of the surface modifier layer formed can be improved. In the present invention, the SP value refers to the solubility parameter. The SP value of a solvent can be calculated using HSPiP (Hassen Solubility Parameters in Practice) software (version 5.0.04). This software calculates the Hildebrand SP value based on the Hansen SP value. The relationship between these values ​​is as follows: Hildebrand SP 2 =tot HSP 2 =δD 2 +δP 2 +δH 2 Here, "tot HSP" refers to the total Hansen Solubility Parameters, "δD" refers to the dispersion term and the van der Waals force, "δP" refers to the polar term and the dipole moment force, and "δH" refers to the hydrogen bond term and the force possessed by water, alcohol, etc.

[0066] The surface modifier solution is not particularly limited, and can be prepared, for example, by dissolving the above-mentioned surface modifier in a solvent.

[0067] The method of applying the surface modifier solution to the surface of the bottom substrate is not particularly limited, and any known application method can be used. Examples of such application methods include bar coater method, comma coater method, dipping method, roll coating method, gravure coating method, knife coating method, air knife coating method, roll knife coating method, die coating method, screen printing method, spray coating method, gravure offset method, and inkjet method. Among these, the bar coater method and comma coater method are preferably used.

[0068] Here, examples of a method for applying a surface modifier solution onto the bottom substrate and forming a surface modifier layer on the bottom substrate include the following methods (1) and (2). (1) A method of applying a surface modifier solution to the entire surface of the bottom substrate to form a surface modifier layer on the entire surface of the bottom substrate (full surface application method); and (2) A method of applying a surface modifier solution to only a portion of the surface of the bottom substrate to form a surface modifier layer only on a portion of the surface of the bottom substrate (spot application method). When manufacturing a container part in which a surface modifier layer is interposed between the container body member (well side wall) and the bottom substrate (well bottom surface), as shown in Figure 3, the full surface coating method (1) above is preferably used. On the other hand, the spot application method (2) above is preferably used when manufacturing a container part in which the container body member (well sidewall) and the bottom substrate (well bottom) are in direct contact, as shown in Figure 2. For example, the container part of Figure 2 can be manufactured by not applying the surface modifier solution to the surface portion of the bottom substrate that will be joined to the container body member in the insert injection molding process described below.

[0069] <<Contact angle>> Here, the contact angle between the bottom substrate and the surface modifier solution is preferably 0° or more, more preferably 5° or more, even more preferably 10° or more, preferably 40° or less, more preferably 38° or less, even more preferably 36° or less, and particularly preferably 34° or less. If the contact angle between the bottom substrate and the surface modifier solution is within the above range, the uniformity of the surface modifier layer can be improved. The contact angle between the bottom substrate and the surface modifier solution can be adjusted, for example, by changing the type of solvent in the surface modifier solution, the material of the bottom substrate, the presence or absence and type of surface treatment of the bottom substrate, etc.

[0070] <<Coating film thickness>> Furthermore, when the surface modifier solution is applied to the bottom substrate, the coating thickness is preferably 1 μm or more, more preferably 5 μm or more, even more preferably 10 μm or more, and preferably 150 μm or less, more preferably 120 μm or less, and even more preferably 100 μm or less. If the coating thickness is 1 μm or more, the cell adhesiveness of the surface modifier layer can be ensured well. On the other hand, if the coating thickness is 150 μm or less, the uniformity of the surface modifier layer formed can be improved.

[0071] The amount of the surface modifier solution applied to the bottom substrate was 0.5 ng / mm 2 It is preferable that the concentration is 0.8 ng / mm or more. 2 More preferably, it is 1.0 ng / mm 2 More preferably, it is 25 ng / mm 2 Preferably, it is 22 ng / mm 2 More preferably, it is 20 ng / mm 2 It is more preferable that the coating amount is equal to or greater than the lower limit. If the coating amount is equal to or greater than the upper limit, the cell adhesiveness of the surface modifier layer can be ensured satisfactorily. On the other hand, if the coating amount is equal to or less than the upper limit, the uniformity of the surface modifier layer formed can be improved.

[0072] <Insert injection molding process> In the insert injection molding step, the container portion is formed by insert injection molding using the bottom substrate with the surface modifier layer obtained in the surface modifier layer forming step.

[0073] <<Insert injection molding>> Insert injection molding can be performed, for example, by the following procedure. First, the bottom substrate with a surface modifier layer obtained in the surface modifier layer formation step is placed in an injection molding mold, and the mold is clamped. Next, molten resin is filled into the mold. Due to the heat of the molten resin, the bottom substrate with a surface modifier layer previously placed in the mold and the molten resin are compatible with each other and welded together to form a single unit. The mold is then cooled to solidify the molten resin, thereby obtaining a container part in which the container body member formed by solidifying the molten resin and the bottom substrate with a surface modifier layer are joined together.

[0074] Here, as described above, by performing insert injection molding using a bottom substrate with a surface modifier layer in which a surface modifier layer is formed over the entire surface of the bottom substrate, it is possible to suitably manufacture a container portion in which a surface modifier layer is interposed between the container body member and the bottom substrate, as shown in Figure 3. On the other hand, by using a bottom substrate with a surface modifier layer in which a surface modifier layer is formed only on a portion of the surface of the bottom substrate and performing insert injection molding so that the molten resin comes into contact with the portion of the bottom substrate on which the surface modifier layer is not formed, it is possible to manufacture a container portion in which the container body member and the bottom substrate are in direct contact, as shown in Figure 2.

[0075] <Other processes> Other steps include, for example, a step of drying the surface modifier solution applied to the surface of the bottom substrate in the surface modifier layer forming step (drying step), a step of assembling a cell culture vessel by combining the container part obtained in the insert injection molding step with other components (assembly step), etc. The method of drying the surface modifier solution on the bottom substrate is not particularly limited and any known method can be used, such as drying with warm air, hot air, or low-humidity air, vacuum drying, or drying by irradiation with infrared rays or electron beams. [Example]

[0076] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples. In the following description, "%" and "parts" representing amounts are based on mass unless otherwise specified. In the examples and comparative examples, various measurements and evaluations were carried out according to the following methods.

[0077] <Contact angle> The contact angle between the surface modifier solution used in the examples and comparative examples and the bottom surface of the well of the microwell plate was determined using a fully automatic contact angle meter "LCD-400S" manufactured by Kyowa Interface Science Co., Ltd., where the radius r and height h of the droplet were measured, and the contact angle θ was calculated by tanθ1=h / r, θ=2θ1→θ=2arctan(h / r) (θ / 2 method). <Presence or absence of a surface modifier layer on the sidewall> In the examples and comparative examples, after applying the surface modifier solution, the microwell plate before drying was observed at 30x magnification using a digital microscope (VHX-6000, manufactured by Keyence Corporation) to check whether the surface modifier solution was attached to the side walls of the wells of the microwell plate. <Cell migration to the sidewall, uniformity of the surface modifier layer> The microwell plates prepared in the examples and comparative examples, in which a surface modifier layer was formed on the bottom surface of the wells, were used as cell culture vessels to carry out cell culture evaluation. Commercially available dopamine neurons (FUJIFILM Cellular Dynamics) were cultured in the above cell culture vessel at a density of 4 × 10 4 After culturing for 3 days, calcium imaging was performed and the cells were observed using a 4x objective lens on a confocal quantitative image cytometer (CellVoyager CQ1, Yokogawa Electric Corporation) to confirm the cell coverage on the well bottom and evaluated according to the following criteria: The higher the cell coverage on the well bottom, the more uniform the surface modifier layer formed on the well bottom. A: Cell coverage is 95% or more in terms of area. B: Cell coverage is 90% or more but less than 95% of the surface area. C: Cell coverage is less than 90% by area. In addition, 1–10 μL / well of cell dissociation reagent (ThermoFisher, StemProAccutase) was added so that only the bottom of the wells of the cell culture vessel was wetted. The detached cells were then washed away with phosphate-buffered saline (PBS). The bottom of the cell culture vessel was observed from the backside of the vessel using a 4x objective lens with an inverted fluorescence phase-contrast microscope (Keyence Corporation, BZ-X710). After confirming that no cells were observed, approximately 100–150 μL / well of cell dissociation reagent was added to impregnate the well sidewalls. If any cells were attached to the well sidewalls, they were dissociated from the well sidewalls. The bottom of the cell culture vessel was then observed from the backside of the vessel using an inverted fluorescence phase-contrast microscope with a 4x objective lens to confirm whether cells were observed. If cells were observed, it was determined that cells had attached to the well sidewalls (that is, cells had migrated to the sidewalls).

[0078] Example 1 <Preparation of cell culture vessels (microwell plates)> An oligopeptide (number of amino acid residues: 29) serving as a surface modifier was dissolved in a 1-propanol aqueous solution (70% by mass of 1-propanol, 30% by mass of pure water) to prepare a 100 μg / mL surface modifier solution. A surface-untreated film consisting of a norbornene-based ring-opening polymer hydrogenated product (ZEONOR® ZF16, manufactured by Zeon Corporation; glass transition temperature: 163°C) prepared by melt extrusion was placed on the stage of an automatic coating machine (PI-1210 manufactured by Tester Sangyo Co., Ltd.). A #6 wire bar was installed in the automatic coating machine, and the surface modifier solution was applied to the film serving as the bottom substrate at a speed of 40 mm / sec. Subsequently, a bottom substrate with a surface modifier layer was obtained by applying 80°C hot air for 1 minute. The resulting bottom substrate with the surface modifier layer was placed in a mold so that the surface modifier layer was exposed to the mold cavity. The mold was then clamped, and insert injection molding was performed to obtain a microwell plate. The container body was made of a hydrogenated norbornene ring-opening polymer (ZEONOR 1420R, manufactured by Zeon Corporation; glass transition temperature: 137°C; weight-average molecular weight: 31,000). Various measurements and evaluations were performed on the resulting microwell plate. The results are shown in Table 1.

[0079] Example 2 A cell culture vessel (microwell plate) was prepared in the same manner as in Example 1, except that an aqueous 1-propanol solution (20% by mass of 1-propanol, 80% by mass of pure water) was used as the solvent for preparing the surface modifier solution instead of the aqueous 1-propanol solution (70% by mass of 1-propanol, 30% by mass of pure water). Various measurements and evaluations were performed. The results are shown in Table 1.

[0080] Example 3 A cell culture vessel (microwell plate) was prepared in the same manner as in Example 1, except that a 1-propanol aqueous solution (90% by mass of 1-propanol, 10% by mass of pure water) was used as the solvent for preparing the surface modifier solution instead of the 1-propanol aqueous solution (70% by mass of 1-propanol, 30% by mass of pure water). Various measurements and evaluations were performed. The results are shown in Table 1.

[0081] Example 4 Except for changing the wire bar size installed in the automatic coating device from #6 to #18, cell culture vessels were produced and various measurements and evaluations were carried out in the same manner as in Example 1. The results are shown in Table 1.

[0082] Example 5 A cell culture vessel (microwell plate) was prepared in the same manner as in Example 1, except that a pilot coater (RK PRINTCOAT INSTRUMENTS LTD., VCML) was used instead of the automatic coating device, and various measurements and evaluations were performed. Specifically, a comma roll and a back roll were installed in the coating device (pilot coater), and the surface modifier solution was applied at a speed of 2 m / min with a gap between the rolls of 300 μm, and then dried in a hot air drying oven at 80°C to obtain a bottom substrate with a surface modifier layer. Using the obtained bottom substrate with a surface modifier layer, a cell culture vessel (microwell plate) was prepared in the same manner as in Example 1, and various measurements and evaluations were performed. The results are shown in Table 1.

[0083] Example 6 A cell culture vessel (microwell plate) was prepared in the same manner as in Example 1, except that a film made of a norbornene-based ring-opening polymer hydride (ZEONOR ZF16, manufactured by ZEON Corporation) used as the bottom substrate was subjected to plasma treatment, and various measurements and evaluations were carried out.

[0084] (Comparative Example 1) <Preparation of cell culture vessels (microwell plates)> An oligopeptide (29 amino acid residues) serving as a surface modifier was dissolved in a balanced salt solution (D-PBS(-), manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) to prepare a surface modifier solution at a concentration of 10 μg / mL. A 96-well plate (well bottom shape: circular, 6.34 mm diameter, sidewall height: 11.7 mm) made of norbornene-based ring-opening polymer hydride (Zeonor 1060R, manufactured by Zeon Corporation) fabricated by the insert molding method described in Example 1 of Japanese Patent Publication No. 7020146 was placed on the stage of a benchtop multipipettor (EDR-384SR, manufactured by Biotec Co., Ltd.), and the prepared surface modifier solution was applied at a volume of 150 μL / well. The well plate was then left to stand at 37°C for 45 minutes. Next, the wells were washed twice by injecting and withdrawing 200 μl of sterile water per well, and then the plate was dried in a cabinet for 1 hour to obtain a microwell plate with a surface modifier layer formed on the bottom of the wells. Various measurements and evaluations were performed on the resulting 96-well plate. The results are shown in Table 1.

[0085] In addition, in Table 1 shown below, "COP" indicates a norbornene ring-opening polymer hydride, "Tg" indicates the glass transition temperature, "Insert" refers to insert injection molding.

[0086] [Table 1]

[0087] Table 1 shows that in Examples 1 to 6, which used cell culture vessels in which a surface modifier layer was formed only on the bottom substrate, migration of cells to the side walls was suppressed. On the other hand, in Comparative Example 1, which used a cell culture vessel in which a surface modifier layer was formed on more than just the bottom substrate, it was found that migration of cells to the side walls could not be suppressed. [Industrial Applicability]

[0088] According to the present invention, it is possible to provide a cell culture vessel capable of suppressing cell migration to the side wall. Furthermore, the present invention can provide a method for producing a cell culture vessel that can suppress cell migration to the side wall. [Explanation of symbols]

[0089] 1. Microwell plate (cell culture vessel) 10 wells (container) 11 well side wall (container body member) 12 Well bottom (bottom substrate) 20 Lid 30 cells 40 Culture Medium 50 Surface modifier layer

Claims

1. A cell culture vessel having a vessel portion for accommodating cells and a culture medium, the container portion includes a container body member and a bottom substrate; a surface modifier layer having cell adhesive properties is formed only on the bottom substrate; The bottom substrate is joined to the container body member so that the surface of the surface modifier layer faces a space for accommodating the cells and the culture medium.

2. The cell culture vessel according to claim 1 , wherein the bottom base material is made of one or more selected from the group consisting of cycloolefin polymer, polyester, polypropylene, and polystyrene.

3. The cell culture vessel according to claim 1 , wherein the bottom substrate is not subjected to a surface treatment.

4. 4. The cell culture vessel according to claim 1, wherein the surface modifier layer contains an oligopeptide.

5. A method for manufacturing a cell culture vessel having a vessel portion for containing cells and a culture medium, comprising: the container portion includes a container body member and a bottom substrate; The manufacturing method includes: A step of applying a surface modifier solution onto the bottom substrate to obtain a bottom substrate with a surface modifier layer formed on the bottom substrate; forming the container portion by insert injection molding using the bottom substrate with the surface modifier layer; A method for manufacturing a cell culture vessel, comprising:

6. The method for manufacturing a cell culture vessel according to claim 5 , which does not include both a step of washing the bottom substrate with the surface modifier layer and a step of washing the vessel part.

7. The method for manufacturing a cell culture vessel according to claim 5 , wherein a contact angle between the surface modifier solution and the bottom substrate is 0° or more and 40° or less.

8. 8. The method for producing a cell culture vessel according to claim 5, wherein the thickness of the coating film when the surface modifier solution is applied to the bottom substrate is 150 μm or less.

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