JP2013099884A - Method for manufacturing cell culture container - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technology for bonding a functional substrate having a functional organic compound layer and container body members while maintaining functions of the functional organic compound layer in a bonding method therefor accompanied by heat and pressure such as ultrasonic welding or injection molding.SOLUTION: In the method, the method includes the following process. A functional substrate (140) comprises a resin-made base material layer (502) and a functional organic compound layer (501). Resin-made container body members (101, 102, 103, 105, and 106) and the functional substrate are bonded to each other by melting and integrating resins by ultrasonic melting, in-mold molding or the like at parts where surfaces of the members are in contact with the surface of the base-material layer of the functional substrate or the peripheral edge (640 or 610) of the functional organic compound layer.

Description

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

  As a method for producing a plastic petri dish suitable for microorganisms and cells, a method according to Patent Document 1 has been proposed. Patent Document 1 discloses a technique in which the bottom inner surface of a petri dish is attached to a plastic film layer in which silicon oxide is deposited as a hydrophilic film on the surface of the film by an insert injection molding method.

Japanese Utility Model Publication No. 5-88299 JP-A-2-21865

  In recent years, for example, Patent Document 2 reports a technique for noninvasively collecting a cell sheet formed into a sheet shape using a temperature-responsive polymer such as polyisopropyl acrylamide. When a film having a hydrophilic polymer film containing a temperature-responsive polymer is bonded to a support member serving as a container by a process involving heat and pressure such as injection molding, the function of the hydrophilic polymer film is deteriorated. It was confirmed.

  The present invention has been made in view of the above circumstances. The present invention is a method for joining a functional substrate having a functional organic compound layer and a container body member with heat and pressure such as ultrasonic welding and injection molding, while maintaining the function of the functional organic compound layer. It aims at providing the technique to join.

The present invention includes the following inventions.
(1) A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
The container body member or a member that is a part of the container body and the functional substrate are melted and integrated at a portion where the surface of the member is in contact with the surface of the fourth region or the surface of the second region of the functional substrate. The said method including the joining process joined by doing.

(2) In the joining step, a resin-made container main body member or a member that is a part thereof, and a functional base, a surface of the member, a surface of a fourth region of the functional base, The surface of the member is in contact with the surface of the two regions and arranged so that the functional organic compound layer of the functional substrate faces the space for containing cells and culture medium. Ultrasonic welding that applies ultrasonic vibration to a portion where the surface of the fourth region or the surface of the second region is in contact with the surface of the functional substrate to melt and integrate the resin at the portion, thereby joining the container body member and the functional substrate. The method of (1) including a process.
(3) The ultrasonic welding process
The member and the functional substrate are arranged and combined so that the surface of the member and the surface of the fourth region of the functional substrate are in contact with each other,
An ultrasonic horn comprising a protruding portion capable of contacting the second region of the functional substrate and having a recessed portion surrounded by the protruding portion, for the combination of the member and the functional substrate, From the side of the member, the protrusion is brought into contact with a portion located on the back side of the second region of the functional base on the surface of the member, and in this state, ultrasonic vibration is applied from the ultrasonic horn. Alternatively, the method includes a step of pressing from the side of the functional substrate so that the protruding portion is in contact with the surface of the second region of the functional substrate, and in that state, applying ultrasonic vibration from the ultrasonic horn. The method of (2).

(4) The ultrasonic welding process
On the welding jig, from above, the functional substrate is placed so that the functional organic compound layer becomes the lower surface, and further the member is placed from above the functional substrate. From the side of the member, the ultrasonic horn is brought into pressure contact with the portion located on the back side of the second region of the functional base on the surface of the member, and in this state, ultrasonic vibration is applied from the ultrasonic horn. The method of (2) or (3) including the process of giving.
(5) A portion of the welding jig on which the functional base is placed includes a protrusion that can contact the second region of the functional base, and a portion surrounded by the protrusion is recessed. The method of (4), having a shape.
(6) The ultrasonic welding step is performed in a state where the removable first protective member is placed on the surface of the functional organic compound layer of the functional substrate, any one of (2) to (5) Method.
(7) The container body member is a resin first member to be bonded to the surface of the fourth region or the second region of the functional substrate, and the region surrounded by the portion to be bonded to the surface is opened. Including at least a portion of a first member having a configured shape,
The joining step includes
An injection mold for molding the first member, the surface of the fourth region of the functional substrate or the second region when the functional substrate is disposed at a predetermined position in the injection mold and clamped The surface of the mold is exposed to the mold space corresponding to the first member in the mold, and the inflow of the molten resin to the surface of the first region and the surface of the third region of the functional base is blocked by the inner wall of the mold. The functional base is placed at the predetermined position inside the injection mold formed in such a manner, the mold is clamped, the molten resin is filled in the mold space, and solidified. (1) The method of (1) including the process of shape | molding the said 1st member with which the base | substrate was integrated.

(8) A cell culture container provided with a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
Inside the injection mold for molding the container main body member or a part thereof, the functional substrate, the functional organic compound layer covers a part of the inner wall surface of the injection mold, and the base material layer The functional substrate is integrated by arranging the mold so that at least a part of the surface on the side is exposed to the mold space of the member, clamping the mold, filling the molten resin in the injection mold, and solidifying it. Forming the formed member,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
The region of the inner wall surface of the injection mold covered with the functional organic compound layer of the functional substrate includes a contact region that contacts the second region of the functional substrate, and a recessed region surrounded by the corresponding contact region, When the molten resin is filled in the injection mold in a state where the functional base is arranged so as to cover the area of the inner wall surface, the second area of the functional base and the contact area of the inner wall surface are in close contact with each other, The molten resin is prevented from flowing into the second region and the first region surrounded by the second region, and a gap is formed between the first region of the functional base and the recessed region of the inner wall surface. Said method.

(9) A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
Inside the injection mold for molding the container body member or a member that is a part of the container body, the functional substrate is a portion including the first region on the surface of the functional substrate on the side of the functional organic compound layer. In a state where the removable second protective member is placed, the second protective member side covers a partial region of the inner wall surface of the injection mold, and at least a part of the surface on the base material layer side is the above-mentioned A container body member integrated with a functional substrate or a part thereof by placing the mold so as to be exposed in the mold space of the member, clamping the mold, filling the molten resin in the injection mold, and solidifying it. The said method including the process of shape | molding the member which is.

(10) A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
Inside the injection mold for molding the container main body member or a member that is a part thereof, the functional substrate is placed on the surface of the functional substrate on the base material layer side including the third region. With the protective member placed, the functional organic compound layer covers a partial region of the inner wall surface of the injection mold, and at least a part of the surface of the third protective member is exposed to the mold space. A mold body, or a member that is a part of the container main body integrated with the functional substrate, is filled with a molten resin in the injection mold and solidified. Including said method.

  According to the present invention, the function of the functional organic compound layer is maintained in a bonding method involving heat and pressure, such as ultrasonic welding and injection molding, between the functional substrate having the functional organic compound layer and the container body member. It is possible to join them.

FIG. 1A shows an example of a cell culture container produced in the present invention. FIG. 1B is a cross-sectional view taken along the line I-I ′. FIG. 1C is a cross-sectional view taken along the line II-II ′. FIG. 2A shows an example of a cell culture container produced in the present invention. FIG. 2B is a sectional view taken along line III-III '. FIG. 3 shows an example of an embodiment in which after the functional substrate is fixed to the container partial member, the container portion is completed by combining other members. FIG. 4 shows an example of an embodiment in which after the functional substrate is fixed to the container partial member, the container portion is completed by combining other members. FIG. 5 shows a cross-sectional view of the functional substrate. FIG. 6A shows the surface 601 of the functional substrate 140 on the functional organic compound layer 501 side. FIG. 6B shows the surface 602 of the functional substrate 140 on the substrate layer 502 side. FIG. 7A shows an example of a container partial member for ultrasonic welding. FIG. 7B is a cross-sectional view taken along the line IV-IV ′. FIG. 8 is a schematic cross-sectional view for explaining an example of a joining process by ultrasonic welding. FIG. 9 is a perspective view for explaining the upper and lower positional relationship of each element in an example of a joining process by ultrasonic welding. FIG. 10 is a schematic cross-sectional view for explaining an example of a joining process by ultrasonic welding. FIG. 11 is a schematic cross-sectional view for explaining an example of a joining process by ultrasonic welding. FIG. 12 is a schematic cross-sectional view for explaining an example of a joining process by ultrasonic welding. FIG. 13 is a schematic cross-sectional view for explaining an example of a joining process by in-mold molding. FIG. 14A shows an example of the first member of the container part to which the functional substrate is bonded. FIG. 14B is a cross-sectional view taken along the line V-V ′. FIG. 15 is a schematic cross-sectional view for explaining an example of a joining process by in-mold molding. FIG. 16 is a schematic cross-sectional view for explaining an example of a joining process by in-mold molding. FIG. 17 is a schematic cross-sectional view for explaining an example of a joining process by in-mold molding. FIG. 18 is a schematic cross-sectional view for explaining an example of a joining process by in-mold molding.

  In the following description, the features of the present invention will be described with reference to the drawings as needed. However, each drawing is a schematic view, and the size and shape of each portion are appropriately exaggerated for easy understanding. It shows.

<Shape of cell culture vessel>
The overall shape of the cell culture container produced in 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. May be.

  An example of a preferred embodiment of the container portion is shown in FIG. 1A includes a bottom portion 101, a side wall portion 102 standing on the periphery of the bottom portion 101, and a top surface portion 103 that is joined to the upper end portion of the side wall portion 102 and is disposed to face the bottom portion 101. At least. A through-hole 104 is formed in a part of the side wall portion 102, and a container portion having a shape called a “flask type” including a neck portion 105 extending from the periphery of the through-hole 104 to the outside of the container portion. A locking portion 106 for locking the lid 110 is formed on the neck portion 105 of the container portion 100, and the lid 110 is detachably mounted through the locking portion. 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 portion 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 100 surrounded by the bottom 101, the side wall 102, and the top surface 103. 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 bottom part 201 and the side wall part 202 standingly arranged in the periphery of the bottom part 201 is mentioned. A functional substrate 210 is fixed to the surface (inner bottom surface) of the bottom portion 201 on the side of the space 220 for accommodating cells and culture medium.

  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 body member is a part composed of the bottom part 101, the side wall part 102, the top surface part 103 and the neck part 105. In the container part 200 of FIG. 2, the container body member is It is a part composed of a bottom part 201 and a side wall part 202.

  The joining step in the present invention is a step of joining the container body member or a member that is a part thereof and the functional substrate. When a member that is a part of the container body member (hereinafter sometimes referred to as “container part member”) is used in the joining step, the container part member and the other member are joined after the functional substrate is joined. To complete the container. The point of dividing the container portion in this way will be described in more detail with reference to FIGS.

  As shown in FIG. 3, in one embodiment for forming the container portion 100 after the joining step, the functional base 140 is provided on the inner bottom surface of the container partial member 301 including the bottom portion 101 and the side wall portion 102 in the joining step. Join. Next, the container portion 100 is formed by joining the top surface member 302 corresponding to the top surface portion 103. The container part member 301 and the top surface 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.

In another embodiment for forming the container part 100 after the joining process shown in FIG. 4, the functionality of the container part member 401 corresponding to the bottom part 101 on the surface facing the space 130 after completion in the joining process is shown. The substrate 140 is bonded. The container part 100 is formed by joining the side wall member 402 corresponding to the side wall part 102 provided with the neck part 105 and the top surface member 302 corresponding to the top surface part 103.
The combination of a plurality of members is not limited to the form shown in FIGS.

<Material of cell culture container>
The material which forms the member of other cell culture containers, such as a container part and a lid | cover, is not specifically limited, The material generally used in cell culture can be used.

  In the method of the present invention, at least the container portion is a resin material that can be joined or injection-molded by ultrasonic welding.

  Examples of the resin constituting the container part 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 resin. , A resin material such as a melamine resin, an epoxy resin, or a vinyl chloride resin, or a resin material containing at least one of the above-described materials subjected to surface hydrophilization treatment. The resin material is particularly preferably a polystyrene resin or a polyethylene terephthalate resin.

<Functional substrate>
As shown in FIG. 5, the cross section along the thickness direction of the functional substrate 140 in the present invention includes at least a resin base layer 502 and a functional organic compound layer 501 disposed on the base layer 502. Prepare. Here, the “base” may be a film or a plate-like body as long as it has a predetermined structure.

  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 a resin base layer by an appropriate method. One or more other layers (for example, a primer layer described later) may be present between the resin 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 resinous substrate layer is used, and if the functional substrate is a film-like, a film-like resinous substrate layer (hereinafter referred to as “film substrate layer”) is used. It is done.

  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 ultrasonic welding or in-mold molding of the present invention.

  The base material layer should just contain the resin material which can form the above-mentioned functional organic compound layer in one surface, and the kind of material is not specifically 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), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic and the like.

  The surface of the part of the base material layer to be joined to 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 preferably a thickness that imparts flexibility, 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 may be, for example, in the range of 0.5 nm to 300 nm, and 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-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 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.

  Bio-related substances that can be covalently bonded to the end of 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 hydrophilic compound layer such as an ethylene glycol chain on the surface of a resin base material layer, the surface 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 forming a covalent bond by reacting with the terminal hydroxyl group of the glycol 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, the manufacturing method of the cell culture container concerning this invention is demonstrated concretely. The bonding step will be described mainly with respect to an embodiment in which the functional base 140 is bonded to the container partial member 301 shown in FIG. 3, but the step is not limited to the embodiment.

<First embodiment>
The first embodiment of the method of the present invention is:
A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
The container body member or a member that is a part of the container body and the functional substrate are melted and integrated at a portion where the surface of the member is in contact with the surface of the fourth region or the surface of the second region of the functional substrate. And a joining step for joining.

  As shown in FIG. 6A, a region of the surface 601 of the functional organic compound layer 501 of the functional substrate 140 that functions in a space for containing cells and a medium is referred to as a first region 610, and its periphery is defined as the first region 610. The surrounding area is referred to as a second area 620. The “first region for functioning in a space for containing cells and a medium” is a region that exhibits a desired function in an environment where cells and / or a medium are present during culture, It is an exposed area. The second region 620 is preferably exposed in the space and exhibits a desired function in the same manner as the first region, but is not limited thereto, and may be not exposed in the space. Good. Then, as shown in FIG. 6B, a region corresponding to the back side of the first region on the surface 602 on the base material layer 502 side of the functional base 140 is referred to as a third region 630 and corresponds to the back side of the first region. The area is referred to as a fourth area 640.

  In the first embodiment of the present invention, in the joining step, the surface of the fourth region 640 or the surface of the second region 620 (preferably, the surface of the fourth region 640) of the functional substrate 140, and the resin container body The resin is melted and integrated (ie, welded) at a portion where the surface of the member or the container partial member comes into contact. Since there is no need to weld the resin members in the first region 610 of the functional substrate 140 and the third region 630 on the back side of the functional substrate 140 that should function in cell culture, the influence of heat and force accompanying welding in the first region The cell culture container can be manufactured while maintaining the function of the first region 610.

  As a method for joining the functional substrate and the resin container main body member or the container partial member, typically, a joining method by ultrasonic welding and a joining method by in-mold molding may be mentioned. In-mold molding means that in the present invention, a functional base is disposed at a predetermined position in an injection mold in which a mold space for manufacturing a container body member or a container partial member is formed, and the mold space is clamped. A method of obtaining a container main body member or a container partial member in which a functional substrate is integrated by filling a resin in a molten state and solidifying the inside. Hereinafter, the joining method by ultrasonic welding and the joining method by in-mold molding will be specifically described.

<First Embodiment / Joint Method by Ultrasonic Welding>
An example in which the functional substrate 140 is joined to the resin container partial member 301 ′ by ultrasonic welding will be described with reference to FIG.

  In ultrasonic welding, a resin member provided with a thin resin projection that functions as an energy director and another resin member are usually brought into contact with each other while pressing the apex of the energy director against the other resin member. While maintaining the state, the energy director is melted by oscillating ultrasonic waves from an ultrasonic horn to vibrate the contact surface, and both members are integrated and joined. As shown in FIG. 7, the bonding portion of the resin container partial member 301 ′ with the fourth region of the functional substrate 140 has a narrow width that functions as an energy director when ultrasonic welding is performed. The protrusion 701 is integrally formed. The lateral width of the protrusion 701 is not limited, but is about 100 to 1000 μm, and the height is about 50 to 1000 μm. It is desirable that the protrusion 701 has a sharp tip.

  As shown in FIG. 8A, the functional substrate 140 is placed in the container partial member 301 ′ fixed to the welding jig 801, and the functional organic is placed on the side of the space 130 (FIGS. 1B and 1C) for containing cells and culture medium. Arranged so that the surface 601 on the compound layer 501 side faces, and is pressed from the functional substrate 140 side so that the protruding portion 811 of the ultrasonic horn 810 contacts the surface of the second region of the functional substrate, In this state, ultrasonic vibration is applied from the ultrasonic horn 810. Accordingly, the protrusion 701 is melted and solidified on the contact surface between the fourth region of the functional base 140 and the container part member 301 ′, and the functional base 140 and the container part member 301 ′ are integrated (FIG. 8B). , C). Finally, the top surface member 302 corresponding to the top surface portion 103 is joined to complete the container portion 100.

  FIG. 8 shows a mode in which the ultrasonic horn 810 is brought close to the functional base 140 side and ultrasonic vibration is applied in a state where the container partial member 301 ′ and the functional base 140 are combined and arranged. Instead, as shown in FIG. 9 and the like, the ultrasonic horn 810 may be approached from the container partial member 301 ′ side to apply ultrasonic vibration.

  As shown in the figure, the ultrasonic horn 810 includes a protruding portion 811 having a shape capable of coming into contact with the second region 620 of the functional base 140, and a portion in which the portion surrounded by the protruding portion is recessed is used. It is preferable (see also the perspective view of FIG. 9). Since the ultrasonic horn having such a shape can selectively contact only the second region 620 of the functional base 140 (or the portion located on the back side of the second region 620 of the container partial member 301 ′), The adverse effect on the function of the first region 610 of the functional substrate 140 due to the shear stress due to the contact of the ultrasonic horn, the pressure for pressing the ultrasonic horn, and the ultrasonic vibration can be reduced.

  In a more preferred embodiment of the ultrasonic welding process, as shown in FIG. 9, the functional substrate 140 is placed on the welding jig 901 from above so that the surface 601 on the functional organic compound layer 501 side becomes the lower surface. The container partial member 301 ′ is further placed from above. Since the outer shape of the welding jig 901 matches the inner surface of the container part member 301 ′, the container part member 301 ′ is fitted to the welding jig 901 with the functional base 140 interposed. Can be held stably. Further, the ultrasonic horn 810 is pressed from the container partial member 301 ′ side so that the ultrasonic horn comes into contact with a portion located on the back side of the second region 620 of the functional base 140 on the surface of the member. In this state, ultrasonic vibration is applied from the ultrasonic horn. A schematic diagram of the cross section at this time is shown in FIG. The container part member 301 ′ is disposed on the welding jig 901 with the functional base 140 interposed (FIG. 10A), and the protruding portion 811 of the ultrasonic horn 810 is pressed from the container part member 301 ′ side. In this state, ultrasonic vibration is applied to melt and integrate (FIG. 10B). According to this mounting order, it is possible to avoid remaining bubbles between the functional base 140 and the container partial member 301 ′. This effect is particularly remarkable when a functional film is used as the functional substrate. The functional substrate (film) 140 is flattened by being pressed by the ultrasonic horn 810 while being sandwiched between the top surface of the welding jig 901 and the bottom plate of the container part member 301 ′, and bubbles are removed. It will be easier.

  In another preferred embodiment of the ultrasonic welding process, the functional substrate 140 is placed as shown in FIG. 11 in place of the welding jig 901 having a top surface that contacts the flat functional substrate 140. A welding jig 1100 having a shape in which a portion includes a protrusion 1101 that can come into contact with the second region 620 of the functional base 140 and a portion surrounded by the protrusion is recessed is used. When this welding jig 1100 is used, the contact surface between the welding jig and the surface 601 on the functional organic compound layer 501 side of the functional base 140 is only the second region 620, and the first Since the region 610 and the welding jig 1100 are not in contact with each other, shear stress is not generated on the surface of the first region 610 during ultrasonic vibration (FIG. 11B), and damage to the function of the first region 610 can be prevented.

  In another preferred embodiment of the ultrasonic welding process, the above-described ultrasonic welding process is performed by placing the removable first protective member 1201 on the surface 601 of the functional substrate 140 on the functional organic compound layer 501 side. To implement. In the embodiment shown in FIG. 12, the first protective member 1201 is interposed between the surface 601 on the functional organic compound layer 501 side of the functional base 140 and the top surface of the welding jig 901, and ultrasonic vibration And the functional base 140 and the container partial member 301 ′ are joined (FIG. 12B). After joining, the first protective member 1201 is removed and the process proceeds to the subsequent steps (FIGS. 12C and 12D). Although not shown, in the embodiment in which the ultrasonic horn is pressed against the functional base 140 as shown in FIG. 8, the ultrasonic welding process is performed with the first protective member similarly placed. Can be implemented. The presence of the first protective member can avoid direct contact between the welding jig or ultrasonic horn and the surface 601 of the functional organic compound 501 layer of the functional substrate 140, so that the functional organic compound layer The shear stress that can lead to damage of the function of 501 can be reduced.

  Although it does not specifically limit as a 1st protective member, Polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene, Medium density polyethylene, vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane, acrylic, silicone resin-based, Teflon, fluororesin-based protective members, or inorganic materials such as aluminum in these protective members A metal or silicon-deposited material can be used. As the first protective member, a film-like or plate-like one can be used. The protective member on which an inorganic metal such as aluminum or silicon is deposited is preferably disposed so that the surface of the deposited layer is in contact with the functional organic compound layer.

<First embodiment / joining method by in-mold molding>
An outline of a method of joining a resin container body member or container part member and a functional substrate by in-mold molding will be described with reference to FIG. In an injection mold that combines a convex mold 1301 and a concave mold 1302 that form a mold space 1304 corresponding to the container part member 301 at the time of mold clamping, the functional base 140 has a surface 601 on the functional organic compound layer 501 side as an injection mold. A part of the inner wall surface is covered, and at least a part (the whole in FIG. 13) of the surface 602 on the base material layer 502 side is disposed so as to be exposed to the mold space 1304 (FIG. 13A), and the mold is clamped. Next, the resin is filled into the mold through the gate 1303 by applying injection pressure (FIG. 13B). After filling, the mold is opened after the resin is solidified, and the container partial member 301 to which the functional substrate 140 is fixed is obtained (FIG. 18C). Finally, the top member 302 is joined to the open end of the container partial member 301 to complete the container unit 100.

  However, in the method shown in FIG. 13, since the high-temperature molten resin comes into contact with the third region located behind the first region 610 of the functional base 140, the function of the first region can be degraded by the heat of the molten resin. There is sex. This possibility is more likely to be avoided when the functional substrate 140 is a thin functional film.

  Therefore, in the first embodiment of the present invention, in-mold molding is performed in the manner shown in FIGS.

  In this example, as shown in FIG. 14, a resin-made first member 1401 in which an opening 1402 is formed is shown. The functional substrate 140 is bonded to the peripheral portion of the opening 1402 via the fourth region 640. The third region 630 of the functional substrate 140 is exposed from the opening 1402. Although not shown, the functional substrate 140 may be joined to the first member 1401 via the second region 620. In this case, the first region accommodates cells and a medium from the opening 1402. It is exposed to the space 130.

  Next, a procedure for fixing the functional base 140 to the resin first member 1401 will be described with reference to FIG.

  An injection mold for molding the first member 1401 is composed of a pair of a convex mold 1501 and a concave mold 1502. When the functional substrate 140 is arranged at a predetermined position in the mold so that the surface 601 of the functional organic compound 501 layer is in contact with the inner wall surface of the mold and the mold is clamped, the surface of the fourth region 640 of the functional substrate 140 Is exposed to the mold space 1506 corresponding to the first member 1401 in the mold. On the other hand, a raised portion 1504 is formed on the inner wall surface of the mold so as to contact the third region 630 of the functional base 140 so as to cover the region when the mold is clamped. The mold is clamped and the molten resin is filled through the gate 1503. At this time, the raised portion 1504 prevents the molten resin from flowing into the surface of the third region. On the other hand, the inflow of the molten resin to the surface 601 of the functional organic compound layer 501 including the first region and the second region is blocked by the convex mold 1501. After filling the resin, the resin is solidified, taken out from the mold, and a member in which the functional base 140 is joined to the first member 1401 through the fourth region is obtained. According to this embodiment, since the high-temperature molten resin does not come into contact with the first region and the third region of the functional base 140, the possibility that the function of the surface of the first region is lowered due to heat can be reduced.

  Although not shown, at the time of mold clamping, a part of the inner wall surface of one injection mold abuts on and covers the entire surface of the functional base 140 including the third region and the fourth region. The top surface of the raised portion formed on a part of the inner wall surface of the other injection mold is in contact with and covers the first region of the functional base 140, and the second region of the functional base 140 is the mold. You may be comprised so that it may be exposed to space. Further, when the mold is clamped, the top surface of the raised portion formed on a part of the inner wall surface of one injection mold is in contact with and covers the first region of the functional base 140, and one injection mold The top surface of the raised portion formed on a part of the inner wall surface of the substrate is in contact with and covers the third region of the functional base 140, and the second region and the fourth region are exposed to the mold space. May be.

<Second embodiment / joining method by in-mold molding>
In the method shown in FIG. 13, since the first region 610 of the functional base 140 and the inner wall surface of the mold are in contact with each other, there is a possibility that the function of the first region is deteriorated due to shear stress at the time of injection molding. In order to reduce this possibility, the following second embodiment of the joining process by in-mold forming is provided.

  In the second embodiment of the joining process by in-mold molding, a functional base is placed inside an injection mold for molding a container body member or a member that is a part thereof, and a functional organic compound layer is an injection mold. Covering a partial region of the inner wall surface of the substrate, and arranging so that at least a part of the surface on the base material layer side is exposed to the mold space of the member, clamping the mold, and injecting the molten resin into the injection mold A step of forming the member integrated with the functional base by filling and solidifying, and the region of the inner wall surface of the injection mold covered with the functional organic compound layer of the functional base is a functional base The molten resin is placed in the injection mold in a state in which the functional base is disposed so as to cover the area of the inner wall surface. When filled, the second region and inner wall of the functional substrate In contact with the second region and the inflow of the molten resin into the second region and the first region surrounded by the second region, and the first region of the functional substrate and the inner wall surface An air gap is formed between the hollow region.

  A specific example of this embodiment is shown in FIG. In this embodiment, a pair of a convex mold 1601 and a concave mold 1602 that form a mold space 1604 corresponding to the container partial member 301 at the time of mold clamping is used as the injection mold. In the convex mold 1601, a contact region 1610 that contacts and contacts the second region 620 when covered with the surface 601 of the functional base 140 on the side of the functional organic compound layer 501 is in contact with the second region 620. Is provided. Further, a recessed region 1611 is formed in a portion surrounded by the contact region 1610 of the convex mold 1601. When the resin is filled, the contact area 1610 and the second area 620 of the functional base 140 are in close contact with each other, and the inflow of the molten resin to the first area surrounded by the second area and the second area is prevented. Is done. Due to the presence of the depression region 1611, the first region 610 of the functional base 140 does not contact the convex mold 1601. For this reason, it can avoid that the function of the 1st field of a functional base falls by shear stress.

<Third embodiment / Join method by in-mold molding>
In the method shown in FIG. 13, since the first region 610 of the functional base 140 and the inner wall surface of the mold are in contact with each other, there is a possibility that the function of the first region is deteriorated due to shear stress at the time of injection molding. As another means for reducing this possibility, the following third embodiment of the joining process by in-mold molding is provided.

In the third embodiment of the bonding process by in-mold molding, a functional substrate is placed inside an injection mold for molding a container body member or a member that is a part thereof, and the functional organic compound of the functional substrate. In a state where the removable second protective member is placed on the layer-side surface including the first region, the second protective member side covers a partial region of the inner wall surface of the injection mold, And, it is arranged so that at least a part of the surface on the base material layer side is exposed in the mold space of the member, clamped, filled with molten resin in the injection mold, and solidified. Including a step of forming a container body member integrated with a conductive substrate or a member that is a part thereof,
It is characterized by.

  A specific example of this embodiment is shown in FIG. In this embodiment, as the injection mold, a pair of a convex mold 1701 and a concave mold 1702 that form a mold space 1704 corresponding to the container partial member 301 at the time of clamping is used. In this embodiment, with the removable second protective member 1710 placed on the surface 601 of the functional substrate 140 on the functional organic compound layer 501 side, the functional substrate is brought into contact with the convex 1701. The mold is clamped and filled with molten resin (FIG. 17B), and injection molding is performed. After the molded product is removed from the mold, the second protective member 1710 is removed to obtain the container partial member 301 to which the functional base 140 is fixed (FIG. 17C). The presence of the second protective member can avoid direct contact between the inner wall surface of the injection mold and the surface 601 of the functional organic compound layer 501 of the functional base 140, which may lead to functional damage. It is possible to reduce shear stress.

  Although it does not specifically limit as a 2nd protection member, Polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene, Medium density polyethylene, vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane, acrylic, silicone resin-based protective members such as Teflon, or these protective members such as aluminum A material on which an inorganic metal or silicon is deposited can be used. As the second protective member, a film-like or plate-like one can be used. The protective member on which an inorganic metal such as aluminum or silicon is deposited is preferably disposed so that the surface of the deposited layer is in contact with the functional organic compound layer.

<Fourth embodiment / Join method by in-mold molding>
In the method shown in FIG. 13, since the high-temperature molten resin is in direct contact with the surface of the functional substrate 140 on the base material layer side, the function of the functional organic compound layer may be lowered depending on the temperature. As another means for reducing this possibility, the following fourth embodiment of the joining process by in-mold molding is provided.

  In the fourth embodiment of the joining process by in-mold molding, the functional substrate is placed inside the injection mold for molding the container body member or a member that is a part of the container body member, and the base layer side of the functional substrate. The functional organic compound layer covers a partial region of the inner wall surface of the injection mold with the third protective member placed on the portion of the surface including the third region, and the third protection A container in which a functional substrate is integrated by arranging and clamping so that at least a part of the surface of the member is exposed to the mold space, filling the molten resin in the injection mold, and solidifying it. The method includes a step of forming a main body member or a member that is a part thereof.

  A specific example of this embodiment is shown in FIG. In this embodiment, a pair of a convex mold 1801 and a concave mold 1802 that form a mold space 1804 corresponding to the container partial member 301 at the time of clamping is used as the injection mold. In this embodiment, the third protective member 1810 is disposed on the surface 602 of the functional substrate 140 on the base material layer 502 side. At this time, the third protective member 1810 is disposed so as to cover at least the third region 630 of the functional substrate 140. In a state in which the functional base 140 is overlapped with the third protective member 1810, the surface 601 on the functional organic compound layer 501 side is brought into contact with the convex 1801, and at least a part of the surface of the third protective member 1810 (see FIG. 18 is arranged so that the whole is exposed to the mold space 1804. The mold is clamped and filled with molten resin (FIG. 18B), and injection molding is performed. In the molded product, the third protective member 1810 is integrated in a state of being sandwiched between the functional base 140 and the container portion member 301 (FIG. 18C). In this embodiment, the presence of the third protective member prevents the molten resin from coming into direct contact with the functional base 140, thereby reducing the influence of heat that can lead to functional damage. it can. This effect is particularly advantageous when the functional substrate 140 is a film-like functional film.

  The third protective member is not particularly limited as long as it can be integrated with the functional substrate 140 and the container part member 301, but preferably polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene, medium density polyethylene, vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyethersulfone, polyethylene naphthalate, polypropylene, urethane, acrylic Examples thereof include a member subjected to surface treatment such as corona treatment or plasma treatment, or a member applied with a heat sealant. Further, a wood fiber sheet such as a cellulose mesh sheet having a high heat insulating effect may be used. As the third protective member, a film-like or plate-like one can be used.

  As described above, the resin base material is formed in at least one region of the inner wall surface of the container portion facing the inner chamber of the resin portion in which the inner chamber for containing the cells and the medium is formed. A functional substrate comprising at least a functional organic compound layer containing a stimulus-responsive polymer disposed on the base material layer, and the surface of the functional substrate on the side of the functional organic compound layer, A region for functioning in the container portion is a first region, a region surrounding the first region is a second region, a surface on the base layer side on the back side of the first region of the functional base is a third region, When the surface on the base material layer side on the back side of the second region of the functional substrate is the fourth region, the container portion, the functional substrate, the surface of the inner wall surface of the container portion, and the fourth region of the functional substrate. In the part where the surface of the surface or the surface of the second region is in contact, the resin was melted and joined, Cells culture vessel is provided.

(Example 1) In-mold molding method
Sample 1
N-Isoproacrylamide was dissolved in isopropyl alcohol to a final concentration of 20% by weight to prepare a coating solution. 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., electron beam irradiation was performed to graft polymerize N-isoproacrylamide, and poly-N-isoproacrylamide 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). Then, after passing through a drying hood at 120 ° C. for 2 m and drying, electron beam irradiation was performed to graft polymerize N-isoproacrylamide, and poly-N-isoproacrylamide 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 like FIG. 6 using the hand press machine. Thus, a trapezoidal functional substrate was obtained.

  A functional substrate is placed on a core mold (convex mold) of an injection molding machine (α-100C, FANUC). A cavity mold (concave mold) with a guide is arranged so that the molten resin does not contact the functional surface of the functional substrate. In-mold molding was performed using polystyrene pellets (PS Japan SGP10) (resin temperature: 220 ° C., mold temperature: 20 ° C.). As a result, a member in which the functional substrate and the bottom plate were joined was produced. Thereafter, the peripheral wall member and the top plate member made of polystyrene resin produced by injection molding were sequentially joined to the previously produced member by ultrasonic welding to obtain a flask-shaped member. 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.

(Example 2) Ultrasonic fusion 1
Using an ultrasonic plastic welding machine (900 series, Branson), a functional substrate similar to that prepared in Example 1 is formed on a metal welding jig, and the functional polymer layer is the surface of the welding jig. Place it so that it touches.

Using a bottom plate member, a peripheral side wall member, and a top plate member made of polystyrene resin for constituting the flask type vessel, a flask type cell culture vessel was prepared according to the following procedure. Specifically, a peripheral side wall member made of polystyrene resin that can be fitted around the welding jig on which the functional base is placed, a position in contact with the peripheral side wall member, and the functional base A bottom plate member made of polystyrene resin in which an energy director is formed at the periphery of the position where is fixed. The peripheral side wall member is fitted and fixed to the welding jig on which the functional base is placed, and the bottom plate member is contacted from above, and the energy director contacts the base layer of the functional base and the end of the peripheral side wall member. Was placed as follows. An ultrasonic horn was pressed from the side of the bottom plate member, and ultrasonic vibration was applied at 20 kHz for 2 seconds to melt the energy director and weld each member. Thereafter, a polystyrene resin top plate member produced by injection molding was joined by ultrasonic welding to obtain a flask-shaped member. Finally, capping was performed to produce a flask having a temperature responsive function on the bottom surface.
No bubbles were found between the functional substrate and the bottom plate member.

(Example 3) Ultrasonic fusion 2
A functional base substrate similar to that of Example 2 and a bottom plate member, a peripheral side wall member, and a top plate member made of polystyrene resin for constituting a flask-type container were prepared.

  Using an ultrasonic plastic welding machine (900 series, Branson), the bottom plate member is fixed on a metal welding jig, the functional substrate is placed on the side of the bottom plate member, and the energy of the bottom plate member It arrange | positioned so that a director may be located in the peripheral part of the surface of a base material layer.

  An ultrasonic horn was pressed from the functional substrate side, and ultrasonic vibration was applied at 20 kHz for 2 seconds to melt the energy director and weld the bottom plate member and the functional substrate. As a result, a member in which the functional substrate and the bottom plate were joined was produced. Thereafter, the peripheral wall member and the top plate member made of polystyrene resin produced by injection molding were sequentially joined to the previously produced member by ultrasonic welding to produce a flask type cell culture vessel. Finally, capping was performed to produce a flask having a temperature responsive function on the bottom surface.

  In this case, a slightly larger number of bubbles were observed between the functional substrate and the bottom plate portion as compared with Example 2, but there was no practical problem.

DESCRIPTION OF SYMBOLS 100,220 ... Container part 120 ... Cell culture container 101-106, 201-202 ... Container main body member 140,210 ... Functional base | substrate 610 ... 1st area | region 620 ... 2nd Region 630 ... Third region 640 ... Fourth region

Claims (10)

A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
The container body member or a member that is a part of the container body and the functional substrate are melted and integrated at a portion where the surface of the member is in contact with the surface of the fourth region or the surface of the second region of the functional substrate. The said method including the joining process joined by doing.   The joining step includes a resin-made container body member prepared in advance or a member that is a part thereof, and a functional substrate, a surface of the member, a surface of a fourth region of the functional substrate, or a second region. The surface of the member is in contact with the surface, and the functional organic compound layer of the functional substrate is arranged on the space side for containing the cells and the medium so that the functional organic compound layer faces. Including an ultrasonic welding step in which ultrasonic vibration is applied to a portion where the surface of the fourth region or the surface of the second region is in contact to melt and integrate the resin at the portion, thereby joining the container body member and the functional substrate. The method of claim 1. Ultrasonic welding process
The member and the functional substrate are arranged and combined so that the surface of the member and the surface of the fourth region of the functional substrate are in contact with each other,
An ultrasonic horn comprising a protruding portion capable of contacting the second region of the functional substrate and having a recessed portion surrounded by the protruding portion, for the combination of the member and the functional substrate, From the side of the member, the protrusion is brought into contact with a portion located on the back side of the second region of the functional base on the surface of the member, and in this state, ultrasonic vibration is applied from the ultrasonic horn. Alternatively, the method includes a step of pressing from the side of the functional substrate so that the protruding portion is in contact with the surface of the second region of the functional substrate, and in that state, applying ultrasonic vibration from the ultrasonic horn. The method of claim 2. Ultrasonic welding process
On the welding jig, from above, the functional substrate is placed so that the functional organic compound layer becomes the lower surface, and further the member is placed from above the functional substrate. From the side of the member, the ultrasonic horn is brought into pressure contact with the portion located on the back side of the second region of the functional base on the surface of the member, and in this state, ultrasonic vibration is applied from the ultrasonic horn. 4. The method of claim 2 or 3 comprising the step of providing.   The site | part in which the functional base | substrate is mounted of the said welding jig is provided with the protrusion part which can contact | abut with the 2nd area | region of a functional base | substrate, and has the shape where the part enclosed by this protrusion part is depressed. The method of claim 4.   The method according to any one of claims 2 to 5, wherein the ultrasonic welding step is performed with a removable first protective member placed on the surface of the functional organic compound layer of the functional substrate. The container body member is a resin-made first member that is bonded to the surface of the fourth region or the second region of the functional substrate, and an area surrounding the portion to be bonded to the surface is opened. Including at least a portion of a shaped first member;
The joining step includes
An injection mold for molding the first member, the surface of the fourth region of the functional substrate or the second region when the functional substrate is disposed at a predetermined position in the injection mold and clamped The surface of the mold is exposed to the mold space corresponding to the first member in the mold, and the inflow of the molten resin to the surface of the first region and the surface of the third region of the functional base is blocked by the inner wall of the mold. The functional base is placed at the predetermined position inside the injection mold formed in such a manner, the mold is clamped, the molten resin is filled in the mold space, and solidified. The method of claim 1, comprising forming the first member with an integrated substrate. A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
Inside the injection mold for molding the container main body member or a part thereof, the functional substrate, the functional organic compound layer covers a part of the inner wall surface of the injection mold, and the base material layer The functional substrate is integrated by arranging the mold so that at least a part of the surface on the side is exposed to the mold space of the member, clamping the mold, filling the molten resin in the injection mold, and solidifying it. Forming the formed member,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
The region of the inner wall surface of the injection mold covered with the functional organic compound layer of the functional substrate includes a contact region that contacts the second region of the functional substrate, and a recessed region surrounded by the corresponding contact region, When the molten resin is filled in the injection mold in a state where the functional base is arranged so as to cover the area of the inner wall surface, the second area of the functional base and the contact area of the inner wall surface are in close contact with each other, The molten resin is prevented from flowing into the second region and the first region surrounded by the second region, and a gap is formed between the first region of the functional base and the recessed region of the inner wall surface. Said method. A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
Inside the injection mold for molding the container body member or a member that is a part of the container body, the functional substrate is a portion including the first region on the surface of the functional substrate on the side of the functional organic compound layer. In a state where the removable second protective member is placed, the second protective member side covers a partial region of the inner wall surface of the injection mold, and at least a part of the surface on the base material layer side is the above-mentioned A container body member integrated with a functional substrate or a part thereof by placing the mold so as to be exposed in the mold space of the member, clamping the mold, filling the molten resin in the injection mold, and solidifying it. The said method including the process of shape | molding the member which is. A cell culture container comprising a container part for containing cells and a medium,
The container part
A resin container body member;
A functional substrate including at least a resin base layer and a functional organic compound layer disposed on the base layer;
The functional substrate is a method for producing a cell culture container in which the functional organic compound layer is fixed on the surface of the container body member so that the functional organic compound layer faces the space for containing cells and a medium,
A region for functioning in the space on the surface of the functional substrate on the side of the functional organic compound layer is defined as a first region, a region surrounding the first region is defined as a second region, and the first region of the functional substrate. When the surface on the back side of the base material layer side is the third region, and the surface of the functional base on the back side of the base material layer side is the fourth region,
Inside the injection mold for molding the container main body member or a member that is a part thereof, the functional substrate is placed on the surface of the functional substrate on the base material layer side including the third region. With the protective member placed, the functional organic compound layer covers a partial region of the inner wall surface of the injection mold, and at least a part of the surface of the third protective member is exposed to the mold space. A mold body, or a member that is a part of the container main body integrated with the functional substrate, is filled with a molten resin in the injection mold and solidified. Including said method.

Images