Method for manufacturing culture container and culture container

Abstract

When manufacturing a culture container in which a culture surface having a plurality of recesses is coated with a cell low-adhesion polymer as a coating agent, the amount of the coating agent used can be reduced. The cell low-adhesion polymer is coated as a coating agent on the surface of a culture substrate in advance, and then the culture substrate is processed to form a culture container in which the surface of the culture substrate coated with the coating agent is used as the culture surface.

Description

Technical Field

[0001] The present invention relates to a cell culture technology, and in particular to a method for manufacturing a culture container subjected to a cell low-adhesion treatment. Background Art

[0002] In recent years, when culturing stem cells such as iPS cells and ES cells, not only methods are used in which cells are allowed to adhere to a culture container for culturing, but also methods are used in which cells are allowed to adhere and agglomerate within a culture container to form cell clusters (spheres, agglomerates) or organoids, thereby culturing cells in a three-dimensional state that is closer to that in vivo.

[0003] In cell culture to form cell clusters, a culture container having a plurality of holes (concave portions) in a culture portion is used, and cell clusters are formed in the concave portions, thereby culturing the cells.

[0004] In order to prevent cells from adhering to the surface of the culture portion (hereinafter referred to as the culture surface) in such a culture container for forming a cell cluster, it is necessary to perform a cell low-adhesion treatment on the culture surface.

[0005] As a treatment for reducing cell adhesion, generally, a coating agent is applied to the culture surface, and a low cell adhesion polymer is used as the coating agent.

[0006] Here, when applying a low-cell-adhesion polymer to a culture container having relatively large wells such as a 96-well plate, the operation of applying the polymer to each well is extremely time-consuming and is a major problem.

[0007] In addition, as a method for uniformly applying a polymer to a hole, there is a method similar to dip coating in which the entire hole is immersed in a polymer solution and the solution is immediately discharged. However, in the coating of the polymer using this method, a larger amount of polymer solution than the amount of polymer applied to the culture surface is required, so there is a problem of increased cost.

[0008] In addition, when applying a low cell adhesion polymer to a culture container having a plurality of very small recesses microfabricated in the culture portion, bubbles enter the recesses and are difficult to remove, making it very difficult to evenly apply the culture surface. For example, when recesses having openings of about several tens of μm to 1 mm are formed on the culture surface, once bubbles are mixed into the recesses, it is basically impossible to remove them.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Laid-Open No. 8-131153

[0012] Patent Document 2: Japanese Patent Application Publication No. 2013-106531 Summary of the invention

[0013] Problems to be solved by the invention

[0014] As a method for applying a coating agent to a culture container having holes, there can be mentioned a cell culture container and a method for manufacturing the same described in Patent Document 1. In this method, the coating agent is injected into a plate having holes and immediately discharged by suction, and then the plate is turned over and air is blown to dry the plate, thereby performing coating.

[0015] However, this method takes time and requires a large amount of polymer solution, so the problem of increased cost cannot be solved. In addition, in this method, it is difficult to uniformly apply the coating agent to a culture container that has been microfabricated with very small recesses.

[0016] In addition, as a method for producing a culture container coated with a coating agent, there can be mentioned a method for producing a cell culture container described in Patent Document 2. In this method, a substrate coated with a coating agent in advance is inserted into a culture container by insert injection molding.

[0017] However, this method is not a technique related to a method of applying a coating agent to a culture surface having a plurality of recesses, and therefore is not a technique capable of solving the above-mentioned problems when a coating agent is uniformly applied to a culture surface having a plurality of recesses.

[0018] Therefore, the present inventors have conducted in-depth research, applied a cell low-adhesion polymer as a coating agent on the surface of a culture substrate in advance, and then processed the culture substrate to form a culture container, thereby successfully reducing the concentration and amount of the coating agent used in the coating. In addition, a culture container with a culture surface having a plurality of very small recesses uniformly coated with a coating agent was successfully obtained, thereby completing the present invention.

[0019] The present invention has been completed in view of the above situation, and its purpose is to provide a method for manufacturing a culture container, which can reduce the amount of coating agent used when manufacturing a culture container in which a cell low-adhesion polymer is coated as a coating agent on a culture surface formed with multiple recesses. The present invention also provides a culture container.

[0020] Means used to solve problems

[0021] In order to achieve the above-mentioned purpose, the manufacturing method of the culture container of the present invention is set as the following method, that is, a cell low-adhesion polymer is preliminarily coated as a coating agent on the surface of a culture substrate, and then the culture substrate is processed to form a culture container with the surface of the culture substrate coated with the coating agent as the culture surface.

[0022] In addition, it is also preferred that the manufacturing method of the culture container of the present invention is set as the following method, that is, the culture substrate is a film or a sheet, the culture container is in a bag shape, and during the processing, a plurality of recessed portions are formed on the surface of the culture substrate by using concave-convex processing, and this surface is set as the culture surface of the culture container.

[0023] In addition, it is also preferred that the manufacturing method of the culture container of the present invention is set as follows: in the processing, after the culture substrate is formed into a bag shape, the concave-convex processing is performed using at least any one of blow molding, vacuum forming, and compressed air molding. It is also preferred that in the processing, the concave-convex processing is performed using heat transfer, and then the culture substrate is formed into a bag shape.

[0024] In addition, the culture container of the present invention has the following structure, that is, it is a culture container with at least one port, after coating the cell low adhesion polymer on the surface of the culture substrate formed by a film or a sheet, it is made into a bag shape with the surface formed with multiple recesses as the culture surface, and there is no cell low adhesion polymer attached in the port and on the opposite side of the culture surface in the culture container.

[0025] Effects of the Invention

[0026] According to the present invention, a method for manufacturing a culture container can be provided, which can reduce the amount of coating agent used when manufacturing a culture container in which a culture surface having a plurality of recesses is coated with a low-cell-adhesion polymer as a coating agent, and can provide a culture container. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graph shows the results of a test to confirm the adhesion of cells to the concave portions of culture containers obtained by a conventional method for producing a culture container using various amounts of a polymer solution.

[0028] Figure 2 This is a diagram showing the results of cell cluster formation when a culture container having recessed portions formed by vacuum compressed air is used using the method for manufacturing a culture container according to an embodiment of the present invention.

[0029] Figure 3 The figure shows a microscope photograph (700 times) of a silicon mold with fine protrusions used for concave-convex processing by heat transfer in the manufacturing method of the culture container according to an embodiment of the present invention, and a microscope photograph (600 times) of a culture surface with fine concave portions obtained by using the silicon mold.

[0030] Figure 4 This is a diagram showing the results of cell cluster formation when a culture container in which recessed portions are formed by heat transfer is used using the method for manufacturing a culture container according to an embodiment of the present invention. DETAILED DESCRIPTION

[0031] Hereinafter, the manufacturing method of the culture container of the present invention and the embodiment of the culture container will be described in detail. However, the present invention is not limited to the specific contents of the following embodiment and examples.

[0032] The method for manufacturing a culture container of this embodiment is characterized in that a low cell adhesion polymer is preliminarily coated as a coating agent on the surface of a culture substrate, and then the culture substrate is processed to form a culture container having the surface of the culture substrate coated with the coating agent as a culture surface.

[0033] The low cell adhesion polymer can be applied to the surface of the culture substrate preferably using, for example, a bar coater or a gravure coater.

[0034] As the culture substrate, a film or a sheet is preferably used.

[0035] As the material of culture substrate, olefin resins such as polyethylene and polypropylene can be used suitably. As other usable materials, polymethylpentene, cyclic olefin polymer, cyclic olefin copolymer, polyvinyl chloride, polyurethane, polymethyl methacrylate, polyester, polyamide, ionomer, ethylene-alpha olefin copolymer, ethylene-vinyl acetate copolymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl methacrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate, polymethyl methacrylate, polydimethylsiloxane, fluororesin, silicone resin, polybutadiene resin, chlorinated polyethylene etc. can be enumerated. In addition, thermoplastic elastomers such as olefin thermoplastic elastomer, vinyl chloride thermoplastic elastomer, styrene thermoplastic elastomer, urethane thermoplastic elastomer, ester thermoplastic elastomer, nylon thermoplastic elastomer can be used. In addition, thermosetting elastomers such as urethane rubber, silicone rubber, and fluororubber, and thermosetting resins such as phenolic resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, urethane resin, and thermosetting polyimide may also be used.

[0036] As the low cell adhesion polymer applied to the surface of the culture substrate, phospholipid polymers, polyvinyl alcohol derivatives, phospholipid-polymer complexes, polyhydroxyethyl methacrylate, polyvinyl alcohol, agarose, chitosan, polyethylene glycol, albumin, etc. can be used. In addition, these can also be used in combination.

[0037] Furthermore, in order to evenly coat the low cell adhesion polymer on the surface of the culture substrate and to give the surface an uneven surface, the initial surface of the culture substrate is preferably smooth.

[0038] The culture container to be formed is preferably a bag-shaped culture container (culture bag) formed of a soft packaging material.

[0039] The culture bag can be formed by bonding the peripheral portions of the lower film (bottom film) and the upper film (top film) together by heat sealing or the like.

[0040] In the culture bag, the surface of the culture substrate coated with the low cell adhesion polymer is formed as the culture surface. Thus, when the culture bag is used to form a cell cluster or culture a single cell, the cell cluster or single cell can be prevented from adhering to the culture surface.

[0041] The culture bag is provided with at least one port. A tube is connected to the port, and a liquid feeding mechanism such as a pump disposed on the tube is used to feed the culture medium, such as injecting the culture medium into the culture bag and discharging the culture medium from the culture bag, through the port.

[0042] As the material of the port, for example, thermoplastic resins such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, and FEP can be used.

[0043] In the method for producing a culture container according to the present embodiment, first, a low cell adhesion polymer is coated on the surface of a culture substrate, and then the culture substrate is processed.

[0044] As the processing of the culture substrate, it is preferable to form a plurality of recesses on the surface of the culture substrate (the upper surface of the bottom side film) by recessed processing. In this case, the surface with the recesses formed is used as the culture surface of the culture container.

[0045] In addition, in the processing of the culture substrate, it is preferred that after the culture substrate is made into a bag shape, at least any one of blow molding, vacuum molding, and compressed air molding is used for concave-convex processing. In addition, as concave-convex processing, vacuum compressed air molding is particularly preferred.

[0046] As vacuum compressed air forming, for example, a culture bag can be arranged in a mold having a plurality of openings of a size corresponding to the recessed portions, a clamp for vacuum compressed air forming can be inserted and pressurized, high-pressure air can be blown into the culture bag, vacuum is drawn from the outside of the mold, and the mold can be heated, thereby forming a plurality of recessed portions in the culture bag.

[0047] In the processing of the culture substrate, it is also preferable to perform concavoconvex processing by heat transfer, and then form the culture substrate into a bag shape.

[0048] Specifically, a cushioning material is arranged on a base material, and a film or sheet as a culture substrate is arranged on the cushioning material. Then, a mold having a plurality of convex portions corresponding to the concave portions is arranged on the culture substrate, and the culture substrate is pressed from above the mold using a heating plate, and heated while applying pressure, thereby forming a plurality of concave portions on the culture substrate.

[0049] As the mold, a silicon mold, a metal mold such as nickel, or the like can be suitably used.

[0050] In this case, in order to facilitate the peeling of the culture substrate from the mold used in the heat transfer, it is preferred that a mold release agent be applied to the surface of the mold facing the culture substrate and then the surface be roughened.

[0051] As the release agent, a fluorine-based release agent or a silicone-based release agent can be preferably used.

[0052] By processing the culture substrate in this manner, a culture bag having a plurality of fine recesses on the culture surface can be manufactured. For example, a culture bag in which the diameter of the circle or inscribed circle of the opening of the recess is 1 μm or more and 10 mm or less can be suitably manufactured.

[0053] The shapes of the plurality of recesses formed on the culture surface of the bottom side film of the culture bag may be, for example, cones such as cones and quadrangular pyramids, hemispherical shapes, or R-shaped shapes.

[0054] In addition, it is also preferred that the shape of the recessed portion formed on the culture surface of the bottom side membrane is set to a shape in which at least a part of the side wall is formed in an approximately vertical shape. Specifically, in these recessed portions, it is preferred that the length of the vertical direction of the approximately vertical portion of the side wall is longer than half of the maximum diameter of the culture object. In addition, it is preferred that the shape of the region where the side wall of the recessed portion is approximately vertical is set to, for example, a cylindrical shape, a quadrangular prism shape, etc.

[0055] By forming the recessed portion of the culture bag in this shape, when the culture medium is fed into the culture bag from the port, even if the flow rate is 1 ml / min or more, culture objects such as cell clusters can be prevented from flying out of the recessed portion and moving to other recessed portions.

[0056] When the culture object is a cell cluster, the depth of the recess is preferably 50 to 500 μm. This is because if the depth of the recess is greater than 500 μm, even if the culture medium is introduced into the culture bag, it may be difficult to fully replace the culture medium in the recess, and the processing difficulty of the culture bag having a recess deeper than 500 μm becomes high. In addition, the size of the cell cluster is about 50 μm to 100 μm for a small cell cluster and about 200 μm to 300 μm for a large cell cluster.

[0057] The shape of the bottom of the recess is not particularly limited, but when the culture object is a cell cluster, it is preferably a cone, a hemispherical, or an R-shaped bottom so that single cells can be easily aggregated and cell clusters can be appropriately formed.

[0058] When the culture object is a single cell, the depth of the concave portion is preferably 5 to 50 μm. This is because the size of a single cell is about 6 μm to 15 μm, and most single cells are about 10 μm.

[0059] The shape of the opening of the recess is not particularly limited, and may be circular or rectangular such as square. In addition, the width of the opening of the recess may be appropriately set in accordance with the size of the culture object.

[0060] For example, when the culture object is a cell cluster, the lower limit of the diameter of the circle or inscribed circle of the opening of the recess can be set to 60 μm or more, 70 μm or more, 80 μm or more, 90 μm or more, 100 μm or more, 110 μm or more, 120 μm or more, 150 μm or more, etc. In addition, the upper limit of the diameter of the circle or inscribed circle of the opening of the recess can be set to 1 mm or less, 900 μm or less, 800 μm or less, 700 μm or less, 500 μm or less, etc.

[0061] In addition, when the culture object is a single cell, the lower limit of the diameter of the circle or the inscribed circle of the opening of the recess can be set to 5 μm or more, 6 μm or more, 8 μm or more, etc. In addition, the upper limit of the diameter of the circle or the inscribed circle of the opening of the recess can be set to 50 μm or less, 40 μm or less, 30 μm or less, etc.

[0062] Furthermore, the plurality of recesses in the culture portion may be arranged in, for example, a zigzag pattern or a lattice pattern.

[0063] In addition, in the processing of the culture substrate, it is preferred to apply the low cell adhesion polymer only to the culture surface of the culture container. This is because if the low cell adhesion polymer is applied to the peripheral portion of the culture surface, the peripheral portion cannot be heat-sealed, and bag making becomes difficult.

[0064] Therefore, it is preferred that a low cell adhesion polymer be applied to the culture substrate after a frame material (shielding material) is disposed on the culture substrate, and a heat-sealable region not coated with the low cell adhesion polymer be provided in the culture substrate.

[0065] In addition, in the processing of the culture substrate, it is also preferred that after the cell low-adhesion polymer is coated in the peripheral portion of the culture surface including the bottom side membrane of the culture container, the cell low-adhesion polymer coated in the peripheral portion is subjected to excimer treatment and ultraviolet irradiation. Thus, the peripheral portion can be heat-sealed. In addition, it is also preferred that the cell low-adhesion polymer coated in the peripheral portion is removed using a solvent.

[0066] Furthermore, it is also preferable to locally apply the low cell adhesion polymer only on the culture surface of the culture container using a bar coater, a gravure coater or the like.

[0067] In addition, it is preferred that the surface of the culture substrate is hydrophilized in advance during the processing of the culture substrate, and then the low cell adhesion polymer is coated on the surface, because the adhesion of the low cell adhesion polymer to the surface of the culture substrate can be improved.

[0068] According to the manufacturing method of the culture container of this embodiment, after the cell low-adhesion polymer is preliminarily coated on the surface of the culture substrate using a rod coater, etc., a culture container having a plurality of recesses formed on the culture surface can be obtained by vacuum compressed air molding, heat transfer, etc.

[0069] Therefore, as will be described later in Examples, the amount of the low cell-adhesion polymer required for coating can be significantly reduced.

[0070] Furthermore, particularly when manufacturing a culture bag in which very small recesses are microfabricated, a culture bag in which a low-cell-adhesion polymer is uniformly coated on the culture surface can be obtained.

[0071] It should be noted that the method for producing a culture container according to this embodiment can also be applied to the case where a coating agent other than a low cell adhesion polymer is used as the coating agent, for example, it can also be applied to the case where a coating agent containing a protein that withstands drying is used.

[0072] In addition, it is also preferred that the culture container manufactured using the present embodiment is set as follows: that is, in the top plate side membrane, a top plate protrusion is provided on the inner side of the culture container, the width of the top plate protrusion is smaller than the width of the opening of the recessed portion formed in the bottom side membrane, and the height of the top plate protrusion is smaller than the minimum diameter of the culture object, and when the top plate protrusion contacts a part of the upper end surface of the culture portion, a gap is formed between the upper end surface and the lower surface of the top plate side membrane.

[0073] If the culture container of this embodiment is configured in this way, the top plate protrusion can be made to contact a part of the upper end surface, and a gap smaller than the size of the culture object can be formed between the upper end surface and the top plate. In this way, it is possible to prevent the movement of the culture object such as cell clusters while allowing liquid substances such as culture medium to pass through the gap.

[0074] The culture container of this embodiment is characterized in that it is a culture container with at least one port, after a cell low-adhesion polymer is coated on the surface of a culture substrate formed by a membrane or a sheet, the above-mentioned surface with multiple recesses formed thereon is made into a bag shape as a culture surface, and there is no cell low-adhesion polymer attached inside the port and on the opposite side of the culture surface in the culture container.

[0075] That is, when a low cell adhesion polymer is injected into a bag-shaped culture container and immediately discharged by suction as in the conventional method, and then the culture container is turned over and dried, the resulting culture container has the low cell adhesion polymer attached to the inside of the port and on the opposite side of the culture surface (the lower surface of the top plate side membrane in the culture container), so there is a problem that an excessive amount of the low cell adhesion polymer is required.

[0076] In contrast, in the culture container of the present embodiment, after the cell low-adhesion polymer is coated on the surface of the culture substrate, a plurality of recesses are formed on the surface by means of convex-concave processing, and the surface is made into a bag shape using the surface as the culture surface. Therefore, it has the characteristic that there is no cell low-adhesion polymer attached inside the port and on the opposite side of the culture surface.

[0077] Therefore, the problem of the conventional method that an excessive amount of the low cell-adhesion polymer is required can be eliminated.

[0078] In the culture container of the present embodiment, the surface density of the low cell adhesion polymer coated on the culture surface is preferably set to 100 μg / cm 2 ~1 μg / cm 2 .

[0079] This is because, as shown in the examples below, it is possible to 2 , 50μg / cm 2 , 15μg / cm 2 , 5μg / cm 2 , 3μg / cm 2 , 2μg / cm 2 The culture container of this embodiment is obtained by coating the low cell adhesion polymer with a relatively small surface density. In addition, when the culture surface is subjected to concavo-convex processing to form hemispherical concave portions, the surface density of the low cell adhesion polymer becomes 1 / 2.

[0080] Example

[0081] Hereinafter, a test conducted to confirm the effect of the culture container and the manufacturing method of the culture container according to the embodiment of the present invention will be described.

[0082] (Test 1)

[0083] First, as a comparative example, a test was performed to confirm the amount of the low cell adhesion polymer required in a conventional method of coating the culture surface with the low cell adhesion polymer after forming a plurality of recesses on the culture surface of a culture bag.

[0084] Specifically, LLDPE (linear low-density polyethylene) was used to prepare a plurality of rectangular films with a long side of 12 cm and a short side of 7.5 cm, and half of the films was used as a bottom film and the remaining half as a top film.

[0085] Then, the bottom side film and the top plate side film are overlapped and the peripheral parts are bonded together by heat sealing to form a culture bag. At this time, the culture bag is bonded together with a port sandwiched on one side to form a culture bag equipped with one port.

[0086] Then, the culture bag was sandwiched and pressurized in a jig manufactured by our company having a mold for forming a plurality of recessed portions with an opening diameter of 2 mm, high-pressure air was blown into the culture bag, and vacuum was drawn from the outside of the mold. The lower mold was heated to 80° C., thereby performing vacuum compressed air molding to form a plurality of hemispherical recessed portions in the culture bag.

[0087] By operating as described above, three culture plates were manufactured, each having a plurality of concave portions densely arranged in a zigzag pattern over the entire surface of the culture plate, and the area of ​​the culture plate was 60 cm. 2 of culture bags.

[0088] Next, a 2% MPC polymer solution was sealed in the obtained culture bag as a low cell adhesion polymer, and the solution was immediately drained, and the culture bag was turned upside down and left to stand to dry.

[0089] At this time, the amount of the MPC polymer solution sealed in the culture bag was set to three types: 1.5 mL, 2 mL, and 3 mL, and each culture bag was prepared.

[0090] Thereafter, each culture bag was sealed with StemFit AK02N (product number RCAK02N, Ajinomoto Co., Ltd.) as a culture medium, and iPS cells (1231A3 strain) were inoculated. After 24 hours, the presence or absence of cell adhesion to the surface of the concave portion was confirmed. The results are shown in Figure 1 middle.

[0091] At this time, it is considered that when the concave portions of the culture bag are appropriately coated with the MPC polymer, the cells do not adhere to the concave portions, and when the concave portions of the culture bag are not appropriately coated with the MPC polymer, the cells adhere to the concave portions.

[0092] like Figure 1 As shown, when the amount of the MPC polymer solution sealed in the culture bag was 1.5 mL, cells adhered to the concave portion. At this time, the total amount of the MPC polymer was 30 mg.

[0093] In contrast, when the amount of the MPC polymer solution sealed in the culture bag was 2 mL and 3 mL, no cell adhesion to the concave portion was observed. At this time, the total amount of the MPC polymer was 40 mg and 60 mg, respectively.

[0094] That is, it can be seen that in order to provide a plurality of recesses as described above, the culture area of ​​the culture portion is 60 cm 2 In order to properly coat the culture bag with a low cell adhesion polymer, conventional methods require a minimum of 2 mL of solution.

[0095] (Test 2)

[0096] Next, an experiment was conducted on the case where a culture bag having a recess formed by vacuum compressed air was manufactured according to the method for manufacturing a culture container of the present embodiment.

[0097] Specifically, LLDPE (linear low-density polyethylene) was used to prepare a plurality of rectangular films with a long side of 12 cm and a short side of 7.5 cm, and half of the films were used as bottom films and the remaining half as top films.

[0098] Thereafter, the area of ​​one side of the bottom side membrane except the peripheral part was coated with MPC polymer as a cell low adhesion polymer using a rod coater (Yasuda Seiki Manufacturing Co., Ltd., ROD. No. 3), and this area was set as the culture surface. The amount of solution used in the coating was 0.5 mL, and the area of ​​the culture surface coated with the cell low adhesion polymer was 60 cm 2 .

[0099] The amount of solution used in the coating was 0.5 mL, and the coating area was 60 cm using a bar coater. 2 The minimum amount required for coating. If the amount of solution is less than this amount, it will be difficult to coat 60cm 2 coating area.

[0100] At this time, five concentrations of the MPC polymer were prepared: 4%, 2%, 0.5%, 0.15%, 0.1%, and 0.067%. Four bottom side membranes coated with the MPC polymer at a concentration of 0.5% were made, and one bottom side membrane each coated with the MPC polymer at the remaining concentrations was made, and these eight membranes were used as examples.

[0101] The total amount of MPC polymer used in the preparation of the bottom side membrane coated with MPC polymer at concentrations of 4%, 2%, 0.5%, 0.15%, 0.1% and 0.067% was 20 mg, 10 mg, 2.5 mg, 0.83 mg, 0.5 mg and 0.33 mg, respectively.

[0102] In addition, one of the bottom films was not coated with the MPC polymer, and this bottom film was used as a comparative example.

[0103] In addition, the surface density of the MPC polymer coated on the culture surface of the bottom membrane used in the examples was measured using a Fourier transform infrared spectrophotometer (VARIAN, product number: FTS7000).

[0104] As a result, the surface density of the MPC polymer of the bottom side film coated with the MPC polymer at a concentration of 4%, 2%, 0.5%, 0.15%, 0.1%, and 0.067% was 100 μg / cm 2 , 50μg / cm 2 , 15μg / cm 2 , 5μg / cm 2 , 3μg / cm 2 , 2μg / cm 2 .

[0105] Then, the top plate side film is overlapped on the bottom side film in such a way that the culture surface of the bottom side film coated with the MPC polymer is arranged inside, and the peripheral part is bonded by heat sealing to form a culture bag. In addition, at this time, the culture bag is bonded with a port sandwiched on one side.

[0106] Thereafter, the culture bag is sandwiched and pressurized in a jig manufactured by our company having a mold for forming recesses of various predetermined sizes, high-pressure air is blown into the culture bag, and vacuum is drawn from the outside of the mold. The lower mold is heated to 80° C., thereby performing vacuum compressed air molding to form a plurality of hemispherical recesses in the culture bag.

[0107] By operating as described above, one culture bag having a plurality of recesses with an opening diameter of 0.5 mm densely arranged in a zigzag pattern on the entire surface of the culture surface, seven culture bags having a plurality of recesses with an opening diameter of 2 mm, one culture bag having a plurality of recesses with an opening diameter of 4 mm, and one culture bag having a plurality of recesses with an opening diameter of 7 mm were manufactured.

[0108] The culture bags having a plurality of recesses with an opening diameter of 2 mm were five culture bags coated with five concentrations of the MPC polymer of the example and one culture bag used as a comparative example without coating the MPC polymer.

[0109] The culture bags having a plurality of recessed portions with opening diameters of 0.5 mm, 4 mm, and 7 mm were each one of the culture bags coated with the MPC polymer at a concentration of 0.5% according to the example.

[0110] The culture bag thus obtained is configured such that a tube is connected to the port of the culture bag, and the culture medium is transported to the culture bag through the tube.

[0111] Thereafter, iPS cells (1231A3 strain) were used to form cell clusters using these culture bags.

[0112] Specifically, the number of cells used in the inoculation was approximately 3.0 × 10 6 cells. In addition, StemFitAK02N (product number RCAK02N, Ajinomoto Co., Ltd.) was used as a culture medium. The above culture medium containing 10 mM Y-27632 (Wako Pure Chemical Industries, Ltd.) was injected into the culture bag, and the cell suspension containing the above iPS cells was injected and left to stand overnight. The results are shown in Figure 2 middle.

[0113] like Figure 2 As shown, the culture bags obtained by the method for manufacturing a culture container according to the present embodiment can appropriately form cell clusters.

[0114] On the other hand, in the culture bag whose culture surface was not coated with the MPC polymer, no cell clusters were formed.

[0115] As described above, in the method for producing a culture container according to the present embodiment, the amount of solution used for coating is 0.5 mL, and the MPC polymer can be coated on the culture surface with an amount smaller than the minimum required 2 mL in the comparative example of Test 1.

[0116] In addition, in the comparative example of Experiment 1, the total polymer amount must be at least 40 mg. In contrast, according to the method for manufacturing a culture container of this embodiment, in the case of a 2% MPC polymer solution with the same concentration as in the comparative example of Experiment 1, the total polymer amount used is 10 mg, and the coating can be performed in an amount of 25% of the total polymer amount in the comparative example.

[0117] In the case of an MPC polymer solution with a concentration of 0.5%, the total amount of polymer used was 2.5 mg, and it was possible to apply it in an amount of 6.25% compared to the total amount of polymer in the comparative example. In the case of an MPC polymer solution with a concentration of 0.15%, the total amount of polymer used was 0.83 mg, and it was possible to apply it in an amount of 2.075% compared to the total amount of polymer in the comparative example. In the case of an MPC polymer solution with a concentration of 0.1%, the total amount of polymer used was 0.5 mg, and it was possible to apply it in an amount of 1.25% compared to the total amount of polymer in the comparative example. In the case of an MPC polymer solution with a concentration of 0.067%, the total amount of polymer used was 0.33 mg, and it was possible to apply it in an amount of 0.825% compared to the total amount of polymer in the comparative example.

[0118] As described above, it is obvious that according to the method for producing a culture container and the culture container of the present embodiment, the amount of polymer used can be significantly reduced.

[0119] Here, in the method for manufacturing a culture container of this embodiment, after the MPC polymer is coated on the smooth bottom side film, a hemispherical concave portion is formed on the culture surface. Therefore, the surface area of ​​the processed concave portion becomes twice the original surface area. That is, the surface density of the MPC polymer on the culture surface is 2 μg / cm 2 In the case of 2 , the value becomes Figure 2 Minimum coating weight for the examples shown.

[0120] (Test 3)

[0121] Next, an experiment was conducted on the case where a culture bag having recessed portions formed therein by heat transfer was manufactured according to the method for manufacturing a culture container of the present embodiment.

[0122] Specifically, LLDPE (linear low-density polyethylene) was used to prepare a plurality of rectangular films with a long side of 12 cm and a short side of 7.5 cm, and half of the films were used as bottom films and the remaining half as top films.

[0123] Thereafter, the area of ​​one side of the bottom side membrane except for the peripheral part was coated with MPC polymer as a low cell adhesion polymer using a rod coater (Yasuda Seiki Co., Ltd., ROD. No. 3), and this area was set as the culture surface. The amount of solution used in the coating was 0.5 mL, and the area of ​​the culture surface coated with the low cell adhesion polymer was 60 cm 2 .

[0124] At this time, the concentration of the MPC polymer was set to 0.5%, and one bottom side film coated with the MPC polymer was prepared. The total amount of the MPC polymer used in the preparation of the bottom side film was 2.5 mg.

[0125] In addition, the surface density of the MPC polymer coated on the culture surface of the bottom membrane used in the examples was measured using a Fourier transform infrared spectrophotometer (VARIAN, product number: FTS7000). The result showed that the surface density of the MPC polymer on the bottom membrane was 15 μg / cm 2 .

[0126] Then, the bottom side film is subjected to heat transfer to be processed into recessed and convex shapes.

[0127] Specifically, a cushioning material is disposed on the base material, and the above-mentioned bottom side film is provided on the cushioning material.

[0128] Then, a silicon mold was placed on the bottom film, and the culture substrate was pressed from above the mold using a hot plate, and heated while being pressed, thereby making the culture surface of the bottom film uneven.

[0129] At this time, the heating temperature of the silicon mold was set to 120° C., and the pressure was applied at 0.4 MPa. In addition, the pressing time was set to 8 seconds.

[0130] In this case, a mold release agent was applied to the silicon mold, and then the concavoconvex processing was performed. A fluorine-based mold release agent (Fluorosurf FG-5084 manufactured by FluoroTechnology Co., Ltd.) was used as the mold release agent.

[0131] Silicon mold Figure 3 As shown in the left photograph of , a silicon mold having fine protrusions of quadrangular pyramids densely arranged in a lattice pattern was used. The opening of the silicon mold had a side of 160 μm and the height of the protrusion was 150 μm.

[0132] In addition, the culture surface of the bottom side membrane made using the mold is as follows Figure 3 As shown in the right photograph of , minute quadrangular pyramidal recesses are densely arranged in a lattice pattern over the entire surface of the culture surface.

[0133] Then, the top plate side film is overlapped on the bottom side film so that the culture surface of the bottom side film coated with the MPC polymer and formed with a plurality of recesses is arranged inside, and the peripheral parts are bonded together by heat sealing to form a culture bag. In addition, at this time, the culture bag is bonded together with a port sandwiched on one side.

[0134] The culture bag thus obtained is configured such that a tube is connected to the port of the culture bag, and the culture medium is transported to the culture bag through the tube.

[0135] Thereafter, iPS cells (1231A3 strain) were used to form cell clusters using the culture bag.

[0136] Specifically, the cells were seeded in such a way that 200 single cells were contained in each concave portion. In addition, StemFit AK02N (Product No. RCAK02N, Ajinomoto Co., Ltd.) was used as a culture medium. The above culture medium containing 10 mM Y-27632 (Wako Pure Chemical Industries, Ltd.) was injected into the culture bag, and the cell suspension containing the above iPS cells was injected and left to stand overnight. The results are shown in Figure 4 middle.

[0137] like Figure 4As shown, a cell cluster of about 80 μm was properly formed in each concave portion.

[0138] In Experiment 3, a 0.5% concentration MPC polymer solution was used, and the total polymer amount was 2.5 mg.

[0139] That is, with respect to the minimum total polymer amount of 40 m as the comparative example of Test 1, sufficient coating was possible with a polymer amount of 6.25%. As is also apparent from this test, the amount of polymer used can be significantly reduced.

[0140] As described above, according to the method for manufacturing a culture container and the culture container according to the embodiment of the present invention, it is obvious that when obtaining a culture container in which a culture surface having a plurality of recesses is coated with a low cell adhesion polymer, the amount of coating agent used can be significantly reduced.

[0141] The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the present invention.

[0142] For example, the size of the culture bag to be manufactured is not limited to the size of the example, and may be appropriately changed to a culture bag of a size capable of forming 500,000 to 1,000,000 cell clusters.

[0143] Industrial Applicability

[0144] The present invention can be suitably used in the case of efficiently producing a large amount of cell clusters of uniform size arranged at equal intervals.

[0145] The contents of the documents described in this specification and the Japanese application specification which is the basis of the Paris priority claim of the present application are incorporated herein by reference in their entirety.

Claims

1. A method for manufacturing a culture container, characterized in that: A low cell adhesion polymer is applied as a coating agent to the surface of a culture substrate in advance, and then the culture substrate is processed to form a culture container having the surface of the culture substrate coated with the coating agent as a culture surface. The surface density of the low cell adhesion polymer coated on the surface was 100 μg / cm 2 ~1 μg / cm 2 , In the processing, a plurality of recessed portions are formed on the surface of the culture substrate by recessed and projected processing, and the surface is used as the culture surface of the culture container.

2. The method for manufacturing a culture container according to claim 1, characterized in that: The culture substrate is a film or a sheet, and the culture container is in a bag shape.

3. The method for manufacturing a culture container according to claim 1, characterized in that: In the processing, after the culture substrate is formed into a bag shape, the concavo-convex processing is performed by at least any one of blow molding, vacuum molding, and compressed air molding.

4. The method for manufacturing a culture container according to claim 1, characterized in that: In the processing, the concavo-convex processing is performed by heat transfer, and then the culture substrate is formed into a bag shape.

5. The method for manufacturing a culture container according to claim 4, characterized in that: The above-mentioned concavoconvex processing is performed after a mold release agent is applied to the mold used in the above-mentioned heat transfer.

6. The method for manufacturing a culture container according to any one of claims 1 to 5, characterized in that: The diameter of the circle or the inscribed circle of the opening of the concave portion is 1 μm or more and 10 mm or less.

7. The method for manufacturing a culture container according to any one of claims 1 to 5, characterized in that: The coating agent is applied only to the culture surface of the culture container.

8. The method for manufacturing a culture container according to any one of claims 1 to 5, characterized in that: After the culture substrate is provided with a frame material, the coating agent is applied, and a heat-sealable region to which the coating agent is not applied is provided in the culture substrate.

9. The method for manufacturing a culture container according to any one of claims 1 to 5, characterized in that: The surface of the culture substrate is subjected to a hydrophilization treatment in advance, and then the coating agent is applied to the surface.

10. A culture container, characterized in that a culture container having at least one port, After coating the surface of the culture substrate formed of a film or sheet with a low cell adhesion polymer, the surface formed with a plurality of recesses is formed into a bag shape as a culture surface, and the low cell adhesion polymer is not attached to the opposite side of the culture surface in the port and the culture container. The surface density of the low cell adhesion polymer coated on the surface was 100 μg / cm 2 ~1 μg / cm 2 .

11. The culture container according to claim 10, characterized in that: The low cell adhesion polymer is selected from phospholipid polymers, polyvinyl alcohol derivatives, phospholipid-polymer complexes, poly(hydroxyethyl methacrylate), polyvinyl alcohol, agarose, chitosan, polyethylene glycol, and albumin.

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