JP2013106531A - Method for manufacturing cell culturing vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for manufacturing a cell culturing vessel that hardly causes a decrease in the function of a functional polymer film when bonding a functional base having a functional polymer film such as temperature responsive polymer to a vessel member by an insert injection molding method.SOLUTION: The method for manufacturing a cell culturing vessel includes: a functional base manufacturing step for forming a functional polymer layer on a base material; a functional base bonding step for disposing the manufactured functional base such that a surface of the functional polymer layer is in contact with an inner wall of a mold, and a surface adjacent to the base material is exposed to a space of a casting mold, and mold clamping the functional base, and then, bonding the functional base and a vessel body member or a partial member thereof by filling and solidifying a molten resin in the space of the casting mold; and a functional base cleaning step for cleaning the functional base bonded in the functional base bonding step.

Description

  The present invention relates to a method for producing a cell culture container for culturing cells.

  In recent years, cells collected from patients in the field of regenerative medicine are cultured in vitro to produce sheet-like cell aggregates (hereinafter referred to as cell sheets) and transplanted into the living body to achieve medical effects. There are attempts to increase it. As a tool for recovering a cell sheet, for example, a tool in which a film in which a temperature-responsive polymer such as poly-N-isopropylacrylamide is fixed on the surface is attached to the bottom of a container such as a petri dish using an adhesive has been reported. (See Patent Document 1).

  When manufacturing a container for culturing cells, as a method for joining the container and the film, there is a method using an insert injection molding method in addition to a method using an adhesive as in Patent Document 1. For example, in Patent Document 2, a petri dish is joined to a plastic film having silicon oxide deposited as a hydrophilic film on the surface by an insert injection molding method.

JP 2010-98799 A Japanese Utility Model Publication No. 5-88299

  The method of joining the container and the film by the insert injection molding method is preferable because it is cheaper and the number of production per unit time is larger than the joining method of sticking the adhesive film to the container using a labeler. . However, when the insert injection molding method as in Patent Document 2 is employed as a means for joining a film provided with a functional polymer film such as a temperature-responsive polymer and a support member serving as a container, the above high It was confirmed that the function of the molecular film was lowered by the heat and pressure of the injection mold.

  The present invention has been made in view of the above circumstances, and when a functional substrate having a functional polymer film such as a temperature-responsive polymer is joined to a container member by an insert injection molding method, the functionality is high. An object of the present invention is to provide a method for producing a cell culture container, in which the function of the molecular film is unlikely to deteriorate.

  By the way, unreacted monomers, oligomers, prepolymers, non-immobilized polymers, etc. remain on the surface of a functional polymer film such as a temperature-responsive polymer formed by a polymerization reaction by irradiation of radiation. Therefore, the functional substrate on which a functional polymer film such as a temperature responsive polymer is formed is washed to remove the residue. The present inventors have found that the above problem can be solved by adjusting the timing of this cleaning, and have completed the present invention. Specifically, the following are provided.

(1) A container part for containing cells and a medium is provided, and the container part has at least a container main body member and a functional polymer layer having a surface having a predetermined function formed on a substrate. A cell culture container, wherein the functional substrate is joined to a container body member such that the surface of the functional polymer layer faces a space for containing cells and a medium. A manufacturing method of
On the base material, a solution containing at least a monomer that can be polymerized by radiation irradiation to form a functional polymer is applied to form a coating film, and then the coating film is irradiated with radiation to form a functional polymer. The functional substrate produced in the functional substrate production step is added to the functional substrate production step of forming the layer, and the injection mold for forming the container body member or the partial member thereof. After the surface is in contact with the inner wall of the mold and the surface on the base material side is exposed in the mold space, and after the mold is clamped, the mold space is filled with a molten resin and solidified. A functional substrate bonding step for bonding the container main body member or its partial member and the functional substrate; and a functional substrate cleaning step for cleaning the functional substrate bonded in the functional substrate bonding step. Cell culture characterized by Method of manufacturing the container.

  According to the method for producing a cell culture container of the present invention, an insert injection molding method is employed when a functional substrate having a functional polymer film such as a temperature-responsive polymer and a container member are joined. However, the function of the functional polymer film is hardly deteriorated.

It is a figure which shows an example of the cell culture container (flask type) manufactured in this invention. 1A is a perspective view, FIG. 1B is a cross-sectional view taken along line II ′, and FIG. 1C is a cross-sectional view taken along line II-II ′. It is a figure which shows an example of the cell culture container (dish type) manufactured in this invention. 2A is a perspective view, and FIG. 2B is a cross-sectional view along III-III ′. It is a perspective view which shows an example which combines a container part member and a functional base | substrate, and completes a flask type container part by combining another member. It is sectional drawing which shows an example of the functional base | substrate in this invention. It is a typical sectional view for explaining an example of joining of a functional base by insert injection molding.

  The features of the present invention will be described below with reference to the drawings. The present invention is not limited to the embodiments described below, and can be implemented with appropriate modifications within a range not departing from the technical idea. Each figure shown below is a diagram schematically, and may be exaggerated as appropriate for easy understanding, and the scale may be different from the actual one.

[Cell culture vessel]
The cell culture container produced in the present invention includes at least a container part for containing cells and a medium, and further includes a lid or the like as appropriate. An example of a preferred embodiment of a cell culture vessel is shown in FIG. A container 100 shown in FIG. 1A includes a bottom 101, a side wall 102 erected on the periphery of the bottom 101, and a top 103 that is joined to the upper end of the side wall 102 and is opposed to the bottom 101. It has. The container portion 100 is provided with a liquid through-hole 104. That is, the container portion 100 has a through hole 104 formed in a part of the side wall portion 102 and includes a neck portion 105 extending from the periphery of the through hole 104 to the outside of the container, and has a so-called “flask type” shape. doing. The neck portion 105 is formed with a locking portion (not shown) for locking the lid 110, and the lid 110 is detachably mounted via the locking portion. The inside of the cell culture vessel 1 is closed by the lid 110, and cell culture can be performed stably. Cells and culture medium can be supplied from the through-hole 104, or the functional substrate 20 peeled off from the upper surface of the bottom 101 can be collected.

  FIG. 1B is a cross-sectional view taken along the line II ′ of the container part 100, and FIG. 1C is a cross-sectional view taken along the line II-II ′ of the container part 100. A space 120 for accommodating cells and a culture medium is formed in a portion of the container 100 surrounded by the bottom 101, the side wall 102, and the top 103. The functional substrate 20 is joined to a part of the inner wall surface facing the space 120 (on the surface of the bottom 101 in the cell culture container shown in FIG. 1). The functional substrate 20 is disposed so that the surface of the functional polymer layer faces the space 120 for accommodating cells and a medium.

  Another example of a preferred embodiment of a cell culture vessel is shown in FIG. A container unit 200 shown in FIG. 2A includes a functional substrate 20. The container part 200 includes a bottom part 201 and a side wall part 202 erected on the periphery of the bottom part 201, and has a so-called “dish type” shape opened upward.

  FIG. 2B is a cross-sectional view taken along the line III-III ′ of the container part 200. The functional substrate 20 is joined to the surface (inner bottom surface) on the space 220 side for accommodating the cells and the medium in the bottom 201 of the container 200. The functional substrate 20 is arranged so that the surface of the functional polymer layer faces the space 220 for accommodating cells and a medium.

  Although FIG. 1 shows a “flask type” cell culture container and FIG. 2 shows a “dish type” cell culture container, the cell culture container produced in the present invention has a shape that can accommodate cells and a medium. For example, a shape called a “beaker type” or a “bottle type” may be used.

  In the present invention, a portion of the container portion excluding the functional substrate is referred to as a “container body member”. For example, in the container part 100 of FIG. 1, the container main body member is a part composed of a bottom part 101, a side wall part 102, a top part 103, and a neck part 105. In the container part 200 of FIG. Is a part composed of a bottom part 201 and a side wall part 202.

  In the production method of the present invention, an insert injection molding method is used so that a functional substrate is disposed on the surface of a container body member or a member that is a part of the container body member (hereinafter sometimes referred to as “container partial member”). The container main body member or the container partial member is bonded to the functional substrate. First, when the container part member and the functional substrate are joined, in the subsequent process, the container part member and another member are joined to complete the container part.

  An example of a method for assembling the container portion of the cell culture container (flask type) produced in the present invention will be described with reference to FIG. First, the container partial member 301 and the functional base 20 are formed by insert injection molding so that the functional base 20 is disposed on the inner bottom surface of the container partial member 301 including the bottom portion 101 and the side wall portion 102. Join. Subsequently, the container part is formed by joining the top member 302 corresponding to the top part 103. The container member 301 and the top member 302 are joined in a liquid-tight manner so that the culture solution does not leak out according to the purpose of cell culture, if necessary.

  The material for forming the container part and the lid in the present invention is not particularly limited, and materials generally used in cell culture can be used. For example, polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, chloride Examples thereof include resin materials such as vinyl resins, resin materials containing at least one of the above-described surface hydrophilized treatment, and inorganic materials such as glass and quartz. Among these, resin materials are preferable. The resin material is preferably a polystyrene resin or a polyethylene terephthalate resin.

<Functional substrate>
An example of a preferred embodiment of the functional substrate in the present invention is shown in FIG. FIG. 4 is a cross-sectional view of the functional substrate cut along the thickness direction. In the functional substrate 20 shown in FIG. 4, a functional polymer layer 22 having a surface having a predetermined function is formed on a substrate 21.

  The functional substrate may be in the form of a film or a sheet. The film-like functional substrate preferably has flexibility so that it can be wound into a roll. This is because mass production by the roll-to-roll method becomes possible.

  The shape of the functional substrate can be arbitrarily selected according to the shape of the region of the member to be joined. For example, the shape may be a triangle, a quadrangle (square, rectangle, parallelogram, rhombus, etc.), a polygon such as a pentagon, a hexagon, a heptagon, an octagon, a circle, or an ellipse.

(Base material)
The base material constituting the functional base is not particularly limited as long as it can form a functional polymer layer described later on one surface and has water resistance that can withstand cell culture. Various materials can be selected and formed according to the functional polymer layer. Typically, the base material is polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE). ), Medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic and the like. Further, it may be a biodegradable polymer such as polylactic acid, polyglycolic acid, polycaprolactan, or a copolymer thereof. Preferred are polyethylene terephthalate, polystyrene, and polycarbonate. Polyethylene terephthalate is preferable in that it can be obtained at a low cost and is a material suitable for mass production. Polystyrene is preferred because it is a material with low cytotoxicity. The film forming method is not particularly limited, and for example, a conventionally known method such as a solution casting method, a melt extrusion method, or a calendar method can be used. Moreover, you may use the commercially available base material previously formed into the sheet form by the said method.

  The surface of the substrate on which the functional polymer layer is formed may be subjected to an easy adhesion treatment. Examples of the easy adhesion treatment include treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, and perfluorooctane sulfonic acid (PFOS).

  The shape of the substrate is not particularly limited, and may be appropriately selected according to the final shape of the functional substrate to be produced. Examples thereof include a sheet shape and a film shape. What follows the shape of the bottom part of the container part joined with a functional base | substrate is preferable. This is because workability is improved. Usually, it is a flat shape. Although the thickness of a base material is not specifically limited, When handling property is considered, Preferably it is 20-500 micrometers, More preferably, it is 50-250 micrometers. Moreover, if it is this range, it is also possible to supply and process in a continuous belt shape.

(Functional polymer layer)
The polymer constituting the functional polymer layer is not particularly limited as long as it has a desired function, but preferably a surface that can be changed from cell adhesiveness to cell nonadhesiveness by a predetermined stimulus. And a stimuli-responsive polymer. Examples of the stimulus responsive polymer include a temperature responsive polymer, a pH responsive polymer, an ion responsive polymer, and a photoresponsive polymer. Among these, a temperature-responsive polymer is preferable because it is easy to give a stimulus.

  As the temperature-responsive polymer, for example, a polymer that exhibits cell adhesion at a temperature for culturing cells and exhibits cell non-adhesion at a temperature at which the produced cell sheet is peeled may be used. For example, a temperature-responsive polymer has improved affinity to surrounding water at temperatures below the critical dissolution temperature, and the polymer takes up water and swells to make it difficult for cells to adhere to the surface (cell non-adhesiveness). It is preferable that the temperature is equal to or higher than the same temperature so that water is desorbed from the polymer and the polymer contracts to easily adhere cells to the surface (cell adhesion). Such a critical solution temperature is called a lower critical solution temperature. It is preferable to use a temperature-responsive polymer having a lower critical solution temperature T of 0 ° C to 80 ° C, more preferably 0 ° C to 50 ° C. This is because the cells can be stably cultured when T is 0 ° C to 80 ° C.

  Specific examples of the temperature-responsive polymer that can be suitably used in the present invention include acrylic polymers and methacrylic polymers, and more specifically, poly-N-isopropylacrylamide (T = 32 ° C.) and poly-N. -N-propylacrylamide (T = 21 ° C), poly-Nn-propylmethacrylamide (T = 32 ° C), poly-N-ethoxyethylacrylamide (T = about 35 ° C), poly-N-tetrahydrofurfuryl Examples include acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = about 35 ° C.), poly-N, N-diethylacrylamide (T = 32 ° C.), and the like. Further, it may be a copolymer obtained by combining two or more monomers for forming these polymers.

  As a monomer for forming these polymers, a monomer that can be polymerized by irradiation with radiation can be used. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, vinyl ether derivatives, and the like. The above may be used. When one type of monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple types of monomers are used in combination, the polymer formed on the substrate is a heteropolymer. Forms are also encompassed by the present invention.

  Further, if necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above-mentioned polymer used in the present invention and another polymer, or a mixture of the polymer of the present invention and another polymer may be used. Moreover, it is also possible to crosslink within a range where the original properties of the polymer are not impaired.

  As the pH responsive polymer, ion responsive polymer, photoresponsive polymer and the like, those suitable for the cell sheet to be produced can be appropriately selected.

  The stimulus-responsive polymer layer is formed by immobilizing the stimulus-responsive polymer on the surface of the substrate so as to have a predetermined thickness. The thickness of the stimulus-responsive polymer layer is, for example, preferably in the range of 0.5 nm to 300 nm, and more preferably in the range of 1 nm to 100 nm. By making the film thickness in the range of 0.5 nm to 300 nm, it is easy to achieve both cell adhesion and peeling. In addition, various additives, such as surfactant, may be mix | blended with the stimulus responsive polymer layer in the range which does not impair the function.

[Method for producing cell culture container]
Unreacted monomers, oligomers, prepolymers, non-immobilized polymers, and the like remain on the surface of a functional polymer layer such as a temperature-responsive polymer formed by a polymerization reaction by irradiation with radiation. These residues are preferably removed from the viewpoint of quality assurance. Therefore, the functional substrate on which a functional polymer layer such as a temperature-responsive polymer is formed is washed to remove the residue. The inventors of the present invention have conventionally placed the functional substrate after washing in the injection mold so that the surface of the functional polymer layer and the inner wall of the injection mold are in contact with each other by an insert injection molding method. The functional substrate and the container main body member or the container partial member have been joined. However, according to this method, the function of the functional substrate has been deteriorated, which is considered to be caused by the heat and pressure of the mold during injection molding. In contrast, according to the present invention, when a functional substrate having a functional polymer film such as a temperature-responsive polymer and a container body member are joined, an insert injection molding method is employed. However, the function of the functional polymer film is hardly deteriorated. Hereinafter, the manufacturing method of the cell culture container of this invention is demonstrated in detail.

  The method for producing a cell culture container of the present invention includes a functional substrate preparation step, a functional substrate bonding step, and a functional substrate cleaning step.

<Functional substrate manufacturing process>
In the functional substrate preparation step, a solution containing at least a monomer that can be polymerized by radiation irradiation to form a functional polymer is applied on the base material to form a coating film, and then the coating film is irradiated with radiation. Thus, a functional substrate is formed by forming a functional polymer layer. Hereinafter, the functional substrate manufacturing process will be specifically described by taking the case where the functional polymer layer is a temperature-responsive polymer layer as an example.

  First, the above-mentioned base material is prepared. The substrate may be a single wafer or a roll. Next, a temperature-responsive polymer layer-forming coating solution is prepared by dissolving the above-mentioned monomer that becomes a temperature-responsive polymer by polymerization in a solvent. And according to a conventional method, this coating liquid is applied to the surface of a base material, and a coating film is formed. Thereafter, the monomers in the coating film are polymerized by irradiation with radiation to form a polymer, and a grafting reaction is caused between the surface of the substrate and the polymer to form a temperature-responsive polymer layer. Thus, the functional substrate in the present invention is produced.

  The solvent is not particularly limited as long as it can dissolve the monomer, but those having a boiling point of 120 ° C. or less, particularly 60 to 110 ° C. under normal pressure are preferable. Specific examples include methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, and water, and these may be used in combination. Other solvents such as 1-pentanol, 2-ethyl-1-butanol, 2-butoxyethanol, and ethylene (or diethylene) glycol or monoethyl ether thereof can also be used. If necessary, other additives may be added to the solution.

  The content of the monomer in the coating liquid is preferably 1 to 70% by weight. In addition to the monomer, the coating composition may contain an oligomer or prepolymer obtained by polymerizing a plurality of monomers. When an oligomer or prepolymer is included, its size is not particularly limited as long as it is a dimer or larger, and preferably has a molecular weight of more than about 3,300 (typically 28 molecular polymers). 700 or more are more preferable.

  The method for coating the surface of the substrate with the coating liquid is not particularly limited, and a conventionally known method can be used. Examples thereof include printing methods such as gravure printing, flexographic printing, and offset printing, and methods using coating such as roll coating, reverse coating, comma coating, knife coating, die coating, and gravure coating.

The coating amount of the temperature-responsive polymer layer may be a coating amount necessary for the grafted polymer to exhibit stimulus responsiveness, for example, 5 to 80 μg / cm ² , and 10 to 50 μg / cm ^2. Is preferred. When the polymer coating amount exceeds 80 μg / cm ² , the cell adhesion may be lowered. Conversely, when the coating amount is less than 5 μg / cm ² , the cell detachability may be lowered.

  Examples of the radiation used for polymerizing the monomer include α rays, β rays, γ rays, electron beams, and ultraviolet rays. In order to produce a desired graft polymer, γ rays and electron beams are preferable in terms of energy efficiency, and electron beams are particularly preferable in terms of productivity. Ultraviolet rays can be used in combination with a suitable polymerization initiator or an anchor agent such as a silane coupling agent. The range of radiation dose is preferably 5 Mrad to 50 Mrad for electron beams, and preferably 0.5 Mrad to 5 Mrad for γ rays. Before and after the irradiation step, the coating film is dried as necessary to remove the solvent.

<Functional substrate bonding process>
In the functional substrate bonding step, the functional substrate produced in the functional substrate production step is applied to the injection mold for forming the container body member or a partial member thereof, and the surface of the functional polymer layer is the mold. The container main body member or the above-mentioned container body or The partial member is bonded to the functional base. Hereinafter, an example of a preferred embodiment of this functional substrate bonding step will be specifically described with reference to FIG.

  FIG. 5 is a schematic cross-sectional view sequentially showing a method of manufacturing a flask-type cell culture container by joining a container partial member and a functional substrate by an insert injection molding method. At the time of mold clamping, the functional base 20 is placed on the functional polymer layer 22 side outermost layer (see FIG. 5) in an injection mold that combines the convex mold 401 and the concave mold 402 to form the mold space 404 corresponding to the container partial member 301. 5, the surface of the functional polymer layer) covers a part of the inner wall surface of the injection mold, and at least a part of the surface of the outermost layer (base material in FIG. 5) (the base material in FIG. 5) In FIG. 5, all are attached so as to be exposed to the mold space 404 (FIG. 5A), and then the mold is clamped. Next, the resin is filled into the mold through the gate 403 by applying injection pressure (FIG. 5B). After filling, the mold is opened after the resin is solidified, and the functional substrate 20 and the container partial member 301 are joined (FIG. 5C).

  In the present invention, residues such as unreacted monomers, oligomers, prepolymers and non-immobilized polymers remaining on the surface of the functional polymer layer may cause the functional polymer layer to be removed from heat and pressure during injection molding. Since it acts like a protective layer for protection, it is thought that the function of the functional polymer layer may be maintained even after injection molding.

<Functional substrate cleaning process>
In the functional substrate cleaning step, the functional substrate bonded in the functional substrate bonding step is cleaned. As described above, on the surface of the functional polymer layer after graft polymerization, there are not only polymer molecules immobilized by covalent bonds, but also free polymers that are not immobilized and unreacted monomers. doing. In the present invention, unlike the conventional method, this cleaning is performed after the functional substrate bonding step. In this washing step, these free polymers and unreacted substances are removed. The cleaning method is not particularly limited, but typically includes immersion cleaning, high-pressure cleaning, rocking cleaning, shower cleaning, spray cleaning, ultrasonic cleaning, and the like. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, and acid / alkali cleaning liquids. The combination of the cleaning method and the cleaning liquid is not particularly limited, and can be appropriately selected according to the functional substrate. The present invention is not limited to performing no cleaning of the functional substrate before the functional substrate bonding step, and within the scope of the effects of the present invention, before the functional substrate bonding step. The functional substrate may be cleaned. In other words, the present invention does not exclude that the functional substrate is cleaned before the functional substrate bonding step.

<Other processes>
Finally, the top member 302 is joined to the open end of the container partial member 301 to complete the container unit 100 (FIG. 5D). Thereafter, the lid is closed, and sterilization treatment such as ethanol sterilization, ethylene oxide gas (EOG) sterilization, ultraviolet irradiation sterilization, and γ-ray irradiation sterilization is performed as necessary. Among these, γ-ray irradiation sterilization is preferable in that all biological species can be killed.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention does not receive a restriction | limiting at all by these description.

[Reference Example 1]
<Preparation of cell culture container>
A polystyrene film (thickness: 50 μm, obtained from Asahi Kasei Chemicals) was prepared as a substrate. One surface of the polystyrene film was subjected to corona treatment to make the surface hydrophilic. Next, N-isopropylamine was dissolved in isopropyl alcohol to a final concentration of 40% by weight to prepare a temperature-responsive polymer layer forming coating solution. On the hydrophilic surface of the polystyrene film, the entire surface of the coating solution was applied by a gravure direct method (application amount: 1.1 g / cm ² , conveyance speed: 5 m / min). Then, after passing through a hot air dryer (2 m) at 40 ° C. and drying, electron beam irradiation (electron beam irradiation dose: 120 kGy) was performed to graft polymerize N-isopropylamine, and the surface of the polystyrene film was polymerized. -N-Isoproacrylamide was immobilized. A trapezoidal functional substrate was obtained by cutting out into a trapezoidal shape using a hand press machine. The surface of the temperature-responsive polymer layer of the obtained functional substrate was immersed in water at 20 ° C. for 4 hours and then dried.

  An injection molding machine (α-100C, manufactured by FANUC) was used to prepare a polystyrene resin bottom member, a peripheral side wall member, and a top member. After the functional substrate obtained above is attached to the bottom member using a double-sided tape, the peripheral side wall member and the top member are joined by ultrasonic welding, and a flask-type cell culture container (hereinafter referred to as flask) Was made.

<Cell peeling test>
The flask produced as described above was sterilized with ultraviolet rays for a predetermined time in a clean bench. Next, bovine vascular endothelial cells were seeded on the functional substrate in the sterilized flask so that the surface cell density was 1.0 × 10 ⁵ cells / cm ^2, and the conditions were 37 ° C. and 5% CO _2. In a CO ₂ incubator for 24 hours. The medium used was 10% FBS-containing DMEM (manufactured by Sigma). The flask was then placed in a CO ₂ incubator at 20 ° C. and 5% CO ₂ . After 20 minutes, the flask was taken out of the CO ₂ incubator, and it was confirmed that a cell sheet was obtained.

[Example 1]
<Preparation of cell culture container>
A polystyrene film (thickness: 50 μm, obtained from Asahi Kasei Chemicals) was prepared as a substrate. One surface of the polystyrene film was subjected to corona treatment to make the surface hydrophilic. Next, N-isopropylamine was dissolved in isopropyl alcohol to a final concentration of 40% by weight to prepare a temperature-responsive polymer layer forming coating solution. On the hydrophilic surface of the polystyrene film, the entire surface of the coating solution was applied by a gravure direct method (application amount: 1.1 g / cm ² , conveyance speed: 5 m / min). Then, after passing through a hot air dryer (2 m) at 40 ° C. and drying, electron beam irradiation (electron beam irradiation dose: 120 kGy) was performed to graft polymerize N-isopropylamine, and the surface of the polystyrene film was polymerized. -N-Isoproacrylamide was immobilized. A trapezoidal functional substrate was obtained by cutting out into a trapezoidal shape using a hand press machine.

  The functional base is placed on a core mold (convex mold) of an injection molding machine (α-100C, manufactured by FANUC), and the surface of the temperature-responsive polymer layer of the functional base and the convex face of the core mold are opposed to each other. Arranged to be. Next, a cavity mold (concave mold) with a guide is arranged and clamped so that the molten resin does not contact the functional surface of the functional substrate, and then a polystyrene pellet (SGP10, manufactured by PS Japan Co., Ltd.) Was used for insert molding (resin temperature: 220 ° C., mold temperature: 20 ° C.). In this way, a container partial member having the functional substrate bonded to the bottom was obtained.

  Next, the surface of the temperature-responsive polymer layer of the functional substrate bonded to the bottom of the container partial member was dipped in water at 20 ° C. for 4 hours and then dried. Thereafter, a top member made of polystyrene resin produced by injection molding was joined to the container partial member by ultrasonic welding to produce a flask-type cell culture container (hereinafter referred to as a flask).

<Cell peeling test>
The flask produced as described above was sterilized with ultraviolet rays for a predetermined time in a clean bench. Next, bovine vascular endothelial cells were seeded on the functional substrate in the sterilized flask so that the surface cell density was 1.0 × 10 ⁵ cells / cm ^2, and the conditions were 37 ° C. and 5% CO _2. In a CO ₂ incubator for 24 hours. The medium used was 10% FBS-containing DMEM (manufactured by Sigma). The flask was then placed in a CO ₂ incubator at 20 ° C. and 5% CO ₂ . After 20 minutes, the flask was taken out of the CO ₂ incubator, and it was confirmed that a cell sheet was obtained.

[Comparative Example 1]
<Preparation of cell culture container>
A polystyrene film (thickness: 50 μm, obtained from Asahi Kasei Chemicals) was prepared as a substrate. One surface of the polystyrene film was subjected to corona treatment to make the surface hydrophilic. Next, N-isopropylamine was dissolved in isopropyl alcohol to a final concentration of 40% by weight to prepare a temperature-responsive polymer layer forming coating solution. On the hydrophilic surface of the polystyrene film, the entire surface of the coating solution was applied by a gravure direct method (application amount: 1.1 g / cm ² , conveyance speed: 5 m / min). Then, after passing through a hot air dryer (2 m) at 40 ° C. and drying, electron beam irradiation (electron beam irradiation dose: 120 kGy) was performed to graft polymerize N-isoproacrylamide, and the surface of the polystyrene film was polycrystallized. -N-Isoproacrylamide was immobilized. A trapezoidal functional substrate was obtained by cutting out into a trapezoidal shape using a hand press machine. The surface of the temperature-responsive polymer layer of the obtained functional substrate was immersed in water at 20 ° C. for 4 hours and then dried.

  The functional base is placed on a core mold (convex mold) of an injection molding machine (α-100C, manufactured by FANUC), and the surface of the temperature-responsive polymer layer of the functional base and the convex face of the core mold are opposed to each other. Arranged to be. Next, a cavity mold (concave mold) with a guide is arranged and clamped so that the molten resin does not contact the functional surface of the functional substrate, and then a polystyrene pellet (SGP10, manufactured by PS Japan Co., Ltd.) Was used for insert molding (resin temperature: 220 ° C., mold temperature: 20 ° C.). In this way, a container partial member having the functional substrate bonded to the bottom was obtained. Thereafter, a top member made of polystyrene resin produced by injection molding was joined to the container partial member by ultrasonic welding to produce a flask-type cell culture container (hereinafter referred to as a flask).

<Cell peeling test>
The flask produced as described above was sterilized with ultraviolet rays for a predetermined time in a clean bench. Next, bovine vascular endothelial cells were seeded on the functional substrate in the sterilized flask by the same method as in Example 1, but no cell sheet was obtained.

DESCRIPTION OF SYMBOLS 1 Cell culture container 2 Cell culture container 20 Functional base | substrate 21 Base material 22 Functional polymer layer 100 Container part 101 Bottom part 102 Side wall part 103 Top part 200 Container part 201 Bottom part 202 Side wall part 301 Container part member 302 Top member

Claims (1)

A container for containing cells and medium;
The container portion includes a container main body member, and a functional substrate on which a functional polymer layer having a surface having a predetermined function is formed on a base material,
The functional substrate is a method for producing a cell culture container, wherein the functional substrate is joined to a container body member so that the surface of the functional polymer layer faces a space for containing cells and a medium,
On the base material, a solution containing at least a monomer that can be polymerized by radiation irradiation to form a functional polymer is applied to form a coating film, and then the coating film is irradiated with radiation to form a functional polymer. A functional substrate manufacturing step of forming a layer;
An injection mold for forming the container main body member or a partial member thereof, the functional substrate produced in the functional substrate production step, the surface of the functional polymer layer in contact with the inner wall of the mold, and After the substrate side surface is exposed to the mold space and clamped, the mold space is filled with a molten resin and solidified, whereby the container body member or its partial member and the function A functional substrate bonding step for bonding the functional substrate;
And a functional substrate cleaning step for cleaning the functional substrate bonded in the functional substrate bonding step.

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