JP5957850B2 - Method for producing cell culture vessel - Google Patents

Description

  The present invention relates to a method for producing a cell culture container, and more particularly to a method capable of efficiently producing a cell culture container of stable quality.

  In recent years, attempts have been made to impart various functionalities to the surface of containers. For example, in Patent Document 1, a temperature-responsive polymer prepared by spreading a solution containing a monomer that becomes a temperature-responsive polymer on the surface of a petri dish and polymerizing the monomer by irradiating it with an electron beam is immobilized. A cell culture support is disclosed. According to this, a sheet-like cultured cell aggregate (hereinafter referred to as a cell sheet) formed using the surface of a container such as a petri dish is peeled from the container without breaking the cell-cell bond. It can be recovered.

  However, the method of individually imparting functionality to the surface of a container such as a petri dish by batch processing is inefficient and is not suitable for mass production. Therefore, after providing functionality to the surface of the sheet-like member by continuous processing and producing a functional sheet, it is possible to improve the production efficiency by fixing it to a container, and various sizes and shapes. Application to a container is also possible.

JP-A-5-192130

  By the way, as a method of fixing a sheet (hereinafter, referred to as a functional substrate) having functionality on the surface to a container, for example, a method using an adhesive may be mentioned. Specifically, an adhesive layer is formed in advance on the surface of the outermost layer opposite to the surface to which the functionality of the functional substrate is imparted, and the functional substrate is placed in the container via this adhesive layer. To fix. According to the method using an adhesive, it is considered that a functional container can be efficiently produced because the functional substrate can be easily fixed to the container. However, for example, in a container intended for culturing cells, safety against cells and stability of quality are strongly demanded, and therefore selection of an adhesive in consideration of this point is required.

  This invention is made | formed in view of the said situation, Comprising: It aims at providing the method for manufacturing efficiently the cell culture container provided with a functional base | substrate with stable quality.

  The inventors of the present invention have conducted research on a method for fixing a functional substrate suitable for a container for culturing cells. As a result, the cross-linking density and the like during the cross-linking reaction vary locally in a cross-linking adhesive. While there is a risk that stable quality may not be obtained due to this, non-crosslinked adhesives satisfy the durability required for cell culture containers and have stable quality cell culture containers. The inventors have found that they can be manufactured efficiently, and have completed the invention. Specifically, the present invention provides the following.

  (1) Manufacture of a cell culture container comprising: a container part comprising at least a bottom part and a side wall part standing on the periphery of the bottom part; and a functional substrate, wherein the functional substrate is adhered to the bottom part of the container part. In the method, the functional substrate includes at least a functional organic compound layer having a surface having a predetermined function on the substrate, and the functional substrate and the bottom portion are interposed via a non-crosslinking adhesive. The above-mentioned method, comprising the step of adhering a functional substrate.

  (2) The container portion further includes a ceiling portion that is joined to the upper end portion of the side wall portion and is disposed to face the bottom portion, and a through-hole that can be plugged in a part of the side wall portion. The method according to (1), wherein an inner chamber for containing cells and a medium is formed inside.

  According to the method of the present invention, a cell culture vessel provided with a functional substrate can be efficiently produced with stable quality.

It is a perspective view which shows an example of the cell culture container (dish type) manufactured with the method which concerns on this invention. It is a perspective view which shows an example of the cell culture container (flask type) manufactured with the method which concerns on this invention. It is sectional drawing which shows an example of a functional base | substrate. It is sectional drawing which shows typically an example of the relationship between the container part and functional base | substrate in the cell culture container (dish type) manufactured with the method which concerns on this invention. It is sectional drawing which shows typically an example of the relationship between the container part and functional base | substrate in the cell culture container (flask type) manufactured with the method which concerns on this invention. It is sectional drawing which shows typically an example of the relationship between the container part and functional base | substrate in the cell culture container (dish type) manufactured with the method which concerns on this invention. It is sectional drawing which shows typically an example of the relationship between the container part and functional base | substrate in the cell culture container (dish type) manufactured with the method which concerns on this invention.

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

[Cell culture vessel]
An embodiment of a cell culture container produced by the method of the present invention will be described.
An example of a preferred embodiment of the container part in the present invention is shown in FIG. In the cell culture container 1 shown in FIG. 1, a so-called “dish type” in which a container part 100 includes a circular bottom part 101 and a side wall part 102 erected on the periphery of the bottom part 101 and is opened upward. It has a shape called “.” Then, one sheet-like functional substrate 20 is bonded to substantially the entire upper surface of the bottom 101 of the container 100 via a non-crosslinking adhesive (not shown). In FIG. 1, the shape of the bottom 101 is a circle, but is not limited to this, and may be any shape such as a rectangle, an ellipse, or a polygon.

  Another example of a preferred embodiment of the container part in the present invention is shown in FIG. The container part 200 shown in FIG. 2 includes a bottom part 201, a side wall part 202 erected on the periphery of the bottom part 201, and a top part 203 that is joined to the upper end part of the side wall part 202 and is opposed to the bottom part 201. It has. The container part 200 is provided with a liquid through hole 204. That is, the container part 200 has a through hole 204 formed in a part of the side wall part 202, and includes a neck part 205 extending from the periphery of the through hole 204 to the outside of the container part, and has a so-called “flask type” shape. Have. One functional substrate 20 is bonded to the bottom 201 of the container 200 via a non-crosslinking adhesive (not shown). A locking part (not shown) for locking the lid 210 is formed on the neck part 205 of the container part 200, and the lid 210 is detachably mounted via the locking part. The inside of the cell culture vessel 2 is closed by the lid 210, and cell culture can be performed stably. Cells and a culture medium may be supplied from the through-hole 204, or the functional substrate 20 peeled from the bottom 201 of the container part 200 may be collected.

  The container part in the present invention may have another shape as long as it has at least a bottom part and a side wall part standing on the periphery thereof and can accommodate cells and a culture medium. Specifically, the container portion may have a shape such as a beaker, a well plate, a bottle, or a tube. Moreover, it is not necessary to have a structure as shown in FIGS. 1 and 2 that can hold cells and culture medium by the container alone, and details will be described later, even if the bottom of the container has an opening. Any structure may be used as long as the functional substrate is attached to the opening to accommodate the cells and the medium.

  The material which forms a container part and a lid | cover is not specifically limited, The material 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, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, chloride Examples thereof include resin materials such as vinyl resins, resin materials containing at least one of the above-described surface hydrophilized treatment, and inorganic materials such as glass and quartz. Among these, resin materials are preferable. The resin material is preferably a polystyrene resin or a polyethylene terephthalate resin.

  1 and 2, one functional substrate is disposed at the bottom of the container, but at least one functional substrate may be disposed at the bottom, for example, a plurality of the same or different functionalities. A functional substrate having the above may be arranged in parallel at the bottom. 1 and 2, the functional substrate is disposed on the upper surface of the bottom of the container, and the size thereof is substantially the same as that of the bottom of the container, but can be disposed on the bottom of the container. Any size is acceptable. Preferably, it is substantially the same as the size of the bottom part of the container part to be arranged. Furthermore, in FIGS. 1 and 2, the shape of the functional substrate is a sheet shape, but may be any shape that conforms to the shape of the bottom of the container portion, and preferably a flat plate shape, a sheet shape, and a film shape. It is preferable that the roll is flexible so that it can be wound up in a long form.

<Functional substrate>
An example of a preferred embodiment of the functional substrate in the present invention is shown in FIG. In the functional substrate 20 shown in FIG. 3, a functional organic compound layer 22 having a surface having a predetermined function is formed on a substrate 21. Hereinafter, the base material and the functional organic compound layer will be described in detail.

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

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

  It is preferable that a base material is a magnitude | size which can be installed in the bottom part of a container part. It is preferable that the shape of a base material is what follows the shape of the bottom part of a container part. A flat plate shape, a sheet shape, and a film shape are preferable. This is because it is easy to culture cells. Although the thickness of a base material is not specifically limited, When handling property is considered, Preferably it is 10-500 micrometers, More preferably, it is 50-250 micrometers. If it is this range, it is also possible to supply and process in a continuous belt shape.

(Functional organic compound layer)
Examples of the functional organic compound layer include a stimulus-responsive polymer layer having a surface that can be changed from cell adhesiveness to cell nonadhesiveness by a predetermined stimulus, and one or more ethylene glycol units (CH ₂ —CH _2). And a hydrophilic compound layer such as an ethylene glycol chain composed of —O).

  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. Of these, a temperature-responsive polymer is preferable in that it can easily give a stimulus.

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

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

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

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

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

Cell adhesion and cell non-adhesion indicate a relative relationship between the degree of cell adhesion in one region and the other region. Cell adhesion means that cells are easily adhered. Cell adhesion is determined by whether or not cell adhesion or extension is likely to occur depending on the chemical or physical properties of the surface. As an index for determining cell adhesion, the cell adhesion spreading rate when cells are actually cultured can be used. The cell adhesive surface is preferably a surface having a cell adhesion extension rate of 60% or more, and more preferably a surface having a cell adhesion extension rate of 80% or more. If the cell adhesion extension rate is high, cells can be cultured efficiently. The cell adhesion extension rate in the present invention is such that cells to be cultured within a seeding density of 4000 cells / cm ² or more and less than 30000 cells / cm ² are seeded on the surface to be measured, and in an incubator at 37 ° C. and a CO ₂ concentration of 5%. It is defined as the ratio of cells that have adhered and spread when cultured for 14.5 hours ({(number of cells adhered) / (number of cells seeded)} × 100 (%)). Cell seeding is suspended in DMEM medium containing 10% FBS, seeded on the measurement object, and then slowly shaken the measurement object on which the cells are seeded so that the cells are distributed as uniformly as possible. It is done by. Furthermore, the measurement of the cell adhesion extension rate is performed after exchanging the medium immediately before the measurement to remove the non-adhered cells. When measuring cell adhesion spread rate, measure the location excluding the location where the cell density tends to be specific (for example, the center of the predetermined area where the density is likely to be high, the periphery of the predetermined area where the density is likely to be low) A place.

  On the other hand, cell non-adhesiveness refers to the property that cells are difficult to adhere. Cell non-adhesiveness is determined by whether or not cell adhesion or extension is unlikely to occur due to chemical or physical properties of the surface. The non-cell-adhesive surface is preferably a surface having a cell adhesion extension rate as defined above of less than 60%, more preferably less than 40%, and even more preferably 5% or less. The surface is preferably 2% or less, and most preferably.

As the hydrophilic compound layer, for example, an ethylene glycol chain composed of one or more ethylene glycol units (CH ₂ —CH ₂ —O) (an ethylene glycol chain composed of a plurality of ethylene glycol units is referred to as a “polyethylene glycol chain”) And a hydrophilic compound layer. The end of the ethylene glycol chain may be blocked with a hydroxyl group, or the other end of the ethylene glycol chain may be covalently linked to another substance such as a biological substance. 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.

  FIG. 4 is a schematic cross-sectional view showing an example of a preferred embodiment in the case where the functional substrate is bonded to the container portion via a non-crosslinking adhesive in the cell culture container having the shape shown in FIG. is there. Cutting is performed along arrows II ′ shown in FIG. In the cell culture container 3 shown in FIG. 4, the functional substrate 20 in which the functional organic compound layer 22 is formed on the substrate 21 is a non-crosslinked adhesive having an area substantially equal to the bottom area of the functional substrate 20. The layer 30 is bonded to a partial region of the upper surface of the bottom 101 of the container 100. FIG. 5 is a schematic cross-sectional view showing an example of a preferred embodiment in the case where the functional substrate is adhered to the container portion in the cell culture container having the shape shown in FIG. Cutting is performed along arrows II-II ′ shown in FIG. Also in the cell culture container 4 shown in FIG. 5, the functional substrate 20 in which the functional organic compound layer 22 is formed on the substrate 21 is non-crosslinked adhesive having an area substantially equal to the bottom area of the functional substrate 20. The adhesive layer 30 is adhered to a partial region of the upper surface of the bottom portion 201 of the container portion 200.

  6 and 7 are schematic cross-sectional views showing another example of the preferred embodiment in which the functional substrate is adhered to the container portion in the cell culture container having the shape shown in FIG. Cutting is performed along arrows II ′ shown in FIG. In the cell culture container 5 shown in FIG. 6, the functional substrate 20 in which the functional organic compound layer 22 is formed on the substrate 21 is in a state in which only the outer peripheral portion is interposed with the non-crosslinking adhesive layer 30. It adheres to a partial region of the upper surface of the bottom 101 of the container 100. According to the embodiment as shown in FIG. 6, since there is no adhesive in the central region, for example, when performing cell observation with a fluorescence microscope, it is difficult to be affected by autofluorescence derived from the adhesive.

  In the cell culture container 6 shown in FIG. 7, the container part 100 has a dish shape similar to the container part shown in FIG. 1, but has a circular opening 103 so as to penetrate substantially the center of the bottom part 101. 1 is different from the container shown in FIG. 1 in that one is formed. The functional substrate 20 is disposed so as to close the opening 103. The functional base 20 is bonded to the lower surface of the bottom 101 of the container 100 with only the outer peripheral portion interposed through the non-crosslinking adhesive layer 30. In FIG. 7, the functional organic compound layer 22 constituting the functional substrate 20 is in contact with the non-crosslinked adhesive layer. The cell culture container 6 shown in FIG. 7 has a structure in which cells and a medium can be held in a form including the container part 100 and the functional substrate 20. According to the embodiment as shown in FIG. 7, since there is no adhesive in the central region, for example, when performing cell observation with a fluorescence microscope, it is difficult to be affected by autofluorescence derived from the adhesive.

  Although FIG. 7 shows an example in which one opening 103 is formed in the approximate center of the bottom 101 of the container 100, the present invention is not limited to this. For example, a plurality of openings may be formed at the bottom of the container. When there are a plurality of openings, each opening may be closed with a separate functional substrate, or a plurality of openings may be closed together with one functional substrate. In FIG. 7, the functional substrate is bonded to the lower surface of the bottom of the container portion. However, the functional substrate may be bonded to the upper surface of the bottom of the container portion to close the opening. Moreover, the shape and size of the opening are not particularly limited, and may be any shape such as a square, a circle, an ellipse, and a polygon. When the container portion has an opening at the bottom as in the cell culture container shown in FIG. 7, the thickness of the functional substrate is preferably a thickness that can ensure supportability on the opening, for example, , 200 μm or more. Further, when the bonding width between the functional substrate and the lower surface of the bottom of the container portion is, for example, 1 mm or more, the functional substrate is stably supported, and it is also preferable from the viewpoint of manufacturing stability.

[Method for producing cell culture container]
The method for producing a cell culture container of the present invention is characterized by including a functional substrate adhesion step in which the functional substrate and the bottom are adhered via a non-crosslinking adhesive. The target for forming the non-crosslinked adhesive layer may be either a functional substrate or a container part. For example, after forming a non-crosslinked adhesive layer for adhering the functional substrate on part or all of the region where the functional substrate of the container portion is installed, the functional substrate is interposed via the non-crosslinked adhesive layer. May be bonded to the bottom of the container portion, or after forming a non-crosslinked adhesive layer on a part or all of the surface of the functional substrate, the functional substrate is bonded via the non-crosslinked adhesive layer. You may adhere | attach the bottom part of a container part.

  Hereinafter, as an example of a preferred embodiment of the method for producing a cell culture container of the present invention, the functional organic compound layer is a stimulus-responsive polymer layer, and the surface of the substrate on which the stimulus-responsive polymer layer is formed A case will be described in which a non-crosslinked adhesive layer is formed on the entire opposite surface, and the functional substrate and the bottom of the container part are bonded via the non-crosslinked adhesive layer.

  First, a container serving as a container part constituting a cell culture container is prepared. The container may be prepared by purchasing a commercially available product having a desired size and shape, or may be prepared by preparing a desired size and shape by an injection molding method or the like. In the case where the final form of the cell culture container does not have an open part necessary for the operation of bonding the functional substrate to the bottom of the container part, a container having the open part is prepared first, After bonding, another member such as a lid may be bonded.

  Next, a functional substrate on which a non-crosslinked adhesive layer is formed in advance is prepared. First, the above-mentioned base material is prepared. The substrate may be a single wafer or a roll. Next, after forming a non-crosslinked adhesive layer on one surface of the substrate, a release layer for protecting the non-crosslinked adhesive layer is formed. A stimulus responsive polymer layer is then formed on the other side of the substrate. In this way, a functional substrate provided with a non-crosslinked adhesive layer in advance is prepared.

  The reason why the non-crosslinked adhesive is selected as the adhesive for bonding the functional substrate and the bottom of the container is as follows. A cross-linking adhesive requires an aging process for cross-linking, and the cross-link density during the cross-linking reaction and the molecular weight of the molecules formed may vary locally. There is a possibility that the cell culture container cannot be produced efficiently. However, since the non-crosslinking adhesive does not crosslink, an aging process is not required, and the production can be performed more efficiently than when a crosslinkable adhesive is used. In addition, since there is no quality variation due to the crosslinking reaction, it is easy to guarantee safety for cell culture.

  The type of the non-crosslinking adhesive is not particularly limited, and examples thereof include natural rubber adhesives such as polyisobutylene adhesives, hot melt adhesives, moisture curable adhesives, and dry adhesives. Is a hot melt adhesive. A hot-melt adhesive is a 100% solid adhesive that does not contain any organic solvent based on a thermoplastic resin. It is solid or semi-solid at room temperature, but is melted by heating to a low viscosity molten state. Then, the material is applied and solidified by cooling to complete the adhesion. Since no solvent is used, a drying step for drying the solvent is unnecessary, and its use is more preferable from the viewpoint of environmental friendliness and productivity. Examples of the hot melt adhesive include, but are not limited to, urethane, polyamide, polyolefin, polyester, ethylene vinyl acetate, styrene butadiene, urethane rubber, and other synthetic rubber adhesives. It is not something.

  For example, in the case of a natural rubber adhesive such as a polyisobutylene adhesive, the non-crosslinking adhesive layer forming method is a non-crosslinking adhesive layer forming coating solution dissolved in a solvent such as an organic solvent. To prepare. Next, according to a conventional method, the coating solution is applied to the outermost surface of the functional substrate opposite to the side on which the functional organic compound layer is formed to form a coating film. Thereafter, the solvent is removed by drying to obtain a functional substrate on which a non-crosslinked adhesive layer is formed. And a functional base | substrate and the bottom part of a container part are adhere | attached through this non-crosslinking-type adhesive bond layer. The solvent is not particularly limited, and a solvent that can dissolve the adhesive may be appropriately selected. The coating method is not particularly limited, and a conventionally known method can be used. Examples thereof include printing methods such as gravure printing, flexographic printing, and offset printing, and methods using coating such as roll coating, reverse coating, comma coating, knife coating, die coating, and gravure coating.

In the case of a hot melt adhesive, the hot melt adhesive is heated and melted, and then the outermost side of the functional substrate opposite to the side on which the functional organic compound layer is formed is formed according to a conventional method. A functional substrate on which a non-crosslinked adhesive layer is formed is obtained by coating the outer surface to form a coating film. And a functional base | substrate and the bottom part of a container part are adhere | attached through this non-crosslinking-type adhesive bond layer. The heating temperature of the hot melt adhesive is usually 160 to 200 ° C. although it depends on the type of adhesive. For coating, a heated roll coater may be used. The application amount of the hot melt adhesive is preferably 10 to 100 g / m ² .

  It is preferable to form a release layer on the non-crosslinked adhesive layer. The release layer means a so-called release sheet made of a release member having peelability and having a function of protecting the surface of the non-crosslinked adhesive layer. The release member is not particularly limited as long as it has the necessary strength and flexibility. For example, a film made of a synthetic resin such as polyethylene terephthalate, polypropylene, polyethylene, or a foamed film thereof, silicone-based, fluorine-based, Examples thereof include those subjected to a release treatment with a release agent such as a long-chain alkyl group-containing carbamate. In general, a synthetic resin film subjected to silicone release treatment is used.

  Examples of the method for forming the stimulus-responsive polymer layer on the other surface of the substrate include the following methods. First, a coating solution for forming a stimuli-responsive polymer layer is prepared by dissolving the above-described monomer that becomes a stimulus-responsive polymer by polymerization in a solvent. Next, according to a conventional method, the coating solution is applied to the surface of the substrate to form a coating film. Thereafter, the monomer in the coating film is polymerized by appropriate means such as irradiation to form a polymer, and a grafting reaction is caused between the surface of the substrate and the polymer, thereby forming a stimulus-responsive polymer layer. Form. In addition, you may form the peeling layer similar to the above on the surface of this stimulus responsive polymer layer as needed.

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

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

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

  The thickness of the stimulus-responsive polymer layer is, for example, preferably in the range of 0.5 nm to 300 nm, more preferably in the range of 1 nm to 100 nm, and most preferably in the range of 10 to 30 nm. By making the film thickness in the range of 0.5 nm to 300 nm, it is easy to achieve both cell adhesion and peeling. In addition, various additives, such as surfactant, may be mix | blended with the stimulus responsive polymer layer in the range which does not impair the function.

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

  The stimulus-responsive polymer layer thus formed may be washed as necessary. On the surface of the stimuli-responsive polymer layer after graft polymerization, not only polymer molecules immobilized by covalent bonds, but also free polymer molecules that are not immobilized or unreacted monomers are present. Conceivable. Washing is preferable because these free polymers and unreacted substances can be removed. Here, the cleaning method is not particularly limited, but typical examples include immersion cleaning, rocking cleaning, shower cleaning, spray cleaning, and ultrasonic cleaning. The cleaning liquid typically includes various water-based, alcohol-based, hydrocarbon-based, chlorine-based, and acid / alkali cleaning liquids.

  The functional substrate produced by the above method is peeled off the release layer on the non-crosslinked adhesive layer, and is adhered and adhered to a predetermined position on the bottom of a container prepared in advance. After bonding the functional substrate and the bottom of the container, sterilization treatment such as ethanol sterilization, ethylene oxide gas (EOG) sterilization, ultraviolet irradiation sterilization, and γ-ray irradiation sterilization is performed as necessary. Among these, γ-ray irradiation sterilization is preferable in that all biological species can be killed. In this way, a cell culture container can be manufactured.

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

Example 1 Production of Cell Culture Container A polystyrene film sheet (OPS sheet, manufactured by Sandick) was prepared as a base material. Non-crosslinked adhesive (Opanol B10SFN, polyisobutylene, manufactured by BASF) was dissolved in toluene to prepare a non-crosslinked adhesive layer forming coating solution. On the surface of the release sheet (SP-PET, manufactured by Mitsui Chemicals, Inc.), which is a peelable protective film, the non-crosslinked adhesive layer forming coating solution is applied so that the film thickness after drying is 10 μm. After coating the entire surface with a coater, it was dried at 100 ° C. for 1 minute using a drying oven. And it laminated on one side of the polystyrene film sheet, and obtained the base material in which the non-crosslinked adhesive layer was formed.

  Next, N-isopropylamine was dissolved in isopropyl alcohol to a final concentration of 20% by weight to prepare a temperature-responsive polymer layer forming coating solution. On the surface opposite to the surface on which the non-crosslinked adhesive layer of the polystyrene film sheet is formed, a temperature-responsive polymer layer-forming coating solution is applied on the entire surface by gravure coating, and then in a hot air dryer at 45 ° C. Was passed through for 15 seconds and dried. Then, an electron beam was irradiated (electron beam irradiation dose: 200 kGy), N-isoproacrylamide was graft-polymerized, and poly-N-isoproacrylamide was immobilized on the surface of the polystyrene film sheet. Thereafter, it was cut into a circle of 32 mmφ, washed with ion exchange water at 5 ° C., and dried to obtain a functional substrate having a temperature responsive polymer layer formed on the surface of a polyethylene terephthalate film substrate.

  A container having a dish shape (corresponding to the container part shown in FIG. 1) was prepared. The cut-out release sheet of the functional substrate was peeled off, and the surface of the non-crosslinked adhesive layer was adhered to the upper surface of the bottom of the container and pasted to produce a cell culture container.

1, 2, 3, 4, 5, 6 Cell culture vessel 20 Functional substrate 21 Base 22 Functional organic compound layer 30 Non-crosslinked adhesive layer 100 Container portion 101 Bottom portion 102 Side wall portion 103 Opening portion 200 Container portion 201 Bottom portion 202 Side wall 203 Top

Claims (1)

A method for producing a cell culture container, comprising a container part comprising at least a bottom part and a side wall part standing on the periphery of the bottom part, and a functional substrate, wherein the functional substrate is adhered to the bottom part of the container part. And
The functional substrate includes at least a layer containing a temperature-responsive polymer, which is an acrylic polymer or a methacrylic polymer, on the substrate, the degree of cell adhesion on the surface changes depending on the temperature,
The container part further includes a top part joined to the upper end part of the side wall part and disposed opposite to the bottom part, and a capped through hole formed in a part of the side wall part. An inner chamber for accommodating the medium is formed inside,
And the functional substrate and the bottom, see containing functional substrate bonding process for bonding through a non-crosslinked adhesive is a hot melt adhesive,
The functional substrate used in the functional substrate bonding step is the non-crosslinked adhesion on the surface of the long base material opposite to the surface on which the layer containing the temperature-responsive polymer is formed. Prepared in advance in a state in which a layer of the agent and a release layer that protects the non-crosslinked adhesive layer are formed,
The functional substrate bonding step includes cutting out the functional substrate prepared in advance, peeling the release layer, and bonding the functional substrate to the bottom .

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