Cell culture container and cell culture method

Abstract

The present invention addresses the problem of increasing the gas permeability of a container body and increasing the shape retention of the container body. A cell culture container (1) is provided with a container body (2) having a cylindrical side wall part (20), an opening part (21) formed on one side in the axial direction of the side wall part (20), and a bottom wall part (22) disposed on the other side in the axial direction of the side wall part (20). The container body (2) is made of silicone rubber. One side in the axial direction is designated as an opening part side, the other side in the axial direction is designated as a bottom wall part side, and a direction orthogonal to the axial direction is designated as a radial direction. The side wall part (20) has: a thick wall part (200) having the largest wall thickness (F) in the radial direction in the side wall part (20); and a thin wall part (201) having the smallest wall thickness (G) in the radial direction in the side wall part (20). The thick wall part (200) is located closer to the bottom wall part than the thin wall part (201).

Description

Cell culture vessels and cell culture methods

[0001] This disclosure relates to cell culture vessels and cell culture methods used for culturing cells in fields such as antibody drugs, regenerative medicine, cellular foods, cosmetics, and genetic engineering.

[0002] As shown in Patent Document 1, the container body of a cell culture vessel is generally made of glass or resin. However, glass and resin container bodies have low oxygen permeability. Also, resin container bodies have low heat resistance. For these reasons, low-cost autoclave sterilization cannot be used.

[0003] In this regard, Patent Document 2 discloses a cell culture vessel comprising a container body made of silicone rubber. The container body comprises a cell culture plate and a thin film sheet. The cell culture plate has a large number of cell culture wells (through holes in the vertical direction). The thin film sheet is adhered to the lower surface of the cell culture plate. The thin film sheet seals the large number of cell culture wells from below. Compared to glass and resin, silicone rubber has high oxygen permeability. Therefore, according to the cell culture vessel in the same document, oxygen can be supplied to the large number of cell culture wells through the container body.

[0004] Japanese Patent Publication No. 6-253815, International Publication No. 2023 / 248620, Brochure

[0005] However, silicone rubber container bodies are more flexible and have lower shape retention compared to glass or resin container bodies. Therefore, the container body is prone to deformation during cell culture. The present disclosure aims to improve both the gas permeability and shape retention of the container body in the cell culture container. Furthermore, the present disclosure aims to improve both the gas permeability and shape retention of the container body during cell culture.

[0006] (1) In order to solve the above problems, the cell culture vessel of the present disclosure is a cell culture vessel comprising a container body having a cylindrical side wall portion, an opening provided in one axial direction of the side wall portion, and a bottom wall portion disposed in the other axial direction of the side wall portion, wherein the container body is made of silicone rubber, and with one axial direction being the opening side, the other axial direction being the bottom wall portion side, and the direction perpendicular to the axial direction being the radial direction, the side wall portion has a thick wall portion with the maximum radial wall thickness of the side wall portion and a thin wall portion with the minimum radial wall thickness of the side wall portion, and the thick wall portion is disposed closer to the bottom wall portion than the thin wall portion. Hereinafter, "radial wall thickness" will be abbreviated as "wall thickness" as appropriate.

[0007] In this configuration, the container body is made of silicone rubber. Therefore, the heat resistance of the container body can be increased. Consequently, low-cost autoclave sterilization can be used for sterilizing the container body. Furthermore, this configuration allows for high gas permeability of the container body.

[0008] Furthermore, in this configuration, the thick wall portion of the side wall is positioned closer to the bottom wall than the thin wall portion. Therefore, compared to cases where the thick wall portion is positioned closer to the opening than the thin wall portion, or where the side wall portion does not have a thick wall portion or a thin wall portion (where the wall thickness of the side wall portion is constant along the entire axial length; that is, the side wall portion does not have a difference in wall thickness), the center of gravity of the side wall portion and thus the container body can be shifted towards the bottom wall portion. Consequently, even though the container body is made of flexible silicone rubber, the shape retention of the container body can be improved. Therefore, the shape of the container body can be stabilized when using the cell culture container (for example, when injecting culture medium into the container body, when transporting the container body, and during cell culture (agitation culture, static culture), etc.). Deformation of the container body can be suppressed.

[0009] (1-1) In any of the above configurations, it is preferable that the cell culture vessel be configured for the culture of animal cells. The culture of animal cells requires a large amount of oxygen. In this respect, according to this configuration, the container body is made of silicone rubber with high gas permeability. For this reason, it is suitable for the culture of animal cells.

[0010] (2) In any of the above configurations, it is preferable that the cell culture vessel be configured for agitation culture. Compared to static culture, cells can be cultured with high efficiency in agitation culture. Among these, agitation culture, in which the vessel itself is shaken without using agitators or stirring bars, can be cultured with even higher efficiency by reducing cell damage due to shear force. However, the vessel body is made of flexible silicone rubber. For this reason, the vessel body is prone to vibration in agitation culture. Consequently, the culture medium in the vessel body (more specifically, the culture medium containing cells; the same applies hereinafter) becomes disturbed, and air bubbles are likely to form in the liquid.

[0011] Thus, using a silicone rubber container for shaking culture presents a new problem: "bubbles tend to form easily in the liquid." It should be noted that the aforementioned Patent Document 2 does not contain any description of this problem.

[0012] In this configuration, the thicker wall portion is positioned closer to the bottom wall portion than the thinner wall portion. This stabilizes the shape of the container body. Consequently, disturbance of the culture medium in the container body during shaking culture can be suppressed. Therefore, the generation of air bubbles in the liquid can be suppressed.

[0013] Thus, with this configuration, even though the container body is flexible, the generation of air bubbles during shaking culture can be suppressed. Therefore, a silicone rubber container body can be used for shaking culture. Consequently, highly efficient cell culture is possible.

[0014] (3) In any of the above configurations, the inner surface of the side wall portion has an inclined section, and the inclined section has an opening side end which is the radially outer end of the inner surface and a bottom wall portion side end which is the radially inner end of the inner surface, and the thick wall portion includes the bottom wall portion side end and the thin wall portion includes the opening side end.

[0015] Here, in the axial cross-section, if the "radial outer end of the inner surface" is a point (actually a line; the same applies hereinafter), then the "opening side end" refers to that point. In the axial cross-section, if the "radial outer end of the inner surface" is a line (extending over a predetermined axial section; actually a surface; the same applies hereinafter), then the "opening side end" refers to the opening side end of that line. Similarly, in the axial cross-section, if the "radial inner end of the inner surface" is a point, then the "bottom wall side end" refers to that point. In the axial cross-section, if the "radial inner end of the inner surface" is a line, then the "bottom wall side end" refers to the bottom wall side end of that line.

[0016] In this configuration, an inclined section is arranged on the inner surface of the side wall. Therefore, it is easy to create thick and thin sections (i.e., differences in wall thickness) in the side wall. Consequently, the center of gravity of the side wall can be shifted towards the bottom wall. Thus, the shape retention of the container body can be improved.

[0017] (3-1) In any of the above configurations, the outer surface of the side wall portion has an inclined section, the inclined section has an opening side end which is the radially inner end of the outer surface and a bottom wall portion side end which is the radially outer end of the outer surface, and the thick wall portion includes the bottom wall portion side end and the thin wall portion includes the opening side end.

[0018] Here, in the axial cross-section, if the "inner radial end of the outer surface" is a point, then the "opening side end" refers to that point. In the axial cross-section, if the "inner radial end of the outer surface" is a line, then the "opening side end" refers to the opening side end of that line. Similarly, in the axial cross-section, if the "outer radial end of the outer surface" is a point, then the "bottom wall side end" refers to that point. In the axial cross-section, if the "outer radial end of the outer surface" is a line, then the "bottom wall side end" refers to the bottom wall side end of that line.

[0019] In this configuration, an inclined section is arranged on the outer surface of the side wall. Therefore, it is easy to create thick and thin sections (i.e., differences in wall thickness) in the side wall. Consequently, the center of gravity of the side wall can be shifted towards the bottom wall. This improves the shape retention of the container body. Furthermore, it increases the design freedom of the inner surface shape of the side wall.

[0020] (4) In the configuration of (3) above, it is preferable that the area of ​​the opening be defined as the opening area and the area of ​​the inner surface of the bottom wall be defined as the bottom area, and that the bottom area be within the range of 65% to 95% of the opening area.

[0021] In this configuration, the bottom area is set to 65% or more of the opening area. Therefore, a larger bottom area can be secured compared to cases where it is less than 65%. Consequently, highly efficient cell culture is possible.

[0022] Furthermore, with this configuration, the base area is set to 95% or less of the opening area. Therefore, compared to the case where it exceeds 95%, the difference in wall thickness between the thick wall section and the thin wall section can be made larger. Consequently, the center of gravity of the side wall section can be shifted more reliably towards the base wall section.

[0023] (5) In the configuration of (3) or (4) above, it is preferable that the angle of inclination of the straight line passing between the opening side end and the bottom wall side end with respect to the axial direction in the axial cross section of the container body be within the range of 1° to 5°.

[0024] In this configuration, the inclination angle is set to 1° or more. Therefore, the difference in wall thickness between the thick wall section and the thin wall section can be increased compared to the case where the angle is less than 1°. Consequently, the center of gravity of the side wall section can be shifted more reliably towards the bottom wall section.

[0025] Furthermore, this configuration sets the inclination angle to 5° or less. Therefore, compared to cases where the angle exceeds 5°, a larger bottom area (the area of ​​the inner surface of the bottom wall) can be secured. Consequently, highly efficient cell culture is possible.

[0026] (6) In any of the above configurations, it is preferable that the JIS hardness of the silicone rubber be within the range of 30° to 60°. In this configuration, the JIS hardness of the silicone rubber (specifically, the hardness measured by a Type A durometer in accordance with JIS K 6253-3:2023) is set to 30° or higher. Therefore, compared to the case where the hardness is less than 30°, it is possible to suppress the container body from becoming excessively flexible. In addition, in this configuration, the JIS hardness of the silicone rubber is set to 60° or lower. Therefore, compared to the case where the hardness exceeds 60°, it is possible to increase the gas permeability of the container body.

[0027] (7) In any of the above configurations, it is preferable that the radial wall thickness of the thick wall portion be within the range of 2.4 mm to 7.8 mm. In this configuration, the radial wall thickness of the thick wall portion is set to 2.4 mm or more. Therefore, compared to the case where it is less than 2.4 mm, the center of gravity of the side wall portion can be shifted more reliably toward the bottom wall portion. In addition, it is possible to suppress the thick wall portion from becoming excessively flexible. In addition, in this configuration, the radial wall thickness of the thick wall portion is set to 7.8 mm or less. Therefore, compared to the case where it exceeds 7.8 mm, the gas permeability of the thick wall portion can be increased.

[0028] (7-1) In any of the above configurations, it is preferable that the radial wall thickness of the thin wall portion be within the range of 1.0 mm to 1.6 mm. In this configuration, the radial wall thickness of the thin wall portion is set to 1.0 mm or more. Therefore, compared to the case where it is less than 1.0 mm, it is possible to suppress the thin wall portion from becoming excessively flexible. Also, in this configuration, the radial wall thickness of the thin wall portion is set to 1.6 mm or less. Therefore, compared to the case where it exceeds 1.6 mm, it is possible to shift the center of gravity of the side wall portion more reliably toward the bottom wall portion. In addition, the gas permeability of the thin wall portion can be increased.

[0029] (8) In any of the above configurations, the oxygen permeability of the container body is 1.0E+04cm ３ / m ２ ・24h・atm or more 1.0E+06cm ３ / m ２・ It is better to adopt a structure within the range of 24 h·atm or less. According to this structure, the oxygen permeability of the container body is 1.0E+04 cm ３ / m ２ ・ It is set to 24 h·atm or more. Therefore, 1.0E+04 cm ３ / m ２ ・ Compared with the case of less than 24 h·atm, the gas permeability of the container body can be increased. Also, according to this structure, the oxygen permeability of the container body is 1.0E+06 cm ３ / m ２ ・ It is set to 24 h·atm or less. Therefore, 1.0E+06 cm ３ / m ２ ・ Compared with the case of exceeding 24 h·atm, it is possible to suppress excessive reduction of the culture solution in the container body.

[0030] (9) In any of the above structures, it is better to adopt a structure further provided with a cap for liquid-tightly sealing the opening. According to this structure, leakage of the culture solution through the opening can be suppressed.

[0031] (10) In the structure of (9) above, the container body further has a protruding portion protruding radially outward from the side wall portion, and further includes a cylindrical cap receiver that is annularly mounted on the side wall portion and attached to the cap. The protruding portion is preferably configured to be sandwiched between the cap and the cap receiver in the axial direction.

[0032] The container body is flexible and has low rigidity. When the rigidity difference between the container body and the cap is large, it is difficult to directly attach the cap to the container body. Therefore, it is difficult to ensure the liquid tightness of the opening.

[0033] In this regard, according to this structure, the cap is attached to the cap receiver. Therefore, there is no need to directly attach the cap to the flexible container body. Thus, it is easy to ensure the liquid tightness of the opening. Also, there is no need to deliberately provide a structure for attaching the cap to the container body. Therefore, the structure of the container body can be simplified.

[0034] (11) In the configuration of (10) above, it is preferable that the cap and the cap receiver are made of a resin having a heat resistance of 120°C or higher. According to this configuration, compared with the case where the heat resistance temperature of the cap and the cap receiver is less than 120°C, high-pressure steam sterilization (autoclave sterilization), which is low-cost, can be adopted for the sterilization treatment of the cap and the cap receiver.

[0035] (12) In the configuration of (10) or (11) above, the cap has a top wall portion and a cylindrical cap side wall portion that protrudes from the top wall portion toward the bottom wall portion and is disposed outside the radial direction of the cap receiver. An external thread portion is disposed on the inner surface of the cap side wall portion, and an internal thread portion is disposed on the outer surface of the cap receiver. It is preferable that the cap receiver is attached to the cap by screwing the internal thread portion onto the external thread portion.

[0036] According to this configuration, the cap receiver can be attached to the cap by screwing the internal thread portion and the external thread portion together. Also, in parallel with the attachment, the protruding portion can be sandwiched by the cap and the cap receiver from the axial direction.

[0037] (13) In any of the above configurations, the container body is disposed between the side wall portion and the bottom wall portion and has a chamfer portion continuous with the bottom wall portion. In the axial cross-section of the container body, it is preferable that the shape of the inner surface of the chamfer portion is a curved shape or a linear shape having a constant curvature. According to this configuration, a chamfer portion is disposed between the side wall portion and the bottom wall portion. Therefore, it is possible to suppress stress concentration at the corner between the bottom wall portion and the side wall portion.

[0038] (14) In any of the above configurations, it is preferable that the axial wall thickness of the bottom wall portion is within the range of 1.0 mm or more and 3.2 mm or less. According to this configuration, the axial wall thickness of the bottom wall portion is set to 1.0 mm or more. Therefore, compared with the case where it is less than 1.0 mm, the center of gravity of the container body can be more reliably shifted to the bottom wall portion side. Also, it is possible to suppress the bottom wall portion from becoming overly flexible. Further, according to this configuration, the axial wall thickness of the bottom wall portion is set to 3.2 mm or less. Therefore, compared with the case where it exceeds 3.2 mm, the gas permeability of the bottom wall portion can be increased.

[0039] (15) In order to solve the above problems, the cell culture method of the present disclosure is characterized by using any of the above cell culture containers. According to this configuration, the center of gravity of the side wall portion and thus the container body can be shifted to the bottom wall portion side. Therefore, the shape retention of the container body can be enhanced. Thus, during cell culture, the shape of the container body can be stabilized.

[0040] (16) In the configuration of (15) above, it is preferable that the liquid volume of the culture solution stored inside the container body is within the range of 70% or more and 100% or less of the internal volume of the container body.

[0041] According to this configuration, since the gas permeability of the container body is high, the liquid volume of the culture solution can be increased. The liquid volume of the culture solution is set to 70% or more of the internal volume of the container body. Therefore, compared with the case where it is less than 70%, a large number of cells can be cultured at once. Also, the liquid volume of the culture solution is set to 100% or less of the internal volume of the container body. Therefore, compared with the case where it exceeds 100%, it is possible to suppress excessive liquid pressure from being applied to the cells.

[0042] According to the cell culture container of the present disclosure, the gas permeability of the container body can be increased and the shape retention of the container body can be enhanced. Also, according to the cell culture method of the present disclosure, during cell culture, the gas permeability of the container body can be increased and the shape of the container body can be stabilized.

[0043] Figure 1 is a vertical cross-sectional view of a cell culture vessel corresponding to one embodiment of the cell culture vessel of this disclosure. Figure 2 is a disassembled vertical cross-sectional view of the same cell culture vessel. Figure 3 is an enlarged view of the area within frame III of Figure 2. Figure 4 is a schematic diagram of a shaking device in which the cell culture vessel is arranged. Figures 5(A) to 5(D) are vertical cross-sectional views of the side wall of cell culture vessels of other embodiments (1 to 4). Figures 6(A) to 6(B) are vertical cross-sectional views of the side wall of cell culture vessels of other embodiments (5 to 6). Figure 7 is a vertical cross-sectional view of the side wall of cell culture vessel of another embodiment (7). Figure 8 is a vertical cross-sectional view of the side wall of a cell culture vessel of Comparative Example 1. Figure 9 is a graph showing the change in the number of cells with respect to the number of days elapsed.

[0044] The embodiments of the cell culture vessel and cell culture method described herein will be explained below.

[0045] <Configuration of the Cell Culture Vessel> First, the configuration of the cell culture vessel of this embodiment will be described. In the following figures, the vertical direction corresponds to the "axial direction" of this disclosure, the upper side corresponds to the "one axial side" or "opening side" of this disclosure, and the lower side corresponds to the "other axial side" or "bottom wall side" of this disclosure. Figure 1 shows a cross-sectional view of the cell culture vessel of this embodiment in the vertical direction (axial direction). Figure 2 shows a disassembled cross-sectional view of the same cell culture vessel in the vertical direction. Figure 3 shows an enlarged view of the area within frame III of Figure 2. As shown in Figures 1 and 2, the cell culture vessel 1 comprises a container body 2, a cap receiver 3, and a cap 4.

[0046] (Container body 2) As shown in Figures 1 and 2, the container body 2 is made of silicone rubber and has a bottomed cylindrical (cup-shaped) form that opens to the top. A culture chamber A is partitioned inside the container body 2. The container body 2 is equipped with a side wall portion 20, an opening 21, a bottom wall portion 22, a protruding portion 23, and a chamfered portion 24.

[0047] As shown in Figure 2, the side wall portion 20 has a cylindrical shape that extends in the vertical direction. As shown in Figure 3, the side wall portion 20 comprises a thick wall portion 200, a thin wall portion 201, and an inclined section 203. The thick wall portion 200 is the part of the side wall portion 20 with the maximum radial wall thickness (horizontal wall thickness, wall thickness in the inward and outward direction of the container body 2). The thin wall portion 201 is the part of the side wall portion 20 with the minimum radial wall thickness. The thick wall portion 200 is located below the thin wall portion 201.

[0048] As shown in Figure 3, the outer surface of the side wall portion 20 has a coaxial cylindrical shape. In contrast, the inner surface (the surface on the culture chamber A side) 202 of the side wall portion 20 has an inclined section 203. The inclined section 203 has a tapered shape (slope shape) that decreases in diameter from the top to the bottom. The inclined section 203 has an opening side end 203a and a bottom wall side end 203b. In the vertical cross-section shown in Figure 3, the upper end and radial outer end 202a of the inner surface 202 has a point shape. The opening side end 203a is located at the radial outer end 202a. The thin wall portion 201 includes the opening side end 203a.

[0049] The lower end and radial inner end 202b of the inner surface 202 is point-shaped. The bottom wall side end 203b is located at the radial inner end 202b. The bottom wall side end 203b is located below the opening side end 203a. The bottom wall side end 203b is located at the boundary between the lower end of the side wall 20 and the upper end of the chamfered portion 24. The thick wall portion 200 includes the bottom wall side end 203b.

[0050] As shown in Figure 2, the bottom wall portion 22 is located below the side wall portion 20. The bottom wall portion 22 is disc-shaped. The opening 21 is provided radially inward from the upper end of the inner surface 202 of the side wall portion 20. The projection portion 23 is flange-shaped (annular). The projection portion 23 protrudes radially outward from the upper end of the outer surface of the side wall portion 20.

[0051] As shown in Figure 3, the chamfered portion 24 is located at the corner between the side wall portion 20 and the bottom wall portion 22. The chamfered portion 24 is continuous with the side wall portion 20 and the bottom wall portion 22. In the vertical cross-section, the inner and outer surfaces of the chamfered portion 24 each exhibit an arc shape with the center O as the center of curvature, that is, a curved shape with a constant curvature (R-chamfer shape).

[0052] (Cap receiver 3) As shown in Figures 1 and 2, the cap receiver 3 is made of resin and has a cylindrical shape that extends in the vertical direction. The cap receiver 3 is fitted around the side wall portion 20. An internal threaded portion 300 is arranged on the outer surface (outer circumference) of the cap receiver 3. The cap receiver 3 abuts against the protrusion portion 23 from below.

[0053] (Cap 4) As shown in Figures 1 and 2, the cap 4 is made of resin and has a bottomed cylindrical shape with an opening on the bottom. The cap 4 liquid-tightly seals the opening 21. The cap 4 comprises a cap side wall portion 40 and a top wall portion 42. The top wall portion 42 is disc-shaped. The top wall portion 42 is vertically opposed to the bottom wall portion 22. The top wall portion 42 abuts the side wall portion 20 and the protruding portion 23 from above.

[0054] The cap side wall portion 40 has a cylindrical shape that extends in the vertical direction. The cap side wall portion 40 protrudes downward from the periphery of the top wall portion 42 (the portion radially outward from the protruding portion 23). The cap side wall portion 40 is located radially outward of the cap receiver 3. An external threaded portion 400 is located on the inner surface (inner circumferential surface) of the cap side wall portion 40.

[0055] The cap receiver 3 is attached to the cap 4 by screwing the inner threaded portion 300 into the outer threaded portion 400. Furthermore, this screwing mechanism holds the protruding portion 23 (container body 2) between the upper top wall portion 42 (cap 4) and the lower cap receiver 3. This screwing mechanism also ensures that the opening 21 is liquid-tightly sealed.

[0056] <Method for Manufacturing Cell Culture Vessels> Next, the method for manufacturing the cell culture vessels of this embodiment will be briefly described. The container body 2 is manufactured, for example, by injection molding. Specifically, first, raw material is injected into the cavity (a cavity with the same shape as the container body 2) of a mold (not shown in the figure) (injection step), then the raw material is hardened in the cavity to form a molded product (hardening step), and then the molded product, i.e., the container body 2, is removed from the mold (release step), thereby manufacturing the container body 2. Similarly, the cap receiver 3 and the cap 4 are each manufactured, for example, by injection molding.

[0057] <Cell Culture Method> Next, the cell culture method of this embodiment will be described. Figure 4 shows a schematic diagram of a shaking device in which the cell culture vessel of this embodiment is arranged. As shown in Figure 4, the cell culture vessel 1 described above is used in the cell culture method of this embodiment.

[0058] The shaking device 9 is located inside an incubator (not shown) set to a predetermined environment (temperature, humidity). The shaking device 9 comprises a main body 90 and a table 91.

[0059] The table 91 can rotate horizontally relative to the main body 90 of the device according to a predetermined program (e.g., trajectory, amplitude, period, time, etc.). When viewed from above, the rotational trajectory of the table 91 resembles the number eight.

[0060] The cell culture method of this embodiment comprises a preparation step and a culture step. In the preparation step, first, as shown in Figures 1 and 2, culture medium B (culture medium (culture culture medium) B containing cells C) is injected into the culture chamber A through the opening 21. Here, a scaffold made of glass, resin, or gelatin (cell adhesion scaffold, not shown in the figure) is placed on the inner surface 220 (upper surface) of the bottom wall 22. Cells C attach to this scaffold and proliferate. For this reason, cells C are placed near the bottom wall 22 of the culture chamber A.

[0061] Next, the cap holder 3 and cap 4 are attached to the container body 2. That is, the opening 21 is sealed liquid-tight. Subsequently, as shown in Figure 4, the cell culture container 1, to which the culture medium B has been injected, is placed on the table 91. Then, the shaking device 9, to which the cell culture container 1 is mounted, is brought into the incubator chamber.

[0062] During the culture process, the shaking device 9 is driven. The cell culture vessel 1, together with the table 91, repeatedly rotates for a predetermined time. This rotational movement agitates the culture medium B in culture chamber A (figure-eight shaking agitation). In this way, the cells C are cultured by agitation.

[0063] <Effects and Effects> Next, the effects and effects of the cell culture container and cell culture method of this embodiment will be described. The container body 2 shown in Figures 1 to 3 is made of silicone rubber. Therefore, the heat resistance of the container body 2 can be increased. Accordingly, low-cost high-pressure steam sterilization can be used for sterilizing the container body 2. In addition, the gas (e.g., oxygen, carbon dioxide, etc.) permeability of the container body 2 can be increased. Furthermore, the silicone rubber of the container body 2 is an FDA (U.S. Food and Drug Administration) certified material. Therefore, it is highly safe.

[0064] As shown in Figure 3, the thick wall portion 200 is positioned below the thin wall portion 201. Therefore, compared to cases where the thick wall portion 200 is positioned above the thin wall portion 201, or where the side wall portion 20 does not have a thick wall portion 200 or a thin wall portion 201 (where the wall thickness of the side wall portion 20 is constant along its entire vertical length; that is, the side wall portion 20 does not have a difference in wall thickness), the center of gravity of the side wall portion 20 and thus the container body 2 can be shifted downwards. Consequently, even though the container body 2 is made of flexible silicone rubber, the shape retention of the container body 2 (the ability to maintain its shape against external forces applied to the container body 2 from the operator, external equipment, culture medium B, etc., including the weight of the container body 2 itself) can be increased. Therefore, the shape of the container body 2 can be stabilized when using the cell culture container 1 (preparation process, culture process, etc.). Deformation of the container body 2 can be suppressed. In addition, because of the low center of gravity, tipping of the cell culture container 1 can be suppressed.

[0065] The cells C shown in Figure 1 are animal cells. Animal cells require a large amount of oxygen to culture. In this respect, the container body 2 is made of highly gas-permeable silicone rubber. Therefore, the cell culture container 1 of this embodiment is suitable for culturing animal cells.

[0066] As shown in Figure 4, the cell culture vessel 1 is for shaking culture. Compared to shaking culture using a stirring blade / stirrer and static culture (of course, the cell culture vessel 1 of this disclosure can be used not only for shaking culture but also for shaking culture using a stirring blade / stirrer and static culture), cells C can be cultured with high efficiency in the case of shaking culture. In particular, if the container body 2 is made of highly gas-permeable silicone rubber, cells C can be cultured with extremely high efficiency. However, while silicone rubber has high gas permeability, it is also flexible. Therefore, in the case of shaking culture, the container body 2 is prone to vibration. Consequently, the culture medium B becomes turbulent (for example, turbulence occurs in the liquid or droplets scatter from the liquid surface), and as a result, bubbles are easily generated in the liquid.

[0067] In this respect, the thick-walled portion 200 is positioned below the thin-walled portion 201. This stabilizes the shape of the container body 2. Therefore, disturbance of the culture medium B during shaking culture can be suppressed. Consequently, the generation of air bubbles in the liquid can be suppressed.

[0068] Thus, according to the cell culture vessel 1 of this embodiment, even though the vessel body 2 is flexible, the generation of bubbles during shaking culture can be suppressed. For this reason, the vessel body 2 made of highly gas-permeable silicone rubber can be used for shaking culture. Consequently, highly efficient culture of cells C is possible. Furthermore, mass production of cells C is possible.

[0069] As shown in Figure 3, an inclined section 203 is arranged on the inner surface 202 of the side wall 20. Therefore, a thick wall section 200 and a thin wall section 201 (i.e., a difference in wall thickness) can be easily set in the side wall 20. Consequently, the center of gravity of the side wall 20 can be easily shifted downwards. Thus, the shape retention of the container body 2 can be improved.

[0070] As shown in Figure 1, the area of ​​the opening 21 is defined as the opening area Sa. The area of ​​the inner surface 220 of the bottom wall 22 is defined as the bottom area Sb. The bottom area Sb is set to 65% or more of the opening area Sa. Therefore, a larger bottom area can be secured compared to the case where it is less than 65%. Consequently, highly efficient culture of cells C is possible.

[0071] Furthermore, the base area Sb is set to 95% or less of the opening area Sa. Therefore, compared to the case where it exceeds 95%, the difference in wall thickness between the thick wall section 200 and the thin wall section 201 can be made larger. Consequently, the center of gravity of the side wall section 20 can be shifted downward more reliably.

[0072] As shown in Figure 3, in the vertical cross-section of the container body 2, the virtual line D extends in the vertical direction. The line E passes through the opening side end 203a and the bottom wall side end 203b. The inclination angle θ of line E with respect to the virtual line D is set to 1° or more. Therefore, the difference in wall thickness between the thick wall portion 200 and the thin wall portion 201 can be increased compared to the case where it is less than 1°. Consequently, the center of gravity of the side wall portion 20 can be shifted downward more reliably.

[0073] Furthermore, the inclination angle θ is set to 5° or less. Therefore, a larger base area Sb can be secured compared to the case where the angle exceeds 5°. Consequently, highly efficient culture of cells C is possible.

[0074] The JIS hardness of the silicone rubber forming the container body 2 shown in Figure 2 (specifically, the hardness measured using a Type A durometer in accordance with JIS K 6253-3:2023) is set to 30° or higher. Therefore, compared to the case where the hardness is less than 30°, it is possible to suppress the container body 2 from becoming excessively flexible. Furthermore, the JIS hardness of the silicone rubber forming the container body 2 is set to 60° or lower. Therefore, compared to the case where the hardness exceeds 60°, it is possible to increase the gas permeability of the container body 2.

[0075] As shown in Figure 3, the radial wall thickness F of the thick wall section 200 is set to 2.4 mm or more. Therefore, compared to the case where it is less than 2.4 mm, the center of gravity of the side wall section 20 can be shifted downward more reliably. In addition, it is possible to suppress the thick wall section 200 from becoming excessively flexible. Furthermore, the radial wall thickness F is set to 7.8 mm or less. Therefore, compared to the case where it exceeds 7.8 mm, the gas permeability of the thick wall section 200 and, consequently, the container body 2 can be increased.

[0076] As shown in Figure 3, the radial wall thickness G of the thin-walled section 201 is set to 1.0 mm or more. Therefore, compared to the case where it is less than 1.0 mm, it is possible to suppress the thin-walled section 201 from becoming excessively flexible. Also, the radial wall thickness G is set to 1.6 mm or less. Therefore, compared to the case where it exceeds 1.6 mm, it is possible to shift the center of gravity of the side wall section 20 more reliably downwards. In addition, the gas permeability of the thin-walled section 201 and, consequently, the container body 2 can be increased.

[0077] The oxygen permeability of the container body 2 shown in Figure 3 is 1.0E+04cm². ３ / m ２ • It is set to 24 hours atm or higher. Therefore, 1.0E+04cm ３ / m ２ Compared to the case where the oxygen permeability is less than 24h·atm, the oxygen permeability of the container body 2 can be increased. The oxygen permeability of the container body 2 is 1.0E+06cm². ３ / m ２ It is set to 24h·atm or less. Therefore, 1.0E+06cm ３ / m ２ Compared to the case where the incubation period exceeds 24 hours atm, this method can suppress the excessive decrease in culture medium B in container body 2.

[0078] As shown in Figures 1 and 2, the cell culture vessel 1 is equipped with a cap 4. This prevents leakage of the culture medium B through the opening 21.

[0079] The silicone rubber container body 2 is flexible and has low rigidity. On the other hand, the resin cap 4 has higher rigidity than the container body 2. Therefore, there is a large difference in rigidity between the container body 2 and the cap 4. Consequently, it is difficult to directly attach the cap 4 to the container body 2. Therefore, it is difficult to ensure liquid-tightness of the opening 21.

[0080] In this regard, as shown in Figures 1 and 2, in the cell culture container 1 of this embodiment, the cap 4 is indirectly attached to the container body 2 by being sandwiched between the cap 4 and the cap receiver 3. The highly rigid cap 4 is screwed onto the highly rigid cap receiver 3. Therefore, there is no need to directly attach the cap 4 to the flexible container body 2. Consequently, it is easier to ensure liquid-tightness of the opening 21. Furthermore, there is no need to add a structure to the container body 2 for attaching the cap 4. Therefore, the structure of the container body 2 can be simplified.

[0081] Furthermore, the resin cap receiver 3 and cap 4 are both highly rigid compared to the silicone rubber container body 2. Therefore, the cap receiver 3 and cap 4 can firmly clamp the protruding portion 23 from above and below. Also, under clamping load, the flexible protruding portion 23 easily deforms elastically along the surface shape of the cap receiver 3 and cap 4. This makes it easier to ensure liquid tightness between the protruding portion 23 and the cap receiver 3, and between the protruding portion 23 and the cap 4.

[0082] The cap 4 and cap receiver 3 shown in Figures 1 and 2 are made of a heat-resistant resin (specifically, PP (polypropylene)) with a heat resistance of 120°C or higher. Therefore, compared to cases where the heat resistance is less than 120°C, low-cost autoclaving can be used for sterilizing the cap 4 and cap receiver 3.

[0083] As shown in Figures 1 and 2, according to the cell culture container 1 of this embodiment, the cap receiver 3 can be easily attached to the cap 4 by screwing together the inner threaded portion 300 and the outer threaded portion 400. In parallel with the attachment, the protruding portion 23 can be clamped from above and below by the cap 4 and the cap receiver 3.

[0084] As shown in Figure 3, the axial wall thickness I of the bottom wall portion 22 (vertical wall thickness, wall thickness in the inward and outward directions of the container body 2) is set to 1.0 mm or more. Therefore, compared to the case where it is less than 1.0 mm, the center of gravity of the container body 2 can be shifted downward more reliably. In addition, it is possible to suppress the bottom wall portion 22 from becoming excessively flexible. Furthermore, the axial wall thickness I is set to 3.2 mm or less. Therefore, compared to the case where it exceeds 3.2 mm, the gas permeability of the bottom wall portion 22 and thus the container body 2 can be increased.

[0085] As shown in Figure 3, a chamfered portion 24 is provided at the corner between the side wall portion 20 and the bottom wall portion 22. This helps to suppress stress concentration at the corner.

[0086] The cell culture method of this embodiment uses the cell culture vessel of this embodiment. As a result, the center of gravity of the side wall portion 20 and thus the container body 2 can be shifted downwards. Therefore, the shape retention of the container body 2 can be improved. Thus, the shape of the container body 2 can be stabilized during preparation and culture processes. In addition, because the center of gravity is low, tipping of the cell culture vessel 1 can be suppressed.

[0087] In the preparation process, the volume of culture medium B stored in culture chamber A is set to 70% or more of the internal volume of culture chamber A. Therefore, compared to the case where the volume is less than 70%, a large amount of cells C can be cultured at once. Furthermore, the volume of culture medium B is set to 100% or less of the internal volume of culture chamber A. Therefore, compared to the case where the volume exceeds 100%, excessive liquid pressure on cells C can be suppressed. Thus, according to the cell culture method of this embodiment, the gas permeability of the container body 2 is high, so the volume of culture medium B can be increased. Therefore, mass production of cells C is possible.

[0088] <Other> The embodiments of the cell culture vessel and cell culture method of this disclosure have been described above. However, the embodiments are not particularly limited to the above forms. Various modified and improved forms can be implemented by those skilled in the art.

[0089] Figures 5(A) to 5(D) show vertical cross-sectional views of the side wall of the cell culture vessel in other embodiments (1 to 4). The same reference numerals are used for parts corresponding to those in Figure 3. Similar to Figure 3, Figures 5(A) to 5(D) show the left portion of the side wall 20. For ease of explanation, a dotted line is drawn at the boundary between the side wall 20 and the protruding portion 23.

[0090] As shown in Figure 5(A), the inclined section 203 has a stepped shape, with the diameter decreasing from the top to the bottom. The radial outer end 202a of the inner surface 202 has a linear shape that extends in the vertical direction. That is, the radial outer end 202a extends over a predetermined vertical section of the inner surface 202. The opening side end 203a is located at the upper end of the linear radial outer end 202a. Similarly, the radial inner end 202b of the inner surface 202 has a linear shape that extends in the vertical direction. That is, the radial inner end 202b extends over a predetermined vertical section of the inner surface 202. The bottom wall side end 203b is located at the lower end of the linear radial inner end 202b.

[0091] As in this embodiment, the inclined section 203 may have a stepped shape. The radial outer end 202a and radial inner end 202b may extend over a predetermined vertical section of the inner surface 202. The straight line E does not have to be a solid line. The straight line E may be a virtual straight line connecting the opening side end 203a and the bottom wall side end 203b.

[0092] As shown in Figure 5(B), the inclined section 203 has a curved shape, decreasing in diameter from the top to the bottom. Similar to the chamfered section 24, the inclined section 203 is curved in a direction that bulges outward in the radial direction.

[0093] As in this embodiment, the inclined section 203 may be curved. As shown by curve H, the inclined section 203 may be curved in a direction that bulges radially inward, opposite to the chamfered section 24.

[0094] As shown in Figure 5(C), the inclined section 203 exhibits a stepped and straight shape, with its diameter decreasing from the top to the bottom. That is, the upper section of the inclined section 203 is a stepped section, and the lower section of the inclined section 203 is a straight section.

[0095] As shown in Figures 5(A) to 5(C), the shape of the inclined section 203 is not particularly limited. It may be stepped, straight, curved, or a composite shape of multiple shapes (stepped, straight, curved, etc.). The position, size, and number of inclined sections 203 on the inner surface 202 are not particularly limited.

[0096] As shown in Figure 5(D), the protrusions 23 shown in Figures 5(A) to 5(C) do not necessarily have to be present. In other words, the presence or absence of protrusions 23 is not limited. Furthermore, the shape, position, size, and number of protrusions 23 are not particularly limited. Regarding the shape of the protrusions 23, when viewed from above, the protrusions 23 may be annular (flange-shaped), arc-shaped, tag-shaped, etc. Regarding the number of protrusions 23, multiple protrusions 23 may be arranged along the circumferential direction of the opening 21 at predetermined intervals. In other words, the protrusions 23 only need to be able to be held between the cap 4 and the cap receiver 3 from above.

[0097] As shown in Figure 5(D), instead of the chamfered portion 24 having a curved (R-shaped chamfer) inner and outer surface as shown in Figures 5(A) to 5(C), a chamfered portion 24 having a straight (C-shaped chamfer) inner and outer surface may be provided. In other words, the shape of the chamfered portion 24 is not particularly limited.

[0098] The chamfered portion 24 may have a chamfer (R chamfer, C chamfer, etc.) applied only to the inner surface, or only to the outer surface, or both the inner and outer surfaces. The presence or absence of the chamfered portion 24 is not limited. The chamfered portion 24 may not be present at all.

[0099] Figures 6(A) to 6(B) show vertical cross-sectional views of the side walls of cell culture vessels in other embodiments (5 to 6). The same reference numerals are used for parts corresponding to those in Figures 5(A) to 5(D).

[0100] As shown in Figure 6(A), an inclined section 205 is provided on the outer surface 204 of the side wall portion 20. On the other hand, the inclined section 203 shown in Figure 5(A) is not provided on the inner surface 202. The inner surface 202 extends in the vertical direction.

[0101] Furthermore, the arrangement and configuration of the inclined section 205 shown in Figure 6(A) is symmetrical in the radial direction (radial direction of the container body 2) to the arrangement and configuration of the inclined section 203 shown in Figure 5(A). For example, the radial inner end 204a of the outer surface 204 (Figure 6(A)) corresponds to the radial outer end 202a of the inner surface 202 (Figure 5(A)), the radial outer end 204b of the outer surface 204 (Figure 6(A)) corresponds to the radial inner end 202b of the inner surface 202 (Figure 5(A)), the opening side end 205a of the inclined section 205 (Figure 6(A)) corresponds to the opening side end 203a of the inclined section 203 (Figure 5(A)), and the bottom wall side end 205b of the inclined section 205 (Figure 6(A)) corresponds to the bottom wall side end 203b of the inclined section 203 (Figure 5(A)).

[0102] As shown in Figure 6(A), the inclined section 205 has a stepped shape, widening in diameter from top to bottom. The radial inner end 204a of the outer surface 204 is linear in shape, extending in the vertical direction. That is, the radial inner end 204a extends over a predetermined vertical section of the outer surface 204. The opening side end 205a is located at the upper end of the linear radial inner end 204a. Similarly, the radial outer end 204b of the outer surface 204 is linear in shape, extending in the vertical direction. That is, the radial outer end 204b extends over a predetermined vertical section of the outer surface 204. The bottom wall side end 205b is located at the lower end of the linear radial outer end 204b.

[0103] In this embodiment, an inclined section 205 is arranged on the outer surface 204. This allows the center of gravity of the side wall 20 to be shifted towards the bottom wall 22. Furthermore, since the inclined section 205 is arranged on the outer surface 204, there is no need to deliberately arrange an inclined section 203 on the inner surface 202 of the side wall 20. Therefore, the design freedom of the shape of the inner surface 202 can be increased. As in this embodiment, the inclined section 205 may be arranged on the outer surface 204 of the side wall 20. The inclined section 205 may also have a stepped shape. The radial inner end 204a and radial outer end 204b may extend over a predetermined vertical section of the outer surface 204. The straight line E does not have to be a solid line. The straight line E may be a virtual straight line connecting the opening side end 205a and the bottom wall side end 205b.

[0104] Alternatively, instead of the inclined section 205 shown in Figure 6(A), an inclined section 203 (with its arrangement and configuration reversed radially) as shown in any of Figures 5(B) to 5(D) may be placed on the outer surface 204.

[0105] As shown in Figure 6(B), the inclined section 203 shown in Figure 5(A) may be arranged on the inner surface 202, and the inclined section 205 shown in Figure 6(A) may be arranged on the outer surface 204. In this case, the configuration of the inclined section 203 is not particularly limited. The configuration of the inclined section 203 may be as shown in any of Figures 5(B) to 5(D). Similarly, the configuration of the inclined section 205 is not particularly limited. The configuration of the inclined section 203 (with the arrangement and configuration reversed radially) may be as shown in any of Figures 5(B) to 5(D).

[0106] The number of inclined sections 203 is not particularly limited. There may be one or more. The location of the inclined sections 203 is not particularly limited. They may be the entire length in the axial direction or just a part of it. The same applies to the inclined sections 205.

[0107] Figure 7 shows a vertical cross-sectional view of the side wall of a cell culture vessel of another embodiment (7). The same reference numerals are used for parts corresponding to those in Figures 5(A) to 5(D). As shown in Figure 7, chamfering may be applied only to the inner surface of the chamfered portion 24. The axial wall thickness (vertical wall thickness, inner-outer wall thickness of the container body 2) I of the bottom wall portion 22 is greater than the radial wall thickness F of the thick wall portion 200. Specifically, the axial wall thickness I is set to exceed 100% of the radial wall thickness F. Therefore, compared to the case where the axial wall thickness I is 100% or less of the radial wall thickness F, the center of gravity of the container body 2 can be shifted downwards. Also, the axial wall thickness I is set to 130% or less of the radial wall thickness F. Therefore, compared to the case where the axial wall thickness I exceeds 130% of the radial wall thickness F, the gas permeability of the bottom wall portion 22 relative to the gas permeability of the side wall portion 20 can be increased. Therefore, the supply and discharge of gas to and from cells C (cells C are attached to scaffolds placed on the inner surface 220) near the inner surface 220 of the bottom wall portion 22 can be improved.

[0108] Furthermore, the settings for the axial wall thickness I and radial wall thickness F shown in Figure 7 can be implemented independently of the thick wall portion 200 and the thin wall portion 201. That is, in a container body 2 having a side wall portion 20 that does not have a thick wall portion 200 or a thin wall portion 201 (a side wall portion 20 in which the wall thickness is the same along the entire length in the vertical direction), the axial wall thickness I of the bottom wall portion 22 may be set to be thicker than the radial wall thickness F of the side wall portion 20. For example, the axial wall thickness I may be set to be more than 100% of the radial wall thickness F. Alternatively, the axial wall thickness I may be set to be 130% or less of the radial wall thickness F. Also, weights may be selectively placed only on the bottom wall portion 22 of the side wall portion 20 and the bottom wall portion 22.

[0109] The method of attaching the cap 4 to the cap receiver 3 shown in Figure 2 is not particularly limited. It may be done by screwing, engaging, or bonding. Alternatively, the cap 4 may be attached to the cap receiver 3 using a separate component (such as a bolt or clamp). Furthermore, a sealing member (such as a sealing ring) may be interposed between the cap receiver 3 and the cap 4.

[0110] The cap 4 and cap receiver 3 may or may not be placed. The cap 4 may be directly attached to the container body by screwing, engaging, or bonding. Doing so eliminates the need for the cap receiver 3, thus reducing the number of parts in the cell culture container 1. It also simplifies the shape of the container body 2 by eliminating the need for the protrusion 23. Furthermore, eliminating the cap receiver 3 and protrusion 23 allows the center of gravity of the container body 2 to be shifted downwards.

[0111] The material of the container body 2 is not particularly limited. The container body 2 may be made of elastomers such as thermosetting elastomer materials such as silicone rubber, natural rubber, isoprene rubber, butadiene rubber, styrene-butadiene rubber, butyl rubber, nitrile rubber, ethylene propylene rubber, chloroprene rubber, acrylic rubber, urethane rubber, and fluororubber, or thermoplastic elastomer materials such as polyamide-based thermoplastic elastomer (TPA), polyester-based thermoplastic elastomer (TPC), olefin-based thermoplastic elastomer (TPO), styrene-based thermoplastic elastomer (TPS), urethane-based thermoplastic elastomer (TPU), and dynamically crosslinked thermoplastic elastomer (TPV). Examples of silicone rubber include liquid-type silicone rubber and millable-type silicone rubber. Examples of the polymer component of silicone rubber include PDMS (polydimethylsiloxane). The container body 2 only needs to have expandability (e.g., rubber elasticity). The container body 2 also only needs to have gas permeability (gas permeability sufficient for cell culture). The material of the cap 4 and cap receiver 3 is not particularly limited. For example, they may be made of resin such as PP, PI (polyimide), or fluororesin, elastomer similar to the container body 2, or metal such as stainless steel.

[0112] The type of culture medium B shown in Figure 1 is not particularly limited. Culture medium B may contain inorganic salts, carbohydrates, amino acids, vitamins, proteins, fatty acids, serum, etc. as appropriate. The culture form is not particularly limited. It may be a suspension culture system (a system in which a single cell C or a cell aggregate consisting of a small number of cells C is suspended in culture medium B) or an adherent culture system (a system in which many cells C are attached in layers to the inner surface 220 or scaffold). The type of cell C is not particularly limited. For example, cell lines (passaged cell lines) include adherent cell lines such as MRC-5, HeLa, Vero, NIH3T3, L929, BHK-21, HEK293, HepG2, BAE-1, and SH-SY5Y. Also, suspension cell lines include CHO, NS0, U937, Namalwa, HL60, WEHI231, YAC1, and U266B1. Other examples include primary cells derived from various tissues directly collected from animals. The method of culturing cell C is not particularly limited. Static culture (two-dimensional culture, monolayer culture), agitated culture (stirrer agitation, rotary agitation, swirling agitation, shaking agitation), suspension culture, three-dimensional culture using a scaffold, etc., are also acceptable. The manufacturing method of each component constituting the cell culture container 1 (container body 2, cap holder 3, cap 4, etc.) is not particularly limited. A manufacturing method appropriate to the material of the component may be adopted.

[0113] Next, a culture test conducted using the cell culture vessel and cell culture method of this disclosure will be described. Figure 8 shows a vertical cross-sectional view of the side wall of the cell culture vessel of Comparative Example 1. The same reference numerals are used for parts corresponding to those in Figure 3. Similar to Figure 3, Figure 8 shows the left portion of the side wall 20. For the sake of explanation, a dotted line is drawn at the boundary between the side wall 20 and the protruding portion 23. The following explanation will be based on Figure 8 and Figures 1 to 4 mentioned above.

[0114] <Specifications of Cell Culture Vessels> First, the cell culture vessels used in the culture test will be described. There are a total of five cell culture vessels: Examples 1 to 3 and Comparative Examples 1 to 2. As shown in Figures 3 and 8, the shape of the chamfered portion 24 differs between Examples 1 to 3 and Comparative Example 1. Specifically, as shown in Figure 3, the inner and outer surfaces of the chamfered portion 24 of Example 1 are arc-shaped with the center O as the center of curvature, that is, curved shapes with a constant curvature (R-chamfered shape). In contrast, as shown in Figure 8, the inner surface of the chamfered portion 24 of Comparative Example 1 is curved, similar to Examples 1 to 3. However, the outer surface of the chamfered portion 24 is not chamfered. Comparative Example 2 is a culture vessel in which a commercially available glass container (500 ml wide-mouth medium bottle manufactured by SIMAX) has an air vent filter (0.2 μm filter manufactured by Merck) attached to the cap via a hollow bolt and silicone tube, allowing gas to be supplied from the top surface of the container. Table 1 shows the specifications and culture results for each cell culture vessel.

[0115] As shown in Table 1, the material of the container body 2 is PDMS for Examples 1-3 and Comparative Example 1. Of these, Examples 1-2 and Comparative Example 1 are made of PDMS-A (Shin-Etsu Chemical Co., Ltd. KE1950-30), Example 3 is made of PDMS-B (Shin-Etsu Chemical Co., Ltd. KE1950-60), and Comparative Example 2 is made of glass. Due to the difference in material, Examples 1-2 and Comparative Example 1 have lower hardness and higher oxygen permeability than Example 3. The oxygen permeability of Comparative Example 2 is lower than that of Examples 1-2 and Comparative Example 1.

[0116] The inclination angles θ of the container body 2 shown in Figures 3 and 8 are, in ascending order, Comparative Example 1 (θ = 0°), Examples 1 and 3 (θ = 1°), and Example 2 (θ = 5°). Due to the difference in the inclination angle θ, the radial wall thicknesses F and G differ between Examples 1 and 3, Example 2, and Comparative Example 1. The axial wall thickness I of the bottom wall portion 22 is the same for all Examples 1 to 3 and Comparative Example 1. The volume (maximum culture medium volume) of Comparative Example 1 (θ = 0°) is 290 ml, the volume (maximum culture medium volume) of Examples 1 and 3 (θ = 1°) is 280 ml, and the volume (maximum culture medium volume) of Example 2 (θ = 5°) is 250 ml.

[0117] <Culture Conditions> Next, we will explain the culture conditions. The culture test was conducted inside an incubator. The environment inside the incubator was set to a temperature of 37°C, a humidity of 95%, and a carbon dioxide concentration of 5%. The culture period was 13 days.

[0118] For all cell culture vessels (Examples 1-3, Comparative Examples 1-2), the target cells C (animal cells, specifically duck hepatocytes (primary, second passage, derived from the liver of a duck fetus), the volume of culture medium B (200 ml), the components of culture medium B (a mixture of basal medium D-MEM (containing high glucose, L-glutamine, phenol red, and sodium pyruvate, Fujifilm Wako Pure Chemical Industries, Ltd.) + 10% FBS (fetal bovine serum, Cosmo Bio Inc.) + 1% PSA (penicillin-streptomycin-amphotericin B suspension, Fujifilm Wako Pure Chemical Industries, Ltd.)), and the scaffold (manufactured by Cesc) were identical. The stirring method was figure-eight shaking. The rotation speed (number of figure-eight movements per minute) was 60 rpm. Under these conditions, culture tests were performed on all cell culture vessels using the shaking device 9.

[0119] <Test Results> Next, we will explain the test results. Regarding the evaluation conditions, in Table 1, for the splashing of liquid during shaking and stirring in the shaking culture evaluation, cell culture container 1 in which culture medium B did not adhere to cap 4 during shaking and stirring was evaluated as "○", and cell culture container 1 in which culture medium B adhered to cap 4 was evaluated as "×". Regarding bubble generation during shaking and stirring, cell culture container 1 in which no bubbles were generated in culture medium B during shaking and stirring was evaluated as "○", and cell culture container 1 in which bubbles were generated in culture medium B was evaluated as "×".

[0120] Regarding liquid splashing, it can be seen that Examples 1 and 3 (θ=1°), Example 2 (θ=5°), and Comparative Example 2 are less prone to liquid splashing than Comparative Example 1 (θ=0°). Regarding bubble generation, it can be seen that Examples 1 and 3 (θ=1°), Example 2 (θ=5°), and Comparative Example 2 are less prone to bubble generation than Comparative Example 1 (θ=0°). Thus, in terms of liquid splashing and bubble generation, the soft PDMS-made Examples 1 to 3 allow for stable shaking and stirring, and exhibit the same level of stability performance as the hard glass-made Comparative Example 2 during shaking and stirring.

[0121] Figure 9 shows the change in cell number over time (culture period). As shown in Table 1 and Figure 9, the cell number (cell proliferation rate) after 7 days from the start of the experiment is, in descending order, Example 1, Example 3, Example 2, Comparative Example 1, and Comparative Example 2. Similarly, the cell number after 13 days from the start of the experiment is, in descending order, Example 1, Example 3, Example 2, Comparative Example 1, and Comparative Example 2. As the number of days increases, the difference in cell number between Examples 1-3 and Comparative Examples 1-2 widens.

[0122] Thus, it can be seen that Examples 1-3 and Comparative Example 1, which are made of PDMS and have high oxygen permeability, can culture cells C more efficiently than Comparative Example 2, which is made of glass and has low oxygen permeability. Furthermore, even though they are all made of PDMS (but of different types), it can be seen that Examples 1 and 3 (θ = 1°) and Example 2 (θ = 5°), which have an inclination angle θ within a predetermined range (1° to 5°), can culture cells C more efficiently than Comparative Example 1 (θ = 0°), which has no inclination angle θ.

[0123] 1: Cell culture vessel, 2: Vessel body, 20: Side wall, 200: Thick wall, 201: Thin wall, 202: Inner surface, 202a: Radial outer end, 202b: Radial inner end, 203: Inclined section, 203a: Opening side end, 203b: Bottom wall side end, 204: Outer surface, 204a: Radial inner end, 204b: Radial outer end, 205: Inclined section, 205a: Opening side end, 205b: Bottom wall side end, 21: Opening, 22: Bottom wall, 220: Inner surface, 23: Protrusion, 24: Chamfered section, 3: Cap holder, 300: Inner threaded section, 4: Cap, 40: Cap side wall, 400: Outer threaded section, 42: Top wall, 9: Shaking device, 90: Device body, 91: Table θ: tilt angle, A: culture chamber, B: culture medium, C: cell, F: radial wall thickness, G: radial wall thickness, I: axial wall thickness, Sa: opening area, Sb: bottom area

Claims

1. A cell culture container comprising a container body having a cylindrical side wall portion, an opening provided in one axial direction of the side wall portion, and a bottom wall portion located in the other axial direction of the side wall portion, wherein the container body is made of silicone rubber, with one axial direction being the opening side, the other axial direction being the bottom wall portion side, and the direction perpendicular to the axial direction being the radial direction, the side wall portion having a thick wall portion with the maximum radial wall thickness of the side wall portion and a thin wall portion with the minimum radial wall thickness of the side wall portion, and the thick wall portion being located closer to the bottom wall portion than the thin wall portion.

2. The cell culture vessel according to claim 1, which is for shaking culture.

3. The cell culture vessel according to claim 1, wherein the inner surface of the side wall portion has an inclined section, the inclined section having an opening side end which is the radially outer end of the inner surface and a bottom wall portion side end which is the radially inner end of the inner surface, the thick wall portion includes the bottom wall portion side end and the thin wall portion includes the opening side end.

4. The cell culture container according to claim 3, wherein the area of ​​the opening is defined as the opening area, and the area of ​​the inner surface of the bottom wall is defined as the bottom area, and the bottom area is within a range of 65% to 95% of the opening area.

5. The cell culture container according to claim 3, wherein, in the axial cross-section of the container body, the inclination angle of the straight line passing through the opening side end and the bottom wall side end with respect to the axial direction is within the range of 1° to 5°.

6. The cell culture vessel according to claim 1, wherein the JIS hardness of the silicone rubber is in the range of 30° to 60°.

7. The cell culture vessel according to claim 1, wherein the radial wall thickness of the thick-walled portion is in the range of 2.4 mm to 7.8 mm, and the radial wall thickness of the thin-walled portion is in the range of 1.0 mm to 1.6 mm.

8. The oxygen permeability of the side wall portion is 1.0E+04cm. ３ / m ２ ・24h・atm or more 1.0E+06cm ３ / m ２ A cell culture vessel according to claim 1, which is included in the range of 24 h·atm or less.

9. The cell culture vessel according to claim 1, further comprising a cap that liquid-tightly seals the opening.

10. The cell culture container according to claim 9, wherein the container body further has a projection that protrudes radially outward from the side wall, and further comprises a cylindrical cap receiver that is fitted around the side wall and attached to the cap, and the projection is held between the cap and the cap receiver from the axial direction.

11. The cell culture container according to claim 10, wherein the cap and the cap holder are made of a resin having heat resistance of 120°C or higher.

12. The cell culture container according to claim 10, wherein the cap has a top wall portion and a cylindrical cap side wall portion that protrudes from the top wall portion toward the bottom wall portion and is arranged radially outward of the cap receiver, an outer thread portion is arranged on the inner surface of the cap side wall portion, an inner thread portion is arranged on the outer surface of the cap receiver, and the cap receiver is attached to the cap by screwing the inner thread portion into the outer thread portion.

13. The cell culture container according to claim 1, wherein the container body is positioned between the side wall portion and the bottom wall portion and has a chamfered portion connected to the bottom wall portion, and in the axial cross-section of the container body, the shape of the inner surface of the chamfered portion is curved or straight with a certain curvature.

14. The cell culture vessel according to claim 1, wherein the axial wall thickness of the bottom wall portion is in the range of 1.0 mm or more and 3.2 mm or less.

15. A cell culture method using the cell culture vessel described in claim 1.

16. The cell culture method according to claim 15, wherein the amount of culture medium stored inside the container body is within the range of 70% to 100% of the internal volume of the container body.

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