JP7593753B2 - Shaking culture device and shaking culture method - Google Patents

Description

The present invention relates to a shaking culture device and a shaking culture method.

2. Description of the Related Art 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 a housing part that contains a flexible culture bag and abuts the culture bag.
Patent Document 2 describes a culture vessel having a protruding baffle extending horizontally inside a cylindrical stirring vessel for containing a liquid content.
Patent Document 3 describes a culture vessel in which the outer shape of the inner bottom surface is circular and a flow path is provided as a concave annular passage arranged concentrically with the bottom surface, which can determine the direction of the flow of the culture medium during shaking culture.

JP 2017-35009 A JP 2019-198850 A Japanese Patent Application Publication No. 9-65876

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-down flow is generated, 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 with mild stirring.
Patent Document 3 describes that animal cells suspended in a culture solution move smoothly in a predetermined flow direction along with the flow of the culture solution, so collisions between animal cells are significantly reduced and damage to the animal cells can be suppressed.

When culturing large amounts of cells, such as stem cells used in regenerative medicine, cells are subjected to shear stress due to stirring or shaking, which inhibits their growth. Simply slowing down the flow rate in order to reduce shear stress results in insufficient mixing of the contents, which inhibits growth.

The present invention was made in consideration of the above circumstances, and aims to provide a shaking culture device and shaking culture method that can reduce shear stress acting on cultures and suppress growth inhibition.

To solve the above problem, the present invention provides a shaking culture device having a curved surface on the bottom surface of a culture vessel, the curved surface being formed so that the height of the curved surface is high at the center of the bottom surface and low at the periphery of the bottom surface.

The bottom surface may be circular and the culture vessel may have a cylindrical side surface above the bottom surface.
The curved surface may be rotationally symmetric about a centre of the base 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 a bottom surface of the outer shell and the inner bag.

The present invention also provides a shaking culture method, which is characterized by performing shaking culture of a culture using the shaking culture device.

According to the present invention, the height of the curved surface formed on the bottom surface of the culture vessel is high at the center of the bottom surface and low at the periphery of the bottom surface, so that the depth of the culture medium is shallow at the center of the bottom surface where the shaking force is less effective, and deep at the periphery of the bottom surface where the shaking force is more effective. This reduces the velocity gradient between the center and periphery of the bottom surface, allowing shaking with less force and reducing shear stress.

FIG. 2 is a perspective view showing the appearance of a culture vessel in the shaking culture apparatus of the first embodiment. FIG. 11 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a second embodiment. FIG. 11 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a third embodiment. FIG. 13 is a cross-sectional view showing the inside of a culture vessel in a shaking culture apparatus according to a fourth embodiment.

The present invention will be described below based on a preferred embodiment with reference to the drawings.

Figure 1 shows the appearance of a culture vessel in a shaking culture device of the first embodiment. This culture vessel 10 is cylindrical and has a curved surface 14 on the bottom surface 13. The height of the curved surface 14 is high at the center of the bottom surface 13 and low 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 may be open.

When shaking culture is performed 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 medium, 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 force is less likely to act, and the depth D2 of the liquid L is deep at the periphery of the bottom surface 13, where the shaking force is more likely to act. This reduces the velocity gradient between the center and periphery of the bottom surface 13, and shaking can be performed with less force, reducing shear stress. The liquid L may be mainly composed of water, for example.

When the bottom surface 13 is flat without the curved surface 14, the flow rate of the liquid L tends to be small in the center of the bottom surface 13 because the shaking power is less likely to act there. In addition, the flow rate of the liquid L tends to be large in the peripheral part 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 shallow in the center of the bottom surface 13, so a moderate flow rate can be obtained even if shaking is performed with a smaller power. Therefore, the flow rate in the peripheral part of the bottom surface 13 tends to be lower than in the conventional case, the radial velocity gradient is reduced, and milder stirring is possible. As a result, even if the distribution of the culture is widely dispersed from the center to the peripheral part of the bottom surface 13, the mixing of the liquid L, which is the content liquid, can be promoted and growth inhibition can be suppressed.

Figure 2 shows the inside of a culture vessel 10A in a shaking culture device of 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 shaking with less power and reducing shear stress.

Figure 3 shows the inside of a culture vessel 10B in a shaking culture device of the third embodiment. This culture vessel 10B has an outer shell 15 with 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 upper 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, 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 shaking with less power and reducing shear stress.

Figure 4 shows the inside of the culture vessel 10C in the shaking culture device of the 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 upper surface 12, and a circular bottom surface 13. The curved member 17 is installed between the bottom surface 13 of the outer shell 18 and the inner bag 16. Since 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, and the depth D2 of the liquid L at the peripheral portion of the bottom surface 13 is deep. This reduces the velocity gradient between the center and peripheral portions of the bottom surface 13, and allows shaking with less power, thereby reducing shear stress.

Next, we will explain the culture vessels 10, 10A, 10B, and 10C in more detail.

The outer shell 15, 18 of the culture vessel 10, 10A or culture vessel 10B may be constructed such that the side surface 11, the top surface 12, and the bottom surface 13 are integrally formed, or may have a seam in the middle of the side surface 11, between the side surface 11 and the top surface 12, between the side surface 11 and the bottom surface 13, etc. The parts on both sides of the seam may be connected to each other. The parts on both sides of the seam may be separable. For example, the side surface 11 and the 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.

When the liquid L is contained in the inner bag 16 in a sealed state, the upper surface 12 may be omitted, or a part of the inner bag 16 may protrude upward from 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 along 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 polygonal columns such as square columns, hexagonal columns, and octagonal columns, or cylindrical shapes. If the inner bag is a gusset bag, it is preferable because it is easy to fold it outside the outer shells 15, 18. If the inner bag 16 is a multi-layered packaging bag such as a double bag or a triple bag, it is preferable because the liquid L is less likely to leak from the inner bag 16.

In addition to the above-mentioned culture vessels 10, 10A, 10B, and 10C, the shaking culture apparatus can be equipped with a shaking device (not shown) that shakes the culture vessels 10, 10A, 10B, and 10C. It is preferable for the shaking device to shake while performing an orbital motion that rotates and moves the center of the bottom surface 13 horizontally on a circular orbit. This causes the liquid surface S to rise to the left and right during stirring, with the center portion becoming concave. In the peripheral portion close to the side surface 11 of the bottom surface 13, the height of the liquid surface S vibrates up and down.

The material of the culture vessels 10, 10A or the material of the outer shells 15, 18 of the culture vessels 10B, 10C is not particularly limited, but preferably has sufficient hardness (rigidity), and examples of such materials include metals such as stainless steel, resins, wood, laminated lumber, and composite materials such as fiber-reinforced plastics. The material of the film that constitutes the inner bag 16 is not particularly limited, but examples of such materials 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).

The inner surface of the side surface 11, bottom surface 13 or curved surface 14 may be a smooth surface. At least a portion of the side surface 11, bottom surface 13 or curved surface 14 may be provided with a curved convex protrusion as described in Patent Document 1. The shape, number, arrangement, etc. of the curved convex protrusion are not particularly limited. The side surface 11 may be provided with a protruding baffle extending in the horizontal direction as described in Patent Document 2. The shape, number, arrangement, etc. of the protruding baffle are not particularly limited.

The inner bag 16 and the curved member 17 may be provided in a state where they are installed at a predetermined position in the outer shells 15, 18, or may be provided as accessories to 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 the inner bag 16 may be combined.

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, 200 L or more. The volume may be 1000 L, 3000 L, 5000 L, etc. A larger volume is 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.

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 medium or atmospheric gas, controlling the pH of the contents, adding additives, etc. The tube may be provided with a filter, flow monitor, flow meter, valve, pump, 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 appropriately selected depending on the purpose of use, etc., and specific examples include radiation such as gamma rays, gas such as ethylene oxide, heating with water vapor, etc.

The above-mentioned culture vessels 10, 10A, 10B, and 10C can be used as shaking culture vessels for batch culture, fed-batch culture, and perfusion culture. The cultures of microorganisms, cells, etc. cultured in the culture process are not particularly limited, but include 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 including mesenchymal stem cells, and animal cells such as differentiated tissue cells. In addition, in the culture process, it is not limited to obtaining cultures of microorganisms, cells, etc. as the target product, but it is also possible to recover products such as enzymes, antibodies, and chemical substances produced by the culture from the culture solution.

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 preferable, and 25 to 37°C is particularly preferable. When the culture is not cultivated and the device is used as an agitator for medium, etc., cooling may be performed. 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 preferable. The viscosity (coefficient of viscosity) of liquid L may be the same as that of water (about 1 mPa·s) or may be higher than that of water.

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 may constitute 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, examples of the shape of the curved surface 14 in a cross section including the central axis of the bottom surface 13 include curved shapes such as a circular arc, an elliptical arc, a parabola, and a hyperbola. The shape may be such that the inclination is greater in the periphery and smaller in the center from the periphery to the center of the bottom surface 13. 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, etc.

The curved surface 14 is not limited to a smooth curved surface without ridgelines, and may be, for example, a shape in which multiple surfaces are connected by radial or concentric ridgelines. 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 stepwise. 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 becomes smaller as the height from the bottom surface 13 increases.

The shape of the stepped curved surface 14 may be rotationally symmetric around the center of the bottom surface 13. Each step constituting the stepped curved surface 14 may be formed concentrically. In the stepped curved surface 14, the surface constituting the concentric ridges may be selected from a horizontal surface, an inclined surface, a vertical surface, a conical surface, a cylindrical surface, and the like. 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 steps. The step members forming 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, and may be a flat surface.

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 different shapes of curved surfaces 14 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 a curved surface 14. By attaching, detaching, or replacing the curved member 17, an appropriate curved surface 14 may be made available depending on the conditions.

The height of the curved surface 14 can be appropriately designed so that the ratio of the depth D1 of the liquid L at the center of the bottom surface 13 to the depth D2 of the liquid L at the periphery of the bottom surface 13 is appropriate. The ratio of the depth D1 at the center to the depth D2 at the periphery 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, or intermediate values thereof.

As shown in FIG. 1, when the diameter of the curved surface 14 is A and the diameter of the bottom surface 13 is B, the diameter A of the curved surface 14 may be approximately the same as the diameter B of the bottom surface 13. The diameter A of the curved surface 14 may be smaller than the diameter B of the bottom surface 13. When the planar shape of the bottom surface 13 or the curved surface 14 is not circular, the maximum diameter such as the major axis or diagonal length can be set as the diameter. The value of the diameter ratio A/B is, for example, 0.1 to 1.0. Specific examples of the value 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, or intermediate values thereof.

The present invention has been described above based on a preferred embodiment, but the present invention is not limited to the above-mentioned embodiment, and various modifications are possible without departing from the gist of the present invention. Modifications include addition, substitution, omission, and other changes to components in each embodiment. In addition, components used in two or more embodiments can be appropriately combined.

When using a combination of a curved member 17 having a curved surface 14 and an outer shell 18 that does not have a 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 in that the curved member 17 is securely fixed to the bottom surface 13 of the outer shell 18, the liquid L can be directly contained inside the outer shell 18 to which the curved member 17 is fixed, without going through the inner bag 16.

A...diameter of curved surface, B...diameter of bottom surface, D1...depth of culture solution at center of bottom surface, D2...depth of culture solution at periphery of bottom surface, G...gas, L...liquid, S...liquid level, 10, 10A, 10B, 10C...culture vessel, 11...side surface, 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 (5)

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 high at the center of the bottom surface and low at the peripheral portion of the bottom surface ;
A shaking culture device characterized in that the culture vessel comprises a flexible inner bag, a curved member having the curved surface, and a metal outer shell that houses the inner bag, and the curved member is installed between the bottom surface of the outer shell and the inner bag . The shaking culture device according to claim 1, characterized in that the bottom surface is circular and the culture vessel has a cylindrical side surface on the bottom surface. The shaking culture device according to claim 1 or 2, characterized in that the curved surface has a rotationally symmetric shape around the center of the bottom surface. 4. The shaking culture apparatus according to claim 1, wherein the inner bag is housed in the outer shell. A shaking culture method, comprising: carrying out shaking culture of a culture using the shaking culture apparatus according to any one of claims 1 to 4 .

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