JP2013107218A - Method for manufacturing cell culture container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture container inexpensively and efficiently without impairing the function of a functional organic compound layer during in-mold forming.SOLUTION: There is provided a method for manufacturing a cell culture container having a container body, on a surface of which a functional base material 140 is fixed so as to allow the functional organic compound layer 402 to face the space side which accommodates a cell and a culture medium. A molding die cavity 504 is formed by an injection molding die obtained by assembling a core molding die 501 which defines the inner wall of the container portion facing a space for accommodating the cell and the culture medium, and a cavity molding die 503 which defines the outer wall of the container portion and has an injection hole 502 for a resin (A). In the molding die cavity 504, the functional base material 140 is arranged in the way that it covers the injection hole 502 and the base material layer 401 side comes into contact with the cavity molding die 503. By filling the resin (A) in the molding die cavity 504 from the injection hole 502, the functional base material 140 is moved so that the functional organic compound layer 402 side contacts to the core molding die 501 together with flowing of the resin (A).

Description

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

  Conventionally, cell culture containers such as petri dishes having a surface suitable for culturing microorganisms and cells are known. For example, in (Patent Document 1), in a petri dish made of plastic, a plastic film layer in which silicon oxide is vapor-deposited on the inner surface of the bottom is pasted by an insert injection molding method (in-mold molding). Is disclosed.

Japanese Utility Model Publication No. 5-88299 (paragraph 0012, FIGS. 1 and 2)

  When manufacturing the petri dish shown in (Patent Document 1), as shown in FIGS. 6A to 6C, first, when the mold for injection molding of the petri dish attached to the injection molding machine is opened, the core mold (male Mold) 601 Adsorbs a vapor-deposited film in which a silicon oxide vapor-deposited layer 611 is provided on one side of a polyester film 610 at a portion corresponding to the bottom inner surface of the petri dish by vacuum-sucking the surface of the vapor-deposited layer 611 onto the mold surface Fix it. 6A and 6B schematically show a state where the surface of the vapor deposition layer 611 is vacuum-sucked. Then, as shown in FIG. 6B, after the mold clamping, the molten polyethylene terephthalate S is melted in the mold space 603 formed by the core mold 601 and the cavity mold (female mold) 602 by the injection molding apparatus. Is injected from the injection hole 604 provided in the cavity mold 602 to mold the petri dish body, and at the same time, a polyester film having a silicon oxide vapor deposition layer 611 on the inner surface of the bottom is thermally bonded so that the surface of the vapor deposition layer 611 is exposed. And paste it. The formed petri dish 620 is taken out by opening an injection mold as shown in FIG. 6C.

  In the above conventional technique, the functional layer such as the vapor deposition layer 611 suitable for cell culture is adsorbed and fixed to the mold surface by vacuum suction, and therefore, the function of the layer may be impaired due to application of suction force or the like. . In particular, in recent years, cell culture containers such as petri dishes having a functional organic compound layer such as a temperature-responsive polymer coated on the surface are known. When such a functional organic compound layer is provided, in-mold The adverse effect of vacuum suction during molding is more serious than when an inorganic vapor deposition layer such as silicon oxide is provided.

  Therefore, an object of the present invention is to provide a method for producing a cell culture container, which can obtain a cell culture container inexpensively and efficiently without impairing the function of the functional organic compound layer during in-mold molding.

  The inventor does not fix the functional substrate including the base material layer and the functional organic compound layer to the mold surface of the core mold, and contacts the cavity mold on the opposite side before filling with the resin. It has been found that the above problems can be solved by arranging and moving the resin to the core mold side by filling the resin, and the invention has been completed. That is, the present invention includes the following inventions.

(1) Provided with a container for containing cells and culture medium,
The container part
A resin container body member;
A functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer;
Have
A method for producing a cell culture container in which a functional substrate is fixed on the surface of a container body member so that the functional organic compound layer faces the space side containing cells and a medium,
A core mold defining an inner wall of the container portion facing a space containing cells and medium;
A cavity mold defining an outer wall of the container portion and having a resin injection hole;
In the mold space formed by the injection mold combined with
The functional substrate is disposed so as to cover the injection hole and the base material layer side is in contact with the cavity mold,
The said manufacturing method including the process of moving a functional base | substrate so that the functional organic compound layer side may contact | connect a core metal mold | die with the flow of resin by filling resin into a casting_mold | template space from the injection hole.

  According to the method for producing a cell culture container of the present invention, the functional organic compound layer is brought into contact with the inner wall surface of the core mold from the cavity mold side using the flow of the resin filled from the injection hole. Therefore, it is not necessary to perform conventional vacuum suction or the like, and damage applied to the functional organic compound layer is reduced. Therefore, it is possible to efficiently obtain a cell culture container in which the function of the functional organic compound layer is well maintained.

It is a perspective view which shows one Embodiment of the cell culture container manufactured in this invention. It is I-I 'sectional drawing of FIG. 1A. It is II-II 'sectional drawing of FIG. 1A. It is a perspective view which shows another embodiment of the cell culture container manufactured in this invention. It is a III-III 'sectional view of Drawing 2A. It is a perspective view for demonstrating the manufacturing process of a cell culture container. It is sectional drawing of a functional base | substrate. It is sectional drawing which shows one Embodiment of the manufacturing method of the cell culture container which concerns on this invention. It is sectional drawing which shows one Embodiment of the manufacturing method of the cell culture container which concerns on this invention. It is sectional drawing which shows one Embodiment of the manufacturing method of the cell culture container which concerns on this invention. It is sectional drawing which shows one Embodiment of the manufacturing method of the cell culture container which concerns on this invention. It is sectional drawing which shows one Embodiment of the manufacturing method of the cell culture container which concerns on this invention. It is sectional drawing which shows the manufacturing method of the conventional cell culture container. It is sectional drawing which shows the manufacturing method of the conventional cell culture container. It is sectional drawing which shows the manufacturing method of the conventional cell culture container.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Each figure is a schematic diagram, and the size and shape of each part are appropriately exaggerated for easy understanding.

<Shape of cell culture vessel>
First, the overall shape of the cell culture container produced according to the present invention will be described.

  The cell culture container produced by 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.

  As shown in FIG. 1, the container portion may have a shape in which a space for containing cells and culture medium is closed by a wall surface, or a shape in which one end of the space is opened as shown in FIG. Also good.

  A preferred embodiment of the container portion is shown in FIGS. 1A-1C. A container portion 100 shown in FIG. 1A includes a bottom portion 101 and a resin-made container body member 103 composed of a side wall portion 102 erected on the periphery of the bottom portion 101 and a bottom portion joined to an upper end portion of the side wall portion 102. 101 is provided at least. A through-hole 105 is formed in a part of the side wall portion 102, and the container portion has a shape called “flask type” including a neck portion 106 that extends from the periphery of the through-hole 105 to the outside of the container portion. A locking portion 107 for locking the lid 110 is formed on the neck portion 106 of the container portion 100, and the lid 110 is detachably mounted through the locking portion 107. A flask type cell culture container 120 is formed by combining the container part 100 and the lid 110.

  FIG. 1B shows a cross-sectional view taken along the line I-I ′ of the container unit 100, and FIG. 1C shows a cross-sectional view taken along the line II-II ′. A space 130 for accommodating cells and a culture medium is formed in a portion of the container portion 100 surrounded by the bottom portion 101, the side wall portion 102, and the top surface member 104. A functional substrate 140 is fixed to a part of the inner wall surface facing the space 130 (the bottom 101 in the embodiment shown in FIG. 1).

  As other embodiment of a container part, as shown in FIG. 2, the dish-shaped container part 200 provided with the container main body member 203 comprised from the side part 202 standing upright at the periphery of the bottom part 201 and the bottom part 201 is mentioned. It is done. A functional substrate 210 is fixed to the surface (inner bottom surface) of the bottom 201 on the side of the space 220 that accommodates cells and culture medium.

  In the present invention, the container body member is formed by injection molding, and at this time, the container body member and the functional substrate are integrated by disposing the functional substrate in the mold space. And the container main body member integrated with the functional base is appropriately joined with other members to form the container portion. For example, as shown in FIG. 3, the container portion shown in FIG. 1A is formed by bonding the top surface member 104 to the container main body member 103 having the functional base 140 fixed to the inner bottom surface. The container body member 103 and the top surface member 104 are joined in a liquid-tight manner so that the culture solution does not leak out according to the purpose of cell culture.

<Material of cell culture container>
The material which forms each member of cell culture containers, such as a container main body member, a top | upper surface member, a neck part, and a lid | cover, is not specifically limited, The material generally used in cell culture can be used. In the present invention, at least the container body member is a resin material that can be injection molded.

  Examples of the resin constituting the container body member include polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, and phenol. Examples thereof include resin materials such as resins, melamine resins, epoxy resins, and vinyl chloride resins, and resin materials including at least one of the above-described surface hydrophilized treatments. The resin material is particularly preferably a polystyrene resin or a polyethylene terephthalate resin.

<Functional substrate>
As shown in FIG. 4, the cross section along the thickness direction of the functional substrates 140 and 210 in the present invention includes at least a base material layer 401 and a functional organic compound layer 402 disposed on the base material layer 401. . Here, the “base” may be a film or a plate-like body as long as it has a predetermined structure. In the container part, the functional substrate is fixed on the surface of the container body member so that the functional organic compound layer faces the space side in which the cells and the medium are accommodated.

  The film-like functional substrate (hereinafter sometimes referred to as “functional film”) is preferably flexible so that it can be wound into a roll. A flexible functional film is preferable because mass production by a roll-to-roll method is easy.

  The functional substrate can be produced by forming a functional organic compound layer on the substrate layer by an appropriate method. One or more other layers (for example, a primer layer described later) may be present between the base material layer and the functional organic compound layer as necessary.

  The shape of the functional substrate can be any shape depending on the shape of the region of the member to be fixed. For example, the shape may be a polygon such as a triangle, a rectangle (rectangle, square, parallelogram, rhombus, etc.), a pentagon, a hexagon, a heptagon, an octagon, a circle, an ellipse, or the like.

<Base material layer>
The substrate layer is appropriately selected according to the final functional substrate. If the functional substrate is plate-like, a plate-like substrate layer is used, and if the functional substrate is film-like, a film-like substrate layer (hereinafter referred to as “film substrate layer”) is used.

  The base material layer and the container main body member can be appropriately selected from materials that can be fused and integrated with each other by in-mold molding in the present invention.

  The material of the base material layer can be appropriately selected from resin materials that can form a functional organic compound layer on one surface, but the type of material is not particularly limited. Typically, as a material for the base layer, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetylcellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE) ), Medium density polyethylene (MDPE), polyvinyl chloride, polyvinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic resin and the like.

  The surface of the portion of the base material layer that is integrated with the container body member may contain a heat-sealable resin material. Since the heat-sealable resin material is melted by contact with a high-temperature molten resin and is solidified with the solidification of the resin, the bonding between the base material layer and the container main body member can be further strengthened.

  The surface of the base material layer on the side on which the functional organic compound layer is formed can be a surface subjected to easy adhesion treatment. “Easy adhesion treatment” refers to treatment with an easy adhesive such as polyester, acrylic ester, polyurethane, polyethyleneimine, silane coupling agent, perfluorooctane sulfonic acid (PFOS), and the like.

  The thickness of the base material layer can be appropriately selected. When the base material layer is a film base material layer, its thickness (when the film base material layer includes an easy adhesion layer in addition to the base material layer, the total thickness of the film base material layer including the easy adhesion layer) Is not particularly limited, but is, for example, 5 to 500 μm, more preferably 20 to 500 μm, and particularly preferably 50 to 250 μm.

<Functional organic compound layer>
The organic compound constituting the functional organic compound layer is not particularly limited as long as it has a desired function, but more preferably it can be changed from cell adhesiveness to cell nonadhesiveness by a predetermined stimulus. And a hydrophilic compound such as an ethylene glycol chain composed of one or more ethylene glycol units (CH ₂ —CH ₂ —O).

  The film thickness of the functional organic compound 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.

  Hereinafter, preferred embodiments of the “stimulus responsive polymer layer” and the “hydrophilic compound layer” will be described.

<Stimulus responsive polymer layer>
The functional organic compound layer is particularly preferably a stimulus-responsive polymer layer. The stimulus-responsive polymer layer is a layer containing a polymer in which the degree of cell adhesion on the surface is changed by a predetermined stimulus. 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 at which cells are cultured 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 to use a material exhibiting a property (cell adhesiveness) that makes the polymer shrink and easily adheres cells to the surface when water is desorbed from the polymer at a temperature equal to or higher than the same temperature. Such a critical solution temperature is called a lower critical solution temperature. A temperature-responsive polymer having a lower critical solution temperature T of 0 ° C. to 80 ° C., more preferably 0 ° C. to 50 ° C. may be used. This is because the cells can be stably cultured when T is 0 ° C to 80 ° C.

  Suitable temperature-responsive polymers include acrylic polymers or methacrylic polymers. Specific examples of suitable temperature-responsive polymers include poly-N-isopropylacrylamide (T = 32 ° C.), poly-Nn-propyl acrylamide (T = 21 ° C.), and poly-Nn-propyl methacrylamide. (T = 32 ° C.), poly-N-ethoxyethyl acrylamide (T = about 35 ° C.), poly-N-tetrahydrofurfuryl acrylamide (T = about 28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T = About 35 ° C.), and poly-N, N-diethylacrylamide (T = 32 ° C.).

  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, and vinyl ether derivatives. More than seeds may be used. When a single monomer is used alone, the polymer formed on the substrate is a homopolymer, and when multiple monomers are used together, the polymer formed on the substrate is a heteropolymer. Both forms are encompassed by the present invention.

  In addition, when it is necessary to adjust T depending on the type of proliferating cell, when it is necessary to enhance the interaction between the coating substance and the cell culture support, and the balance between the hydrophilicity and hydrophobicity of the cell support is adjusted. If necessary, other monomers other than those described above may be further added for copolymerization. Further, a graft or block copolymer of the above 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 and the ion responsive polymer, those suitable for the cell sheet to be prepared can be appropriately selected.

  The stimulus-responsive polymer layer is prepared by preparing a coating composition containing a monomer that is polymerized to form a target stimulus-responsive polymer and an organic solvent that can dissolve the monomer. A coating film is formed by coating on the surface of the layer, and then the monomer in the coating film is polymerized by an appropriate means such as radiation irradiation to form a polymer. And a grafting reaction between them.

<Hydrophilic compound layer>
As another embodiment of the functional organic compound layer, hydrophilicity such as an ethylene glycol chain composed of one or more ethylene glycol units (an ethylene glycol chain composed of a plurality of ethylene glycol units can be referred to as a “polyethylene glycol chain”). A layer of a functional compound. The terminal of the ethylene glycol chain may be in a form blocked with a hydroxyl group, or the terminal of the ethylene glycol chain may be in a form in which another substance such as a biological substance is linked by a covalent bond. A layer containing an ethylene glycol chain whose end is blocked with a hydroxyl group can provide a hydrophilic surface to which cells are difficult to adhere.

  Examples of biological substances that can be covalently bonded to the end of an ethylene glycol chain include antigens, antibodies, DNA, RNA, peptides, hormones, enzymes, cytokines, sugar chains, lipids, coenzymes, enzyme inhibitors, cells, and other functions. The protein which has is included. Furthermore, the low molecular weight compound which has affinity with such a biological substance, and a high molecular compound are also contained in the range of a biological substance.

  In order to immobilize a layer of a hydrophilic compound such as an ethylene glycol chain on the surface of the base material layer, it can be physically adsorbed on the surface of the base material layer in advance, A primer layer containing polysiloxane containing a functional group capable of reacting with a terminal hydroxyl group to form a covalent bond in the side chain is provided. The functional group on the side chain of the polysiloxane is preferably a glycidyl group or an epoxy group. The primer layer can be formed by applying a silanol compound having a desired side chain to the surface of the base material layer, and performing condensation polymerization on the surface to convert it into polysiloxane.

  Next, a functional group of the primer layer is reacted with a hydroxyl group of polyethylene glycol in which two or more ethylene glycol or ethylene glycol units are repeated to form a covalent bond, thereby immobilizing the ethylene glycol chain. At this time, ethylene glycol or polyethylene glycol containing a catalytic amount of concentrated sulfuric acid is brought into contact with the primer layer. A layer containing ethylene glycol chains whose ends are blocked with hydroxyl groups is formed in this way.

  Furthermore, if necessary, at least one functional group capable of forming a covalent bond with another substance is directly or indirectly linked to one end of the ethylene glycol chain. The method for introducing the functional group is not particularly limited.

<Method for producing cell culture container>
Hereinafter, an embodiment of a method for producing a cell culture container according to the present invention will be described with reference to FIGS. 5A to 5E. The example of FIGS. 5A to 5E shows the case where the container portion 100 of FIG. 1B is formed, but when the cell culture container shown in FIGS. 2A and 2B and other types of cell culture containers are manufactured. It can be similarly applied to.

  First, as shown in FIG. 5A, in order to manufacture a cell culture container, a core mold (male type, convex type) 501 that defines an inner wall of the container part 100 facing the space 130 for containing cells and a medium, An injection mold is used, which is composed of a cavity mold (female mold, concave mold) 503 that defines the outer wall of the container 100 and has an injection hole 502 for the resin A. A mold space 504 corresponding to the shape of the container body member is formed by clamping the core mold 501 and the cavity mold 503 in combination. In this wooden mold space 504, the functional base 140 is disposed so as to cover the injection hole 502 and so that the base material layer 401 side is in contact with the cavity mold 502. Thereby, the functional organic compound layer 402 side of the functional base 140 is exposed toward the inside of the mold space 504.

  Subsequently, as shown in FIG. 5B, the mold space 504 is filled with the resin A melted from the injection hole 502. Then, the functional substrate 140 is separated from the inner wall surface of the cavity mold 503 by the resin A flowing in the mold space 504 so that the functional organic compound layer 402 side of the functional substrate 140 is in contact with the inner wall surface of the core mold. In other words, the functional base 140 moves so as to be positioned on the inner bottom surface of the container main body member 103. Since the functional substrate is fixed to the surface of the container main body member facing the space for containing the cells and the culture medium, it is not necessary to provide a conventional means for adsorbing the functional substrate to the core mold. The organic compound layer can be maintained well without damaging the organic compound layer.

  Thereafter, as shown in FIG. 5C, filling of the mold space 504 with the resin A is completed, and the resin is solidified. After the resin A is solidified, the mold is opened to obtain the container body member 103 in which the functional substrate 140 is integrated (FIG. 5D). Finally, as shown in FIG. 5E, the top surface member 104 is joined to the open end of the container body member 103 to complete the container portion 100.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to this.

Example 1
Sample 1
A coating solution was prepared by dissolving N-isopropylacrylamide in isopropyl alcohol to a final concentration of 40% by weight. The coating liquid was applied onto the film using a wire bar on the surface of the polystyrene film (obtained from Asahi Kasei Chemicals Co., Ltd.) having a thickness of 50 μm, which was hydrophilized by corona treatment (1.4 g / m ² ). Then, after drying with hot air at 40 ° C., N-isopropylacrylamide was grafted by irradiation with an electron beam, and poly-N-isopropylacrylamide was immobilized on the film surface.

Sample 2
The coating liquid was applied onto the film by a gravure direct method (1.1 g / cm ² , conveyance speed: 5 m / cm) on a 50 μm-thick polystyrene film (obtained from Asahi Kasei Chemicals Corporation) by corona treatment. min). Thereafter, after passing through a dry hood at 40 ° C. for 2 m and drying, electron beam irradiation was performed to graft polymerize N-isopropylacrylamide, and poly-N-isopropylacrylamide was immobilized on the film surface.

  The produced Sample 1 and Sample 2 were cut into a sheet having an appropriate size, and then immersed in water for 4 hours and dried. And it cut out in the trapezoid shape using the hand press machine. Thus, a trapezoidal functional substrate was obtained.

  A functional substrate was placed in contact with the cavity mold of an injection molding machine (α-100C, FANUC), and in-mold molding was performed using polystyrene pellets (PS Japan SGP10) (resin temperature: 220 ° C., mold) Temperature: 20 ° C). Thereby, a member in which the functional substrate and the container main body member were integrated was produced. A polystyrene resin top surface member produced by injection molding was joined to this member by ultrasonic welding to obtain a flask-shaped container portion. Finally, capping was performed to produce a flask-type cell culture container (hereinafter referred to as “flask”) having a temperature-responsive bottom surface.

The flask was subjected to ultraviolet sterilization for a predetermined time in a clean bench, and bovine aortic vascular endothelial cells were adjusted to have a surface cell density of 1 × 10 ⁵ cells / cm ² and seeded in the prepared flask. The medium used was DMEM containing 10% FBS (manufactured by Sigma), and the culture was performed in a CO ₂ incubator at 37 ° C. and 5% CO ₂ for 48 hours. Thereafter, the flask was placed in an incubator under 20 ° C. and 5% CO 2 conditions. After 20 minutes, the product was removed from the incubator at 20 ° C. The cell sheet formed on the culture surface of the temperature-responsive film was peeled off.

100, 200 Container part 101, 201 Bottom part 102, 202 Side wall part 103, 203 Container body member 104 Top surface member 105 Through hole 106 Neck part 107 Locking part 120 Cell culture container 130, 220 Space 140, 210 Functional substrate 401 Base material Layer 402 Functional organic compound layer 501 Core mold 502 Injection hole 503 Cavity mold 504 Mold space 601 Core mold 602 Cavity mold 603 Mold space 604 Injection hole 610 Polyester film 611 Deposition layer 620 Petri dish A Resin S Polyethylene terephthalate

Claims (1)

A container for containing cells and medium;
The container part
A resin container body member;
A functional substrate comprising at least a base material layer and a functional organic compound layer disposed on the base material layer;
Have
A method for producing a cell culture container in which a functional substrate is fixed on the surface of a container body member so that the functional organic compound layer faces the space side containing cells and a medium,
A core mold defining an inner wall of the container portion facing a space containing cells and medium;
A cavity mold defining an outer wall of the container portion and having a resin injection hole;
In the mold space formed by the injection mold combined with
The functional substrate is disposed so as to cover the injection hole and the base material layer side is in contact with the cavity mold,
The said manufacturing method including the process of moving a functional base | substrate so that the functional organic compound layer side may contact | connect a core metal mold | die with the flow of resin by filling resin into a casting_mold | template space from the injection hole.

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