Shaking culture device and shaking culture method

Abstract

To provide a shaking culture device and a shaking culture method that reduce the share stress acting on a culture, and can suppress the growth inhibition.SOLUTION: A culture vessel 10 has a curved face 14 on a bottom 13. The curved face 14 is formed so that the height of the curved face 14 is higher in the center of the bottom 13 and lower at the periphery of the bottom 13.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a shaking culture apparatus and a shaking culture method. [Background technology]

[0002] Various substances useful as pharmaceuticals, foods, cosmetics, and raw materials for these products are produced by culturing microorganisms, insect cells, plant cells, animal cells, and the like. Patent document 1 describes a single-use cell culture device in which a baffle consisting of a curved, convex protrusion is provided on part of the inner surface of the housing part that contains a flexible culture bag and that abuts against the culture bag. Patent Document 2 describes a culture vessel having a protruding baffle extending horizontally inside a cylindrical stirring vessel for containing liquid contents. Patent document 3 describes a culture vessel in which the outer shape of the inner bottom surface is circular and a channel is provided as a concave annular passage concentrically arranged with respect to the bottom surface, which can determine the direction of the flow of the culture medium during shaking culture. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-35009 [Patent Document 2] Japanese Patent Application Publication No. 2019-198850 [Patent Document 3] Japanese Patent Application Publication No. 9-65876 Summary of the Invention [Problem to be solved by the invention]

[0004] Patent Document 1 describes that when part of the culture solution collides with the baffle, the direction of the swirling flow is changed and an up-and-down flow occurs, thereby enabling efficient mixing so that the culture solution becomes uniform. Patent Document 2 describes that in order to suppress the shear acting on the contents, it is possible to mix the contents uniformly with high stirring efficiency even when stirring mildly. Patent Document 3 describes that animal cells suspended in a culture medium move smoothly in a predetermined flow direction along with the flow of the culture medium, thereby significantly reducing collisions between animal cells and preventing damage to the animal cells.

[0005] When culturing large amounts of cells, such as stem cells used in regenerative medicine, stirring or shaking can cause shear stress on the cells, inhibiting their growth. Simply reducing the flow rate to reduce shear stress results in insufficient mixing of the contents, inhibiting growth.

[0006] The present invention has been made in consideration of the above circumstances, and an object of the present invention is to provide a shaking culture apparatus and a shaking culture method that can reduce shear stress acting on a culture and suppress growth inhibition. [Means for solving the problem]

[0007] In order to solve the above problem, the present invention provides a shaking culture device characterized in that the bottom surface of the culture vessel has a curved surface, and the curved surface is formed so that the height of the curved surface is higher at the center of the bottom surface and lower at the periphery of the bottom surface.

[0008] The bottom surface may be circular, and the culture vessel may have a cylindrical side surface on the bottom surface. The curved surface may have a shape that is rotationally symmetrical around the center of the bottom surface. The culture vessel may include an outer shell having the curved surface, and a flexible inner bag housed in the outer shell. The culture vessel may comprise a flexible inner bag, a curved member having the curved surface, and an outer shell that houses the inner bag, and the curved member may be positioned between the bottom surface of the outer shell and the inner bag.

[0009] The present invention also provides a shaking culture method, which comprises performing shaking culture of a culture using the shaking culture apparatus. [Effects of the Invention]

[0010] According to the present invention, the height of the curved surface formed on the bottom of the culture vessel is higher at the center of the bottom and lower at the periphery of the bottom, so that the depth of the culture medium is shallow at the center of the bottom, where the shaking power is less likely to act, and is deeper at the periphery of the bottom, where the shaking power is more likely to act, thereby reducing the velocity gradient between the center and periphery of the bottom, allowing shaking with less power and reducing shear stress. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 2 is a perspective view showing the appearance of a culture vessel in the shaking culture apparatus of the first embodiment. [Figure 2] FIG. 10 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a second embodiment. [Figure 3] FIG. 10 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a third embodiment. [Figure 4] FIG. 10 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a fourth embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, the present invention will be described based on preferred embodiments with reference to the drawings.

[0013] FIG. 1 shows the appearance of a culture vessel in a shaking culture apparatus according to the first embodiment. This culture vessel 10 is cylindrical and has a curved surface 14 on its bottom surface 13. The height of the curved surface 14 is higher at the center of the bottom surface 13 and lower at the periphery of the bottom surface 13. The bottom surface 13 of the culture vessel 10 is circular. Furthermore, the culture vessel 10 has a cylindrical side surface 11 on the bottom surface 13. Above the side surface 11, the top surface 12 of the culture vessel 10 may be closed or open.

[0014] When a culture is cultured with shaking using a shaking culture device equipped with the culture vessel 10, in a stationary state, the liquid level S of the liquid L, such as a culture solution, is above the curved surface 14. Gas G is present above the liquid level S. According to the culture vessel 10, the depth D1 of the liquid L is shallow at the center of the bottom surface 13, where the shaking power is less likely to act, and the depth D2 of the liquid L is deep at the peripheral portion of the bottom surface 13, where the shaking power is more likely to act. This reduces the velocity gradient between the center and peripheral portions of the bottom surface 13, allowing shaking with less power and reducing shear stress. The liquid L may be composed mainly of water, for example.

[0015] When the bottom surface 13 is flat without the curved surface 14, the flow rate of the liquid L tends to be low at the center of the bottom surface 13 because the shaking power is less likely to act there. Meanwhile, the flow rate of the liquid L tends to be high at the periphery of the bottom surface 13 because the shaking power is more likely to act there. When the curved surface 14 is provided, the depth D1 of the liquid L is shallower at the center of the bottom surface 13, so a moderate flow rate can be obtained even when shaking is performed with less power. Therefore, the flow rate at the periphery of the bottom surface 13 tends to be lower than in the past, reducing the radial velocity gradient and enabling milder stirring. This promotes mixing of the liquid L, which is the content liquid, and suppresses growth inhibition, even if the culture is widely dispersed from the center to the periphery of the bottom surface 13.

[0016] 2 shows the interior of a culture vessel 10A in a shaking culture apparatus according to the second embodiment. This culture vessel 10A has a cylindrical side surface 11, a circular top surface 12, and a bottom surface 13 with a circular periphery. A curved surface 14 is formed integrally with the bottom surface 13. As with the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the center of the bottom surface 13 of the culture vessel 10A is shallow, and the depth D2 of the liquid L at the periphery of the bottom surface 13 is deep. This reduces the velocity gradient between the center and periphery of the bottom surface 13, allowing for shaking with less power and reducing shear stress.

[0017] FIG. 3 shows the interior of a culture vessel 10B in a shaking culture apparatus according to a third embodiment. This culture vessel 10B includes an outer shell 15 having a curved surface 14 and a flexible inner bag 16 housed in the outer shell 15. The outer shell 15 has a cylindrical side surface 11, a circular top surface 12, a bottom surface 13 with a circular outer periphery, and the curved surface 14. As with the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the center of the bottom surface 13 of the culture vessel 10B is shallow, while the depth D2 of the liquid L at the periphery of the bottom surface 13 is deep. This reduces the velocity gradient between the center and periphery of the bottom surface 13, enabling shaking with less power and reducing shear stress.

[0018] FIG. 4 shows the interior of a culture vessel 10C in a shaking culture apparatus according to a fourth embodiment. This culture vessel 10C includes a flexible inner bag 16, a curved member 17 having a curved surface 14, and an outer shell 18 that houses the inner bag 16. The outer shell 18 has a cylindrical side surface 11, a circular top surface 12, and a circular bottom surface 13. The curved member 17 is installed between the inner bag 16 and the bottom surface 13 of the outer shell 18. Because the outer shell 18 does not have a curved surface 14 on the bottom surface 13, the bottom surface 13 may be flat. As with the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the center of the bottom surface 13 of the culture vessel 10C is shallow, while the depth D2 of the liquid L at the periphery of the bottom surface 13 is deep. This reduces the velocity gradient between the center and periphery of the bottom surface 13, enabling shaking with less power and reducing shear stress.

[0019] Next, the culture vessels 10, 10A, 10B, and 10C will be described in more detail.

[0020] The outer shell 15, 18 of the culture vessel 10, 10A or 10B may have a side surface 11, top surface 12, and bottom surface 13 that are integrally formed, or may have a seam along the side surface 11, between the side surface 11 and top surface 12, or between the side surface 11 and bottom surface 13. The portions on both sides of the seam may be joined to each other. The portions on both sides of the seam may be separable. For example, the side surface 11 and bottom surface 13 may be an integral vessel body, and the top surface 12 may be a lid that can be opened and closed relative to the vessel body.

[0021] When the liquid L is contained in the inner bag 16 in a sealed state, the top surface 12 may be omitted, or a portion of the inner bag 16 may protrude above the upper end of the side surface 11. The inner bag 16 may be overlapped on the inside of the curved surface 14 and deformed to conform to the cross-sectional shape of the curved surface 14. It is preferable that the liquid L contained in the inner bag 16 does not come into contact with the outer shells 15, 18. The shape of the inner bag 16 is not particularly limited, but examples include a polygonal prism such as a square prism, a hexagonal prism, or an octagonal prism, or a cylindrical shape. A gusseted bag is preferable because it can be easily folded outside the outer shells 15, 18. A multi-layered packaging bag such as a double-layered bag or a triple-layered bag is preferable because it makes it less likely for the liquid L to leak from the inner bag 16.

[0022] In addition to the above-described culture vessels 10, 10A, 10B, and 10C, the shaking culture apparatus may also include a shaking device (not shown) that shakes the culture vessels 10, 10A, 10B, and 10C. The shaking device preferably performs shaking while performing an orbital motion in which the center of the bottom surface 13 rotates and moves horizontally on a circular orbit. This causes the liquid surface S to rise on both sides during stirring, with the center portion becoming concave. In the peripheral portion near the side surface 11 of the bottom surface 13, the height of the liquid surface S oscillates up and down.

[0023] The material of the culture vessels 10, 10A or the outer shells 15, 18 of the culture vessels 10B, 10C is not particularly limited, but preferably has sufficient hardness (rigidity), and examples thereof include metals such as stainless steel, resins, wood, laminated lumber, and composite materials such as fiber-reinforced plastics. The material of the film constituting the inner bag 16 is not particularly limited, but examples thereof include thermoplastic resins such as polystyrene, polyamide, polyester, and polyolefin, and laminates containing at least one of these resins. The inner bag 16 may be single-use (disposable).

[0024] The inner surfaces of the side surface 11, bottom surface 13, or curved surface 14 may be smooth. At least a portion of the side surface 11, bottom surface 13, or curved surface 14 may be provided with curved convex protrusions as described in Patent Document 1. The shape, number, arrangement, etc. of the curved convex protrusions are not particularly limited. The side surface 11 may be provided with horizontally extending protruding baffles as described in Patent Document 2. The shape, number, arrangement, etc. of the protruding baffles are not particularly limited.

[0025] The inner bag 16 and the curved member 17 may be provided in a state where they are installed in a predetermined position in the outer shells 15, 18, or may be provided as an accessory part of the outer shells 15, 18, or may be provided separately from the outer shells 15, 18. The culture vessels 10, 10A that can be used without the inner bag 16 may be used as the outer shells 15, 18 and combined with the inner bag 16.

[0026] The volume of the culture vessels 10, 10A, 10B, 10C is preferably 1 L or more. Preferably, the volume is 5 L or more, 10 L or more, 20 L or more, 50 L or more, 100 L or more, or 200 L or more. The volume may be 1000 L, 3000 L, 5000 L, etc. Larger volumes are more suitable for large-volume stirring. When liquid L is contained in the inner bag 16, the capacity of the inner bag 16 is preferably compatible with the volume of the culture vessels 10B, 10C. The contents of the inner bag 16 contained in the outer shells 15, 18 preferably include the liquid L and gas G.

[0027] The inner bag 16 may be provided with one or more tubes, hoses, outlets, injection ports, cocks, caps, spouts, etc. for the purpose of adding or removing contents such as culture media or atmospheric gases, controlling the pH of the contents, adding additives, etc. The tubes may also be provided with filters, flow monitors, flow meters, valves, pumps, etc. It is preferable that the inner bag 16 and its accessories are sterilized before use as necessary for purposes such as culture or hygiene. Sterilization means can be selected appropriately depending on the purpose of use, etc. Specific examples include radiation such as gamma rays, gas such as ethylene oxide, heating with water vapor, etc.

[0028] The above-described culture vessels 10, 10A, 10B, and 10C can be used as shaking culture vessels for batch culture, fed-batch culture, and perfusion culture. The cultured microorganisms, cells, and other cultured products in the culture process include, but are not limited to, fungi, bacteria, viruses, yeast, algae, insect cells, plant cells, animal cells, CHO (Chinese Hamster Ovary) cells for biopharmaceutical production, HeLa cells, COS cells, iPS cells for regenerative medicine, stem cells such as mesenchymal stem cells, and animal cells such as differentiated tissue cells. Furthermore, the culture process is not limited to obtaining cultured microorganisms, cells, and other cultured products; it is also possible to recover products such as enzymes, antibodies, and chemical substances produced by the culture from the culture medium.

[0029] The temperature of the contents, such as Liquid L, can be adjusted using a temperature adjustment function such as a heater. The temperature during cultivation depends on the type of culture, but for example, in the case of animal cells, 4 to 40°C is preferred, and 25 to 37°C is particularly preferred. When the device is used as an agitator for culture media without culturing the culture, it may be cooled. Examples of cooling temperatures include 4 to 20°C. Temperature management can be performed by feedback control using the measured value of a thermometer. The thermometer may be a contact thermometer, but a non-contact thermometer such as a stationary or attached type is preferred. The viscosity (coefficient of viscosity) of Liquid L may be similar to that of water (approximately 1 mPa·s) or may be higher than that of water.

[0030] The shape of the curved surface 14 may be rotationally symmetric around the center of the bottom surface 13. Examples of rotationally symmetric shapes that can form the curved surface 14 include curved surfaces such as a sphere, an ellipsoid of revolution, a paraboloid of revolution, and a hyperboloid of revolution. As shown in Figures 2 to 4, the shape of the curved surface 14 in a cross section including the central axis of the bottom surface 13 may be a curved shape such as an arc, an elliptical arc, a parabola, or a hyperbola. The bottom surface 13 may have a shape in which the slope is greater at the periphery and smaller at the center from the periphery to the center. The entire bottom surface 13 may be the curved surface 14. The shape of the portion of the bottom surface 13 other than the curved surface 14 is not particularly limited, and may be, for example, a horizontal surface, an inclined surface, or the like.

[0031] The curved surface 14 is not limited to a smooth curved surface without ridgelines, and may have a shape in which multiple surfaces are connected by radial or concentric ridgelines, for example. A ridgeline is a line that forms a boundary between multiple different surfaces. The ridgelines included in the curved surface 14 may be straight or curved. The change in height of the curved surface 14 from the periphery toward the center of the bottom surface 13 may be continuous or stepped. In a cross section including the central axis of the bottom surface 13, the curved surface 14 may be curved in a stepped manner. An example of a stepped curved surface 14 is a stacked shape in which the diameter of each step decreases as the height from the bottom surface 13 increases.

[0032] The shape of the stepped curved surface 14 may be rotationally symmetric around the center of the bottom surface 13. The steps constituting the stepped curved surface 14 may be formed concentrically. In the stepped curved surface 14, the surfaces between the concentric ridges may be selected from horizontal, inclined, vertical, conical, cylindrical, and other surfaces. The stepped curved surface 14 may be integrated with the bottom surface 13 or may be composed of a curved member 17 including multiple steps. The stepped curved member 17 may also be composed of step members divided into individual steps. The step members constituting each step of the stepped curved surface 14 may have, for example, a cylindrical or truncated conical shape with different diameters. The upper or lower surface of the step member is not limited to the curved surface 14, but may also be flat.

[0033] The curved member 17 may be composed of multiple members in which the curved surface 14 is divided radially, concentrically, etc. Curved members 17 with curved surfaces 14 of different shapes may be installable on the outer shells 15, 18. A curved member 17 may be installable on the bottom surface 13 having the curved surface 14. Another curved member 17 may be installable on a curved member 17 having the curved surface 14. By attaching, detaching, or replacing the curved member 17, an appropriate curved surface 14 may be used depending on the conditions.

[0034] The height of the curved surface 14 can be appropriately designed so that the ratio between the depth D1 of the liquid L at the center of the bottom surface 13 and the depth D2 of the liquid L at the peripheral portion of the bottom surface 13 is appropriate. The ratio between the depth D1 at the center and the depth D2 at the peripheral portion is not particularly limited, but the value of the depth ratio D1 / D2 can be, for example, 0.01 to 0.99. Specific examples of the value of the depth ratio D1 / D2 include 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.99, and intermediate values ​​thereof.

[0035] As shown in FIG. 1, when the diameter of curved surface 14 is A and the diameter of bottom surface 13 is B, diameter A of curved surface 14 may be approximately the same as diameter B of bottom surface 13. Diameter A of curved surface 14 may be smaller than diameter B of bottom surface 13. When the planar shape of bottom surface 13 or curved surface 14 is not circular, the maximum diameter such as the major axis or diagonal length can be used as the diameter. The diameter ratio A / B may be, for example, 0.1 to 1.0. Specific examples of the diameter ratio A / B include 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 0.95, 0.99, 1.0, and intermediate values ​​thereof.

[0036] Although the present invention has been described above based on preferred embodiments, the present invention is not limited to the above-described embodiments and various modifications are possible without departing from the spirit of the present invention. Modifications include addition, substitution, omission, and other changes to components in each embodiment. Furthermore, components used in two or more embodiments can be combined as appropriate.

[0037] When a curved member 17 having a curved surface 14 is used in combination with an outer shell 18 having no curved surface on the bottom surface 13, it is also possible to omit the inner bag 16 and use the outer shell 18 to which the curved member 17 is fixed as a culture vessel. If there is no problem, for example, because the curved member 17 is securely fixed to the bottom surface 13 of the outer shell 18, the liquid L can be placed directly inside the outer shell 18 to which the curved member 17 is fixed, without using the inner bag 16. [Explanation of symbols]

[0038] A...diameter of curved surface, B...diameter of bottom surface, D1...depth of culture solution at the center of the bottom surface, D2...depth of culture solution at the periphery of the bottom surface, G...gas, L...liquid, S...liquid level, 10, 10A, 10B, 10C...culture vessel, 11...side, 12...top surface, 13...bottom surface, 14...curved surface, 15...outer shell with curved surface, 16...inner bag, 17...curved member, 18...outer shell without curved surface.

Claims

1. The culture vessel has a curved surface on the bottom surface, and the curved surface is formed so that the height of the curved surface is high at the center of the bottom surface and low at the peripheral portion of the bottom surface; The culture vessel comprises a flexible inner bag and a metal outer shell that houses the inner bag; The outer shell has a curved surface on the bottom surface, The shaking culture device is characterized in that the inner bag is overlapped on the inside of the curved surface of the bottom surface of the outer shell and deformed to form a curved surface.

2. 2. The shaking culture apparatus according to claim 1, wherein the bottom surface is circular, and the culture vessel has a cylindrical side surface on the bottom surface.

3. 3. The shaking culture apparatus according to claim 1, wherein the curved surface has a shape that is rotationally symmetrical around the center of the bottom surface.

4. 4. The shaking culture apparatus according to claim 1, wherein the inner bag is housed in the outer shell.

5. A shaking culture method, comprising: culturing a culture by shaking using the shaking culture apparatus according to any one of claims 1 to 4.

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