JP2013116073A - Manufacturing method for cell culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for a cell culture vessel that can decrease the adhesion of a foreign matter to the bottom or the like of a container, and can prevent the generation of a bubble when a functionality base is fixed.SOLUTION: The manufacturing method is for a cell culture vessel that includes a container section that accommodates a cell and a medium, wherein the container section includes at least: a container body member 103; and a functionality base 140 including at least: an adhesive layer 503 disposed on the bottom of the container body member 103; a base material layer 502 arranged on the adhesive layer 503; and a functionality organic compound layer 501 arranged on the base material layer 502, wherein the functionality base 140 is floated on a liquid L in a state the adhesive layer 503 is turned to the upper side, to the container body member 103 in which the side that accommodates a cell and a medium is turned to the lower side, the functionality base 140 is brought up by the liquid L, and thereby the container body member 103 and the functionality base 140 are connected.

Description

  The present invention relates to a method for producing a cell culture container on which a functional substrate having a functional organic compound layer is fixed.

  A cell culture vessel such as a petri dish having a surface coated with a functional organic compound layer such as a temperature-responsive polymer is described in (Patent Document 1). By using this cell culture container, it is possible to easily peel off and recover from the cell support without destroying the cultured / proliferated cells simply by changing the temperature. However, as described in (Patent Document 1), it is necessary to perform a surface treatment by batch treatment separately on a cell culture container such as a petri dish to provide a functional compound layer, which requires a lot of labor, There is still room for improvement from the point of view.

  Therefore, as in (Patent Document 2) and the like, a functional substrate having a functional compound layer on the surface and having a film-like or plate-like shape is separately prepared, and the functional substrate is adhered to the bottom surface of the container. A technique for forming a target cell culture vessel by fixing it via an agent or an adhesive has been proposed. According to this technique, workability is enhanced as compared with the case where the functional organic compound layer is provided by batch processing of the cell culture containers individually.

JP-A-2-21865 JP 2010-98799 A JP 2009-154904 A JP 2004-134020 A

  For example, (Patent Literature 3) and (Patent Literature 4) disclose a sticking device for sticking stickers to a desired product. In order to fix a separately prepared functional substrate to the bottom of the container, as shown in (Patent Document 3) and (Patent Document 4), a label is attached to suck and hold the surface of a label piece (functional substrate). It is conceivable to use an applicator (labeler). In this case, the functional substrate is lowered while holding the functional substrate on the inner bottom surface of the container placed so as to open upward. Then, suction is released and a functional substrate is attached. Alternatively, the functional substrate is manually attached on the bottom surface of the container.

  As described above, when a functional substrate is approached from above and fixed on the inner bottom surface of the container with respect to the container placed in a normal state, there is minute dust or foreign matter on the inner bottom surface of the container. Then, there is a problem that the part becomes bubbles after fixing the functional substrate, which hinders cell observation and the like.

  In addition, when the functional substrate is held by a labeler, the functional organic compound layer side of the functional substrate is sucked and held, so that the functional organic compound layer is partially destroyed by the labeler's suction force. There is also a risk that functions such as cell culture may be impaired. Even when the functional substrate is manually pasted, the contact with the functional organic compound layer increases in the pasting process, and functions such as cell culture may be similarly impaired.

  Furthermore, the surface of the separately prepared functional substrate is subjected to surface cleaning as necessary in order to maintain the state of the functional organic compound layer satisfactorily. In that case, the cleaning may be performed before the functional substrate is fixed on the bottom surface of the container, or may be performed after the functional substrate is fixed on the bottom surface of the container. By separately providing such a washing step, there is a concern that the overall production efficiency of the cell culture container is lowered.

  Therefore, an object of the present invention is to provide a method for producing a cell culture container that can reduce the adhesion of foreign matters to the bottom surface of the container when fixing a functional substrate and prevent the generation of bubbles. Also, a method for producing a cell culture container that can maintain the function of the functional organic compound layer satisfactorily without bringing into contact with another member that impairs the functional organic compound layer when fixing the functional substrate. The purpose is to provide. Furthermore, it aims at providing the manufacturing method of the cell culture container excellent in the manufacturing efficiency which does not require a washing | cleaning process separately.

  The inventors of the present invention hold the functional substrate in a state of floating in a liquid, and arrange an inverted container with the cell and culture medium side facing downward above the functional substrate, and the functional substrate with respect to the container. It has been found that the above-mentioned problems can be solved by bringing the joints close to each other from below. That is, the present invention includes the following inventions.

(1) A method for producing a cell culture container comprising a container part for containing cells and a medium,
The container part
A container body member;
A functional base provided at least with an adhesive layer, a base material layer disposed on the adhesive layer, and a functional organic compound layer disposed on the base material layer, provided on the bottom surface of the container body member;
Having at least
Floating a functional substrate on the liquid with the adhesive layer facing upward;
The functional substrate is moved with the adhesive layer facing upward relative to the container body member with the cell and medium containing side facing downward, and the container body member and the functional substrate are bonded via the adhesive layer. And a process of
The said manufacturing method containing.
(2) The production of the cell culture container according to (1) above, wherein the container body member and the functional substrate are joined by moving the functional substrate by spraying the liquid toward the bottom surface of the container body member. Method.
(3) The manufacturing method of the cell culture container as described in said (1) or (2) in which a liquid contains the washing | cleaning component for a functional organic compound layer.

  According to the method for manufacturing a cell culture container according to the present invention, the functional substrate is moved in a state where the adhesive layer is directed upward with respect to the container body member with the side containing the cells and the medium facing downward. Because of the bonding, foreign matter hardly adheres to the bottom surface of the container body member facing downward, and adhesion of the foreign matter to the surface of the adhesive layer can be reduced by the container body member existing above when the functional substrate is stuck. . Therefore, it is possible to prevent the generation of bubbles due to the foreign matter after the functional substrate is fixed.

  In addition, since the functional substrate is floated on the liquid and the adhesive layer is held upward, the functional organic compound layer has a separate member unlike the conventional technique in which the surface of the functional organic compound layer is held by suction. There is no contact, and the function of the functional organic compound is not impaired.

  Furthermore, by spraying the functional substrate toward the container main body member with the liquid, the functional organic compound layer can be washed with the liquid at the same time as the container main body member and the functional substrate are joined. Therefore, it is not necessary to separately provide a washing step, and the production efficiency of the cell culture container is improved. In particular, when the liquid contains a cleaning component, the functional organic compound layer can be more effectively cleaned.

It is a perspective view which shows one Embodiment of the cell culture container manufactured by 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 for demonstrating the manufacturing process of the cell culture container shown to FIG. 1A-FIG. 1C. It is a perspective view for demonstrating the manufacture process of another embodiment of the cell culture container manufactured by this invention. It is a perspective view which shows another embodiment of the cell culture container manufactured by this invention. FIG. 4B is a cross-sectional view taken along the line III-III ′ of FIG. 4A. It is sectional drawing of a functional base | substrate. It is a perspective view for demonstrating one Embodiment of the manufacturing method of the cell culture container of this invention. It is sectional drawing for demonstrating one Embodiment of the manufacturing method of the cell culture container of this invention. It is sectional drawing for demonstrating one Embodiment of the manufacturing method of the cell culture container of this invention. It is sectional drawing for demonstrating one Embodiment of the manufacturing method of the cell culture container of this invention. It is sectional drawing for demonstrating another embodiment of the manufacturing method of the cell culture container of this invention. It is sectional drawing for demonstrating another embodiment of the manufacturing method of the cell culture container of this invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

<Shape of cell culture vessel>
First, the overall shape of the cell culture container produced in the present invention will be described.
The cell culture container produced according to the present invention includes at least a container part for containing cells and a medium, and further includes a lid and the like as appropriate.

  As shown in FIGS. 1A to 1C, the container portion may have a shape in which a space for containing cells and a medium is closed by a wall surface, or one end of the space as shown in FIGS. 4A and 4B. The shape may be open.

  A preferred embodiment of a cell culture vessel is shown in FIGS. 1A-1C. A container unit 100 shown in FIG. 1A includes a container body member 103 composed of a bottom part 101 and a side wall part 102 erected on the periphery of the bottom part 101, and a bottom part 101 joined to an upper end part of the container body member 103. And a top surface member 104 disposed to face each other. 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 part 107 for locking the lid 110 is formed on the neck part 106 of the container part 100, and the lid 110 is detachably mounted via the locking part 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 ′. The container main body member 103 composed of the bottom 101 and the side wall 102 of the container 100 includes an opening 108 that is defined by the bottom 101 and the side wall 102 and opened upward. The opening 108 is closed by joining the top surface member 104, and as a result, a space 130 for accommodating cells and a medium is formed. A functional substrate 140 is fixed to the bottom surface of the container main body member 103 on the opening 108 side (that is, on the bottom 101 facing the space 130).

  In order to form the container part 100, first, as shown in FIG. 2, the functional base 140 is joined to the inner bottom surface of the container body member 103 composed of the bottom part and the side wall part, and then the container body member 103 is joined to the container body member 103. The top member 104 is joined.

  FIG. 3 shows a manufacturing process of another embodiment of a flask-type container similar to FIGS. 1A to 1C. In this embodiment, the container main body member 109 is formed in a flat plate shape, and the functional substrate 140 is fixed on the bottom surface (surface) of the flat plate main body member 109. And the side wall member 111 and the top | upper surface member 104 are joined with respect to the container main body member 109 to which the functional base | substrate 140 was fixed, and the container part which has the same outer shape as FIG. 1A-FIG. 1C is obtained. In FIG. 3, it goes without saying that either the joining process of the container body member 109 and the side wall member 111 and the joining process of the side wall member 111 and the top surface member 104 may be performed first.

  As another embodiment of the container part, as shown in FIGS. 4A and 4B, a dish-shaped container part including a container body member 203 composed of a bottom part 201 and a side wall part 202 standing on the periphery of the bottom part 201. 200. The container body member 203 includes an opening 204 that is defined by the bottom 201 and the side wall 202 and is opened upward, and the opening 204 functions as a space 220 for containing cells and a culture medium. A functional base 210 is fixed to the bottom surface of the container main body member 203 on the opening 204 side (that is, on the bottom 201 facing the space 220).

  The functional substrates 140 and 210 include an adhesive layer 503 including an adhesive or an adhesive, a base material layer 502 disposed on the adhesive layer 503, and a functional organic compound layer 501 disposed on the base material layer 502. Are attached to the bottom portions 101 and 201 of the container body member or the flat container body member 109 shown in FIG.

<Material of cell culture container>
The material forming the members of the cell culture container such as the container main body member, the side wall member, the top surface member, the neck and the lid is not particularly limited, and materials generally used in cell culture can be used. For example, polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride A resin material such as a resin, a resin material containing at least one of the above-described surface hydrophilized treatment, and an inorganic material such as glass or quartz are preferable, but a resin material is preferable. The resin material is preferably a polystyrene resin or a polyethylene terephthalate resin.

  The container body member is preferably made of a transparent material through which the cultured cells can be observed from outside the container.

<Functional substrate>
As shown in FIG. 5, the cross section along the thickness direction of the functional substrates 140 and 210 in the present invention is an adhesive layer 503, a base material layer 502 disposed on the adhesive layer 503, and the base material layer 502. At least a functional organic compound layer 501 disposed thereon is provided. The functional substrate is typically a transparent functional substrate. 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 manufactured by forming the adhesive layer 503 and the functional organic compound layer 501 on the base material layer 502 by an appropriate method. Between the base material layer 502 and the functional organic compound layer 501, one or more other layers (for example, a primer layer described later) may be present 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 base material layer 502 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 502 only needs to include a material capable of forming the above-described functional organic compound layer 501 on one surface, and the type of the 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 other inorganic materials such as glass and quartz. A resin material is preferred.

  The surface of the base material layer 502 on which the functional organic compound layer 501 is formed can be a surface that has been 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 502 can be selected as appropriate. 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 imparting flexibility, for example, 5 to 500 μm, more preferably 20 to 500 μm, and particularly preferably 50 to 250 μm. By setting it as said film base material layer, the responsiveness of the functional base | substrate with respect to the jet of the liquid mentioned later can be improved.

<Functional organic compound layer>
The organic compound constituting the functional organic compound layer 501 is not particularly limited as long as it has a desired function, but more preferably, the organic compound can be changed from cell adhesiveness to cell nonadhesiveness by a predetermined stimulus. Examples include stimuli-responsive polymers having a possible surface, and hydrophilic compounds such as ethylene glycol chains composed of one or more ethylene glycol units (CH ₂ —CH ₂ —O).

  The film thickness of the functional organic compound layer 501 may be, for example, in the range of 0.5 nm to 300 nm, and particularly 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 501 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 a resin base material layer, it can be physically adsorbed on the surface of the resin base material layer in advance. Then, a primer layer containing polysiloxane containing a functional group capable of forming a covalent bond by reacting with the terminal hydroxyl group of the ethylene 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.

<Adhesive layer>
The pressure-sensitive adhesive layer 503 includes an adhesive or a pressure-sensitive adhesive. The adhesive and the pressure-sensitive adhesive are not particularly limited as long as they can join the base layer of the functional substrate and the container main body member, respectively. Specifically, the adhesive includes a polyacrylate adhesive, Cyanoacrylate adhesives, polyester adhesives, polyamide adhesives, polyimide adhesives, epoxy adhesives, polyurethane adhesives, methacrylic adhesives, rubber adhesives, silicone adhesives, inorganic adhesives Examples of such adhesives include acrylic adhesives, urethane adhesives, silicone adhesives, and rubber adhesives. These adhesives or pressure-sensitive adhesives are applied on the base material layer 502 to form the pressure-sensitive adhesive layer 503.

  In the case of manufacturing a functional substrate, the order of the step of forming the functional organic compound layer 501 and the step of forming the pressure-sensitive adhesive layer 503 containing an adhesive or pressure-sensitive adhesive on the surface of the base material layer 502 is particularly limited. Not. For example, (1) A base material in which an adhesive layer is formed and the surface of the adhesive layer is covered with a release sheet and protected as necessary is prepared, and the surface of the base material on which the adhesive layer is not formed is prepared. A functional organic compound layer may be laminated, or (2) after laminating a functional organic compound layer on one surface of the base material, an adhesive layer is formed on the other surface of the base material, and if necessary, sticky The surface of the layer may be protected by covering with a release sheet. The “base material in which an adhesive layer is formed and the surface of the adhesive layer is covered and protected by a release sheet as necessary” used in (1) above may be produced independently or purchased commercially. Also good.

<Method for producing cell culture container>
Next, the manufacturing method of the cell culture container of this invention is demonstrated.
A first embodiment of the manufacturing method of the present invention will be described with reference to FIGS. 6A to 6D. In addition, although the following description is a thing about the case where the cell culture container shown to FIG. 1A-FIG. 1C is manufactured, the cell culture container shown to FIG. 4A and 4B and the cell culture container of another form are manufactured. The same applies to the case. 6B to 6D correspond to the cross-sectional view of FIG. 1B.

  First, as shown in FIG. 6A, the functional substrate 140 is floated on a certain amount of liquid L placed in a storage tank or the like with the adhesive layer facing upward. The functional organic compound layer side of the functional substrate 140 is in a state facing the liquid L. At this time, the functional substrate 140 is held from the periphery by a holding member 300 having an appropriate shape so that the functional substrate 140 does not move irregularly on the liquid surface, and the functional substrate is placed at a predetermined position on the liquid surface. 140 is preferably stabilized. Part or all of the holding member 300 may be movable up and down with respect to the liquid level. In that case, the functional substrate 140 may be conveyed in a state of being floated on the liquid surface by the liquid flow, and the holding member 300 may be appropriately positioned.

  As the type of the liquid L, any liquid can be used as long as the functional organic compound layer 501 is insoluble and does not impair its function. Specific examples include water, alcohols such as methanol, ethanol, isopropanol, and propanol, and mixtures thereof. In particular, a low molecular alcohol having 3 or less carbon atoms is preferable from the viewpoint of easy drying. Further, if necessary, the liquid L may contain a cleaning component for keeping the surface of the functional organic compound layer 501 clean by removing impurities attached to the functional organic compound layer 501 by dissolving the impurities. it can. Such cleaning components include, but are not limited to, surfactants such as phospholipids and fatty acid sodium. The content of the cleaning component varies depending on the type of the cleaning component and the like, but can be contained in the liquid L, for example, about 0.01 to 1% by weight. However, in general, the liquid L itself such as water or alcohol can serve as a cleaning function for the functional organic compound layer without particularly adding a cleaning component.

  Subsequently, as shown in FIG. 6B, the functional base body 140 in a state of being floated on the liquid L is positioned below the container body member 103 with the side of the opening 108, which is a side for containing cells and culture medium, facing downward. . In order to position the functional base 140 under the container main body member 103, the functional base 140 is transported on the liquid L to the container main body member 103 whose position is fixed. The functional substrate 140 may be held, or the container body member 103 may be transported to the functional substrate 140 fixed on the liquid surface. Alternatively, both the container main body member 103 and the functional substrate 140 may be transported, and the transporting operation may be stopped so that the relative positions of the two match. When the functional base 140 is transported, the functional bases that are formed in advance so as to have a shape and area suitable for the bottom surface of the container main body member 103 may be transported one by one. For example, the functional base 140 is wound in a roll shape. The long functional substrate may be conveyed and cut into a predetermined shape and area immediately before reaching the lower part of the container body member.

  When a release sheet is further provided on the adhesive layer 503, the release sheet may be released after the functional substrate 140 is positioned under the container body member 103, or the release sheet may be removed. After peeling, the functional substrate 140 may be positioned under the container body member 103. Since the container body member 103 covers the upper space of the functional substrate like an umbrella by peeling the release sheet after the functional substrate 140 is positioned below the container body member 103, the foreign substance / Garbage is less likely to adhere.

  Next, as illustrated in FIG. 6C, for example, a jet La of the liquid L is generated upward using equipment such as a nozzle provided in the liquid L. With this jet La, the functional substrate 140 is spouted toward the bottom surface of the container body member 103 on the side containing the cells and the medium, and the functional substrate 140 is moved with the adhesive layer 503 facing upward. The functional base 140 is pressed against the bottom surface of the container body member 103 by the pressure of the jet La, and the container body member 103 and the functional base body 140 are joined. The jet La may be generated after first bringing the container main body member 103 and the functional base 140 close to an optimum distance.

  In this embodiment, since the functional substrate 140 is brought close to the container body member 103 with the side of the opening 108 that accommodates the cells and the medium facing downward, foreign matter adheres to the bottom surface of the container body member 103. Hard to do. In addition, since the upper space of the functional substrate moving upward for bonding is covered with the container main body member, it is difficult for foreign matter to adhere to the adhesive layer 503. As a result, bubbles caused by foreign matters generated at the interface between the bonded container body member and the functional substrate are reduced.

  Further, since the functional substrate 140 is supported in a state of being floated on the liquid L, unlike the conventional case where the entire surface of the functional organic compound layer is sucked and held, the functional substrate 140 is separated from the functional organic compound layer 501. A member does not contact and a strong stress is not added and the function of a functional organic compound layer is not impaired.

  Furthermore, by making the functional base 140 float on the liquid L, or by spraying the functional base 140 toward the bottom surface of the container body member 103 with the liquid, the functional organic by the liquid L (jet La) is simultaneously obtained. The compound layer 501 can be cleaned. Therefore, it is not necessary to separately provide a washing step, and the production efficiency of the cell culture container is improved. In particular, when the liquid L contains a cleaning component, the cleaning effect of the liquid L can be further enhanced.

  Thereafter, as shown in FIG. 6D, the cell culture container can be manufactured by joining the top surface member 104 to the container body member 103. 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. Further, before bonding the top member 104, the bonded functional organic compound layer 501 may be appropriately dried.

  7A and 7B show another embodiment of the manufacturing method of the present invention. This embodiment corresponds to the case where the functional substrate 140 is bonded to the bottom surface of the flat plate-like container body member 109 as shown in FIG. The configurations and effects are the same as those of the embodiments of FIGS. 6A to 6D described above except that a plate-like container body member 109 having no side wall is used instead of the container body member 103 in the embodiment of FIGS. 6A to 6D. It is the same.

  That is, as shown in FIG. 7A, the functional substrate 140 is changed to the liquid L with the adhesive layer 503 facing upward at the lower part of the flat container body member 109 with the cell and medium containing side facing downward. Float. Then, as shown in FIG. 7B, the functional base 140 is spouted up by the jet La with the adhesive layer 503 facing upward toward the bottom surface of the container body member 109, and the functional base 140 is put in the container by the pressure of the jet La. By pressing against the main body member 109, the container main body member 109 and the functional substrate 140 can be joined via the adhesive layer 503.

  The plate-like container body member 109 to which the functional substrate 140 is fixed can manufacture a cell culture container by joining the sidewall member 111 and the top surface member 104 as shown in FIG.

  6C and 7B, the two jets La are drawn below the functional base 140, but the mode of these jets is conceptually shown and of course is not limited to this. Is not to be done. For example, a large number of jets can be generated so as to uniformly hit the entire surface of the functional substrate 140.

  Further, the means for moving the functional base 140 with the adhesive layer 503 facing upward relative to the container body member 103 may not be based on the jet of the functional base 140 by the jet La. For example, the functional substrate is floated on the liquid with the adhesive layer facing upward, and the container body member is brought close to contact with the floating functional substrate until the container body member and the functional substrate are joined. Also good. Further, the means for raising the functional base 140 is not limited to the above, and mechanical means such as an elevating pin having an elastic body at the tip may be used.

  The holding member 300 is not limited to the mode illustrated in FIG. 6A and the like as long as the functional base 140 can be held at a predetermined position on the liquid surface. As an aspect other than FIG. 6A, for example, the opposing side wall of the storage tank containing the liquid L narrows along the shape of the functional base, so that the side wall functions as a holding member, and the functional base is sandwiched and held by the side wall. Cases can be mentioned.

100, 200 Container part 101, 201 Bottom part 102, 202 Side wall part 103, 109, 203 Container body member 104 Top surface member 105 Through hole 106 Neck part 107 Locking part 108, 204 Opening part 110 Lid 111 Side wall member 130, 220 Space 140 , 210 Functional substrate 300 Holding member 501 Functional organic compound layer 502 Base material layer 503 Adhesive layer L Liquid La Jet

Claims (3)

A method for producing a cell culture container comprising a container part for containing cells and a medium,
The container part
A container body member;
A functional base provided at least with an adhesive layer, a base material layer disposed on the adhesive layer, and a functional organic compound layer disposed on the base material layer, provided on the bottom surface of the container body member;
Having at least
Floating a functional substrate on the liquid with the adhesive layer facing upward;
The functional substrate is moved with the adhesive layer facing upward relative to the container body member with the cell and medium containing side facing downward, and the container body member and the functional substrate are bonded via the adhesive layer. And a process of
The said manufacturing method containing.   The method for producing a cell culture container according to claim 1, wherein the bonding of the container body member and the functional substrate is performed by moving the functional substrate by spraying the liquid toward the bottom surface of the container body member.   The manufacturing method of the cell culture container of Claim 1 or 2 in which the liquid contains the washing | cleaning component for a functional organic compound layer.

Images