JP2023114106A - Method for producing culture vessel, and culture vessel - Google Patents

Abstract

To provide a method for producing a culture vessel that enables the production of a culture vessel which has a culture surface uniformly and stably coated with a cell-adhesive material, while reducing the amount of the cell-adhesive material required for coating, so as to give the culture vessel suitably usable for mass culture of adherent cells.SOLUTION: A cell-adhesive material is preliminarily applied as a coating agent onto a culture substrate. Then, the surface of the culture substrate coated with the coating agent is processed to form a culture vessel having the processed culture substrate surface as the culture surface. By the processing, a plurality of grooves consisting of mountainous and valley-shaped portions are formed on the surface side of the culture substrate coated with the coating agent. The grooves have a nearly V-shaped cross-section perpendicular to the direction in which the grooves extend, and the surface of the culture substrate having the grooves formed thereon serves as the culture surface of the culture vessel.SELECTED DRAWING: None

Description

TECHNICAL FIELD The present invention relates to cell culture technology, and more particularly to a method for manufacturing culture vessels for adherent cells.

In recent years, in fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy, there has been a demand for efficient and large-scale culture of cells and tissues in an artificial environment.
When adherent cells such as iPS cells are adhered to a culture portion for culturing cells in a culture vessel and cultured in large quantities, the yield is limited by the surface area of the culture portion. Therefore, it would be beneficial if the yield of adherent cells could be increased by manufacturing a culture vessel with a large surface area of the culture portion.

As a culture vessel having a large surface area of such a culture section, for example, a multistage culture vessel is commercially available in which a plurality of culture sections are stacked in multiple stages to form a culture vessel, and a medium can be passed between the culture sections. (Corning (R) Cell Stack, etc.). By using such a culture vessel, a large amount of adherent cells can be cultured.

It has also been proposed to increase the surface area of the culture portion by forming a three-dimensional structure in the culture portion of the culture vessel. A culture unit with such a three-dimensional structure can secure a larger culture area with a similar footprint compared to a normal flat culture unit, so it can be used in an incubator with limited space. Excellent considering.

Japanese Unexamined Patent Application Publication No. 2020-137438

By the way, in recent years, culture vessels pre-coated with proteins or polymers having cell adhesiveness have been commercially available (such as laminin-coated culture dishes manufactured by Corning). When using such a culture vessel, it is necessary to apply a separate coating agent to the culturing part of the culture vessel according to the cells to be cultured, since the adhesiveness of cells differs depending on the coated material. Met.

However, when a spin coating method known as a general coating method is used, it is difficult to evenly apply a coating agent to the culturing portion having a three-dimensional structure, resulting in unstable culturing performance. In addition, there is a problem that the loss of the coating agent is large, and if an expensive coating agent is used, the cultivation cost increases.

Moreover, in order to culture a large amount of cells in a space-saving manner, as described above, it is desirable to manufacture a culture vessel provided with a culture portion having a three-dimensional structure that allows cells to be cultured at high density. However, there is a problem that it is very difficult to appropriately apply the cell adhesive material to the culture part having such a three-dimensional structure.

In other words, it is important to uniformly and stably apply the cell-adhesive material to the culture part so that the adherent cells can adhere to the culture part sufficiently. However, when the cell-adhesive material is applied to the culture part having a three-dimensional structure, it is difficult to apply the material uniformly, and there is a problem that the application cannot be stably performed. There is also a problem that the amount of cell-adhesive material used for coating is increased. Furthermore, there is also a problem that uneven coating may cause cytotoxicity in the culture vessel.

Therefore, the present inventors conducted extensive research, applied a cell-adhesive material to a culture substrate, then processed the culture substrate to obtain a culture surface having a three-dimensional structure capable of culturing adherent cells at high density. By manufacturing a culture vessel with, it is possible to obtain the culture surface uniformly and stably coated with the cell adhesive material, and reduce the amount of cell adhesive material required for coating made it possible to

In addition, in the processing, by forming a substantially V-shaped groove as a three-dimensional structure that allows the adherent cells to be cultured at high density, it is possible to efficiently culture the adherent cells in the resulting culture vessel. .
Furthermore, by such a culture vessel manufacturing method, it has become possible to reliably prevent the formation of material pools in the substantially V-shaped grooves.

Here, in Patent Document 1, a culture substrate is coated with a low-cell-adhesive polymer, and then the culture substrate is processed to prepare a culture vessel whose culture surface is the surface of the coated culture substrate. A method for manufacturing a forming culture vessel is described.
However, since this culture vessel is for floating and culturing spheres, the culture surface is coated with a low-cell-adhesive polymer instead of a cell-adhesive material.

In addition, this culture vessel does not have a culture surface with substantially V-shaped grooves, but has a culture surface with a large number of wells for culturing spheres and the like. Although such wells can increase the culture area, they are not suitable for culturing adherent cells, as will be described later.
Furthermore, since this culture vessel is coated with a low cell adhesion polymer for use in suspension culture, cytotoxicity due to coating unevenness is unlikely to occur. Prevention of the formation of is neither described nor suggested.

The present invention has been made in view of the above circumstances. It is an object of the present invention to provide a method for producing a culture vessel that can reduce the amount of cell adhesive material required for , and that can be suitably used for large-scale culture of adherent cells, and the culture vessel.

In order to achieve the above object, the method for producing a culture vessel of the present invention comprises coating a cell adhesive material as a coating agent on a culture substrate in advance, and then coating the surface of the culture substrate coated with the coating agent. and forming a culture vessel using the surface of the processed culture substrate as a culture surface.
Moreover, it is preferable that the culture substrate is a film or a sheet, and the culture vessel is bag-shaped.
Furthermore, it is preferable that the method for producing a culture vessel of the present invention is a method in which the cell adhesive material is at least one of collagen, gelatin, polylysine, polyethyleneimine, laminin, and fibronectin.

Further, in the method for manufacturing a culture vessel of the present invention, in the processing, a plurality of grooves having mountain-like portions and valley-like portions on the surface side of the culture substrate coated with the coating agent, the grooves It is preferable to form a groove having a substantially V-shaped cross section perpendicular to the direction in which the groove extends, and use the surface of the culture substrate with the groove formed as the culture surface of the culture vessel.
Further, it is preferable that the method of manufacturing a culture vessel of the present invention is a method of processing the inclination angle of the substantially V-shaped side surface of the groove to 80 degrees or less.
Furthermore, in the method of manufacturing a culture vessel of the present invention, the surface area of the groove is 2 to 5.67 times the surface area of the culture substrate before processing after coating with the cell adhesive material. is preferred.

In addition, it is preferable that the method for manufacturing a culture vessel of the present invention is a method that does not form a material reservoir made of the cell adhesive material in the groove.
Further, in the method for manufacturing a culture vessel of the present invention, in the processing, after forming the culture substrate into a bag shape, the formation of the substantially V-shaped groove is performed by at least one of blow molding, vacuum molding, and pressure molding. It is preferable to set it as the method of performing according to.

Moreover, it is preferable that the method for manufacturing a culture vessel of the present invention is a method in which, in the processing, the uneven processing is performed by thermal transfer, and then the culture substrate is formed into a bag shape.
In addition, it is preferable that the method for manufacturing a culture vessel of the present invention is a method of applying a mold release agent to a mold used in the heat transfer, and then performing the uneven processing.

Further, the culture vessel of the present invention is a culture vessel provided with at least one port, wherein the culture surface of the culture vessel is provided with a plurality of grooves consisting of mountain-shaped portions and valley-shaped portions, and the grooves extend in the direction in which the grooves extend. The groove has a substantially V-shaped vertical cross section, and the surface of the groove is provided with a coating layer of a cell-adhesive material having a uniform thickness.
In addition, it is preferable that the culture vessel of the present invention has a configuration in which the groove does not have a material reservoir made of the cell adhesive material.

According to the present invention, in the production of a culture vessel, a culture vessel having a culture surface uniformly and stably coated with a cell adhesive material can be obtained, and the cell adhesive material necessary for coating can be used. It is possible to provide a method for producing a culture vessel that can reduce the amount and can be suitably used for mass culture of adherent cells, and a culture vessel.

Result (B) of coating of the cell adhesive material by the method for manufacturing a culture vessel according to the embodiment of the present invention, and result (A) of coating when the cell adhesive material is coated after processing the culture substrate. It is an explanatory view about the difference from. A state of coating of a cell adhesive material in a substantially V-shaped groove formed by a method for manufacturing a culture vessel according to an embodiment of the present invention, and a well provided in a method for manufacturing a culture vessel according to an embodiment of the present invention. FIG. 10 is an explanatory diagram showing the coating state of the cell-adhesive material when a culture surface is applied. A photograph (B) of a substantially V-shaped groove and a groove of the same shape in a culture vessel according to an embodiment of the present invention, and a photograph (A) of a well composed of a quadrangular prism and a quadrangular pyramid in a conventional culture vessel. be. FIG. 4 is an explanatory view showing the state of substantially V-shaped grooves formed in the culture substrate by vacuum forming or pressure forming in the culture vessel manufacturing method according to the embodiment of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, the manufacturing method of the culture container of this invention and embodiment of a culture container are described in detail. However, the present invention is not limited to the specific contents of the following embodiments.
In the method for producing a culture vessel of the present embodiment, the culture substrate is coated with a cell adhesive material as a coating agent in advance, and then the surface of the culture substrate coated with the coating agent is processed. The method is characterized by forming a culture vessel having a surface of a culture substrate as a culture surface.

First, FIG. 1 shows the difference between the coating result of the cell-adhesive material obtained by the culture vessel manufacturing method of the present embodiment and the coating result when the cell-adhesive material is applied after processing the culture substrate. will be described with reference to
As shown in FIG. 1(A), a culture substrate 100 such as a film is first subjected to processing such as transfer molding, vacuum molding, and air pressure molding to expand the surface area of the culture substrate 100, and then culture. There is a method of coating the surface of the substrate 100 with the cell adhesive material 200 .
In the figure, grooves composed of mountain-like portions and valley-like portions and having a substantially V-shaped cross section perpendicular to the direction in which the grooves extend are formed on one surface side of the culture substrate 100 by processing. example is shown.

In this method, the cell-adhesive material 200 cannot be uniformly applied to the surface of the culture substrate 100, and cannot be applied stably. In addition, there is a problem that the amount of the cell adhesive material required for coating is increased, and uneven coating may cause toxicity to adherent cells.

Specifically, a material pool 201 made of the cell adhesive material 200 is formed at the bottom of the substantially V-shaped groove. As shown in FIG. 1, the material pool means a portion where the cell adhesive material is thickly accumulated at the bottom of the substantially V-shaped groove. For this reason, there was a possibility that toxicity to adherent cells would occur at the bottom of the groove.
Moreover, the coating layer made of the cell adhesive material 200 on the side surface of the substantially V-shaped groove becomes thin. Therefore, there is a possibility that adherent cells cannot be sufficiently adhered to the side surfaces of the groove.

On the other hand, as shown in FIG. 1(B), in the method for manufacturing a culture vessel of the present embodiment, a cell adhesive material 20 is first applied to a culture substrate 10 . Thereby, a coating layer made of the cell adhesive material 20 is formed on the surface of the culture substrate 10 .
Next, the surface area of the culture substrate 10 is expanded by subjecting the obtained culture substrate 10 to processing such as transfer molding, vacuum molding, and pressure molding. The figure shows an example in which substantially V-shaped grooves are formed by processing on the surface side of the culture substrate 10 on which the coating layer is applied.

According to this method, the cell adhesive material 20 can be uniformly applied to the surface of the culture substrate 10, and can be stably applied. Moreover, since the coating is performed before the surface area of the culture substrate 10 is expanded, the amount of the cell adhesive material 20 required for coating can be reduced.

Furthermore, according to this method, the coating layer made of the cell adhesive material 20 is uniformly formed on the side surface of the groove without forming a material pool of the cell adhesive material 20 at the bottom of the substantially V-shaped groove. can be formed.
Therefore, according to the culture vessel manufacturing method of the present embodiment, the problem of cytotoxicity of the culture vessel to adherent cells can be resolved. In addition, since the coating layer made of the cell adhesive material 20 can be formed with a uniform thickness on the surface of the approximately V-shaped groove, the problem that adherent cells cannot sufficiently adhere to the side surfaces of the groove can be resolved. It is possible.

In the culture vessel manufacturing method of the present embodiment, it is preferable to use a film or a sheet as the culture substrate.
Olefin-based resins such as polyethylene and polypropylene can be suitably used as the material for the culture substrate. Other usable materials include polymethylpentene, cyclic olefin polymer, cyclic olefin copolymer, polyvinyl chloride, polyurethane, polymethyl methacrylate, polyester, polyamide, ionomer, ethylene-α-olefin copolymer, and ethylene-vinyl acetate copolymer. Polymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl methacrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate, poly Examples include methyl methacrylate, polydimethylsiloxane, fluorine resin, silicone resin, polybutadiene resin, and polyethylene chloride. Thermoplastic elastomers such as olefinic thermoplastic elastomers, vinyl chloride thermoplastic elastomers, styrene thermoplastic elastomers, urethane thermoplastic elastomers, ester thermoplastic elastomers, and nylon thermoplastic elastomers can also be used. Thermosetting elastomers such as urethane rubber, silicone rubber, and fluororubber; Resins can also be used.

In the method for manufacturing the culture vessel of the present embodiment, the initial surface of the culture substrate is preferably smooth in order to uniformly coat the cell adhesive material on the culture substrate and process the surface.

In addition, in the method for manufacturing a culture vessel of the present embodiment, prior to coating the cell adhesive material on the culture substrate, the culture substrate is surface-treated (hydrophilic treatment), and then the surface is coated with the cell adhesive material. may be coated. As the surface treatment, corona treatment, excimer treatment, or the like can be suitably performed.
Thereby, the adhesiveness of the cell adhesive material to the surface of the culture substrate can be improved.

In the culture vessel manufacturing method of the present embodiment, the culture vessel to be formed is preferably a bag-like culture vessel (culture bag) made of a soft packaging material.
The culture bag can be formed by bonding the peripheral portions of the lower film (bottom side film) and the upper film (top plate side film) by heat sealing or the like.
In the culture bag, the surface of the culture substrate coated with the cell-adhesive material is formed as a culture surface, and adherent cells are cultured by adhering to this culture surface.

The culture bag is provided with at least one port. A tube is connected to this port, and a liquid delivery means such as a pump provided in the tube transfers the medium, such as injecting the medium into the culture bag and discharging the medium from the culture bag, through the port. done.
Thermoplastic resins such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, and FEP (perfluoroethylene propene copolymer) can be used as materials for the port.

In the method for manufacturing the culture vessel of the present embodiment, it is preferable to use at least one of collagen, gelatin, polylysine, polyethyleneimine, laminin, and fibronectin as the cell adhesive material.
In addition, the cell adhesive material used in this embodiment is not limited to these, and other materials may be used as long as they allow adherent cells to adhere to the culture surface.

Examples of cell adhesion proteins that can be used as cell adhesion materials include, in addition to those described above, vitronectin, entactin, osteopontin, thrombospondin, netrin, tenascin, nidogen, fibrinogen, elastin, proteoglycan, and laminin. Its fragment (Nippi iMatrix-511) and the like are included.

Examples of cell-adhesive polymers that can be used as cell-adhesive materials include, in addition to those described above, polyacrylic acid, polymethacrylic acid, polyvinylsulfuric acid, polystyrenesulfonic acid, polyallylamine, polyethyleneimine, polyvinyl Acetal, chondroitin sulfate, dermatan sulfate, dextran sulfate, keratan sulfate, heparan sulfate, hyaluronic acid, chitin and the like.
Furthermore, as the cell adhesive material, poly-N-isopropylacrylamide (PIPAAm), which is a temperature-responsive polymer, and other stimulation-responsive polymers such as pH-responsive polymers and photo-responsive polymers may be used. may be used in combination.

In addition, in the method for manufacturing the culture vessel of the present embodiment, the cell-adhesive material can be suitably applied to the surface of the culture substrate using, for example, a bar coater, a gravure coater, a spray coater, a spin coater, or the like. .

Further, in the culture substrate manufacturing method of the present embodiment, a plurality of grooves having mountain-like portions and valley-like portions are formed on the surface side of the culture substrate coated with the coating agent, It is preferable to form grooves having a substantially V-shaped cross section perpendicular to the direction in which the grooves extend, and use the surface of the culture substrate on which the grooves are formed as the culture surface of the culture vessel.

Also, a substantially V-shaped groove may be formed on the opposite side of the surface of the culture substrate coated with the coating agent. As will be described later, in the present embodiment, when a culture vessel is produced by vacuum forming or pressure forming, molding is performed while the culture substrate is pressed against a mold, so the culture substrate is coated with a coating agent. Approximately V-shaped grooves are formed on both the surface side and the opposite side.

In addition, in the method for manufacturing a culture vessel according to the present embodiment, it is preferable to process the inclination angle of the substantially V-shaped side surface of the groove to 80 degrees or less.
When the inclination angle of the substantially V-shaped side surface of the groove is 60 degrees, the surface area of the groove is double the surface area of the corresponding region without the groove. Similarly, when the inclination angle is 65 degrees, the surface area of the groove is 2.37 times larger, and when the inclination angle is 70 degrees, the surface area of the groove is 2.92 times larger. In this case, the surface area of the groove becomes 3.86 times, and when the inclination angle is 80 degrees, the surface area of the groove becomes 5.67 times. The relationship between the inclination angle and the surface area of the groove is the same whether or not the coating layer is formed on the surface of the groove.

Therefore, from the viewpoint of culture area, the larger the inclination angle, the better. On the other hand, the greater the inclination angle, the lower the machining performance, such as chipping at the tip of the machining die and deformation of the tip of the workpiece. When the inclination angle is 83 degrees, the surface area of the groove becomes 8.21 times. The limit was generally to produce a working mold with the same inclination angle of 80 degrees and use it to create a workpiece.
Therefore, in the method for manufacturing a culture vessel of the present embodiment, the surface area of the grooves is preferably 2 to 5.67 times the surface area of the culture substrate before processing after coating with the cell adhesive material.

In addition, in the method for manufacturing the culture vessel of the present embodiment, the depth of the substantially V-shaped groove (vertical distance from the apex of the mountain-like portion to the plane passing through the lowest point of the valley-like portion) is 10 μm to 1000 μm. About 50 μm to 500 μm is more preferable.

Here, in the method for manufacturing a culture vessel of the present embodiment, it is preferable to machine a substantially V-shaped groove on one surface side of the culture substrate, and to machine a plurality of wells on one surface side of the culture substrate. The reason why it is not preferable to do so will be described with reference to FIGS. 2 and 3. FIG.

The left side of FIG. 2 shows a cross-sectional illustration of a state in which the culture substrate 10 is coated with the cell adhesive material 20 . In addition, an explanatory cross-sectional view showing a state in which wells composed of a plurality of quadrangular prisms and quadrangular pyramids are formed on the surface side coated with the cell adhesive material 20 is shown in the upper right, and the cell adhesive material 20 is coated. An explanatory cross-sectional view showing a state in which a plurality of substantially V-shaped grooves are formed on the front surface side is shown in the lower right.

In addition, FIG. 3(A) is a micrograph of the surface of the culture substrate in which wells composed of a plurality of quadrangular prisms and quadrangular pyramids are formed, and FIG. Figure 2 shows a photomicrograph of the surface of the culture substrate. Although no coating layer is formed on the surface of the culture substrate in these photographs, the shape of each well and the shape of the approximately V-shaped groove are generally the same as those with a coating layer. .

As shown in the upper right of FIG. 2, when processing such wells on the surface of the culture substrate, the coating layer in the vertical direction is stretched, resulting in a coating layer of uniform thickness on the surface of the culture substrate. cannot be formed. In addition, when hemispherical wells are processed on the surface of the culture substrate, it is difficult to apply uniformly because the top surface portion that is not formed in the wells remains.
On the other hand, as in the method for manufacturing a culture vessel of the present embodiment, when forming substantially V-shaped grooves on the surface of the culture substrate, a coating layer having a uniform thickness is formed on the surface of the culture substrate. It is possible to

In addition, in the method for manufacturing a culture vessel according to the present embodiment, a plurality of grooves can be processed in parallel and linearly on the surface of the culture substrate, and the apex portions of the mountain-like portions can be formed linearly. That is, as shown in the photograph of FIG. 3(B), it is possible to form a line-shaped substantially V-shaped groove on the surface of the culture substrate.
Therefore, according to the culture vessel manufacturing method of the present embodiment, it is possible to obtain a culture vessel in which adherent cells grow along the line-shaped grooves, so that the culture area can be effectively increased.

On the other hand, when multiple wells are formed as shown in FIG. 3(A), the adherent cells do not spread and proliferate on the downward slope, so the adherent cells cultured in one recess spread and proliferate in the adjacent recess. never. In addition, seeding cells into each of all the recesses is undesirable from the viewpoint of growth efficiency. Therefore, when culturing adherent cells, it is not preferable to form a plurality of wells on the surface of the culture substrate.

Further, in the processing of the culture substrate, after forming the culture substrate into a bag shape, it is preferable to form substantially V-shaped grooves by at least one of blow molding, vacuum molding, and pressure molding. Particular preference is given to carrying out by molding.
As the vacuum pressure molding, for example, a culture bag is placed in a mold in which convex portions having a size corresponding to a plurality of substantially V-shaped grooves are formed, and the culture bag is sandwiched between jigs for performing vacuum pressure molding and pressurized to culture. A plurality of substantially V-shaped grooves can be formed in the culture bag by blowing high-pressure air into the bag, vacuuming from the outside of the mold, and heating the mold.
The heating temperature at this time is preferably set to a temperature at which the cell adhesive material does not dissolve, for example, it is preferably room temperature to about 100.degree.

In the present embodiment, when a culture vessel is produced by vacuum forming or pressure forming, molding is performed while the culture substrate is pressed against a mold. Therefore, as shown in FIG. Approximately V-shaped grooves are formed on both the surface side where the coating agent 20 is applied and the surface side where the coating agent 20 is not applied.

Further, when a highly heat-resistant cell adhesive material is used, in the processing of the culture substrate, a substantially V-shaped groove may be formed by thermal transfer, and then the culture substrate may be formed into a bag shape. good. In this case, a cushioning material is placed on the base material, and a film or sheet as a culture substrate is placed on the cushioning material. Next, a mold having protrusions corresponding to a plurality of substantially V-shaped grooves is placed on the culture substrate, and a heating plate is used to press the culture substrate from above the mold while applying pressure. By heating, a plurality of substantially V-shaped grooves can be formed in the culture substrate. As the mold, a silicon mold, a metal mold such as nickel, or the like can be preferably used.
At this time, in order to facilitate the peeling of the culture substrate from the mold used in thermal transfer, a release agent is applied to the surface of the mold facing the culture substrate, and then a substantially V-shaped groove is processed. is preferred. As the mold release agent, a fluorine-based mold release agent or a silicon-based mold release agent can be preferably used.

The culture vessel obtained by the method for manufacturing a culture vessel of the present embodiment can be suitably used for culturing adherent cells.
Examples of adherent cells to be cultured include iPS cells, neural stem cells, embryonic stem cells, hepatocytes, pancreatic islet cells, cardiomyocytes, corneal endothelial cells and the like.

The culture vessel of the present embodiment is a culture vessel having at least one port, a groove formed of peaks and valleys on the culture surface of the culture vessel, and a cross section perpendicular to the direction in which the groove extends. A substantially V-shaped groove is provided, and a coating layer of a cell-adhesive material is provided on the surface of the groove with a uniform thickness.
Further, the groove is characterized by not having a material reservoir made of a cell-adhesive material.

That is, as described above, according to the culture vessel manufacturing method of the present embodiment, after coating the culture substrate with a coating layer made of a cell adhesive material, the surface side of the culture substrate coated with the coating layer In order to form a culture vessel whose surface serves as a culture surface by processing a substantially V-shaped groove on the surface, the culture vessel of this embodiment has a cell adhesive material on the surface of the substantially V-shaped groove on the culture surface is provided with a coating layer of uniform thickness.

As described above, the culture vessel of the present embodiment has the cell adhesive material coated evenly on the culture surface, so that adherent cells can be favorably cultured.
Furthermore, according to the culture vessel manufacturing method of the present embodiment, it is possible to reliably prevent the formation of material pools made of the cell adhesive material in the substantially V-shaped grooves. Therefore, the culture vessel of the present embodiment has no risk of cytotoxicity when culturing adherent cells.

The present invention is not limited to the above embodiments, and it goes without saying that various modifications can be made within the scope of the present invention.
For example, rather than processing substantially V-shaped grooves in parallel in a straight line, it is possible to make appropriate changes such as processing them in concentric circles or concentric ellipses.

INDUSTRIAL APPLICABILITY The present invention can be suitably used for the efficient production of a large amount of adherent cells.

10 culture substrate 20 cell adhesive material

Claims (12)

A cell adhesive material is applied as a coating agent to a culture substrate in advance, and then the surface of the culture substrate coated with the coating agent is processed, and the surface of the processed culture substrate is used as a culture surface. A method for manufacturing a culture vessel, which comprises forming a culture vessel to 2. The method for producing a culture vessel according to claim 1, wherein the culture substrate is a film or sheet, and the culture vessel is bag-shaped. 3. The method for producing a culture vessel according to claim 1, wherein said cell adhesive material is at least one of collagen, gelatin, polylysine, polyethyleneimine, laminin, and fibronectin. In the processing, a plurality of grooves having peaks and valleys on the surface of the culture substrate coated with the coating agent, wherein the cross section perpendicular to the direction in which the grooves extend is substantially V-shaped. 4. The method for manufacturing a culture vessel according to any one of claims 1 to 3, wherein grooves are formed on the culture substrate, and the surface of the culture substrate on which the grooves are formed is used as the culture surface of the culture vessel. 5. The method of manufacturing a culture vessel according to claim 4, wherein the inclination angle of the substantially V-shaped side surface of the groove is 80 degrees or less. 6. The method for producing a culture vessel according to claim 5, wherein the surface area of the groove is 2 to 5.67 times the surface area of the culture substrate before processing after coating with the cell adhesive material. The method for manufacturing a culture vessel according to any one of claims 1 to 6, characterized in that the groove is not formed with a material pool of the cell-adhesive material. 1-, wherein in the processing, after forming the culture substrate into a bag shape, the formation of the substantially V-shaped groove is performed by at least one of blow molding, vacuum molding, and air pressure molding. 8. The method for manufacturing the culture vessel according to any one of 7. The method for manufacturing a culture vessel according to any one of claims 1 to 7, wherein in the processing, the uneven processing is performed by heat transfer, and then the culture substrate is formed into a bag shape. 10. The method for manufacturing a culture vessel according to claim 9, wherein the unevenness processing is performed after a release agent is applied to the mold used in the thermal transfer. A culture vessel comprising at least one port,
The culture surface of the culture vessel is provided with grooves consisting of mountain-like portions and valley-like portions and having a substantially V-shaped cross-section perpendicular to the direction in which the grooves extend, and the surfaces of the grooves are coated with a cell-adhesive material. A culture vessel characterized by having a layer with a uniform thickness. 12. The culture vessel according to claim 11, wherein said groove does not have a material reservoir made of said cell adhesive material.