Shaking culture apparatus and shaking culture method

Abstract

The present invention provides a shaking culture apparatus and a shaking culture method that can reduce shear stress acting on cultured materials and suppress growth inhibition. [Solution] The culture vessel 10 has a curved surface 14 on its bottom surface 13, and the curved surface 14 is formed such that its height is higher in the center of the bottom surface 13 and lower in the peripheral part of the bottom surface 13.

Description

Technical Field

[0001] The present invention relates to a shaking culture device and a shaking culture method.

Background Art

[0002] Various substances useful as pharmaceuticals, foods, cosmetics, and raw materials thereof 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 composed of a curved convex protrusion is provided on a part of a contact surface that contacts a culture bag on the inner surface of a housing part that houses a flexible culture bag. Patent Document 2 describes a culture vessel having a protruding baffle provided horizontally inside a cylindrical stirring vessel containing a liquid content. Patent Document 3 describes a culture vessel in which the outer shape of the inner bottom surface of the culture vessel is circular, and a flow path is provided as a concave annular path provided concentrically with the bottom surface, which can define the direction of the flow of the culture solution during shaking culture.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] Patent Document 1 describes that when a part of the culture solution collides with the baffle, the direction of the swirling flow is reversed to generate an up-and-down flow, and the culture solution can be efficiently mixed so as to be uniform. Patent Document 2 states that, in order to suppress the shear acting on the contents, it is possible to mix them uniformly with high stirring efficiency even with mild stirring. Patent Document 3 states that animal cells suspended in a culture medium move smoothly in a predetermined flow direction along the flow of the culture medium, thereby significantly reducing collisions between animal cells and suppressing damage to the animal cells.

[0005] When culturing large quantities of cells, such as stem cells used in regenerative medicine, stirring or shaking can cause shear stress, inhibiting cell growth. Simply reducing the flow rate to mitigate shear stress will result in insufficient mixing of the contents, thus inhibiting cell growth.

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

[0007] To solve the aforementioned problems, the present invention provides a shaking culture apparatus characterized in that the bottom surface of the culture vessel has a curved surface, and the height of the curved surface is higher in the center of the bottom surface and lower in the peripheral part of the bottom surface.

[0008] The bottom surface may be circular, and the culture vessel may have cylindrical sides 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 comprise an outer shell having the curved surface and a flexible inner bag housed within the outer shell. The culture container comprises a flexible inner bag, a curved member having a curved surface, and an outer shell that houses the inner bag, wherein the curved member may be placed between the bottom surface of the outer shell and the inner bag.

[0009] Furthermore, the present invention provides a shaking culture method characterized by performing shaking culture of a culture using the aforementioned 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 in the center of the bottom and lower in the periphery. As a result, the depth of the culture medium is shallower in the center of the bottom where the shaking power is less effective, and deeper in the periphery where the shaking power is more effective. This reduces the velocity gradient between the center and periphery of the bottom, allowing for shaking with less power and thus reducing shear stress. [Brief explanation of the drawing]

[0011] [Figure 1] This is a perspective view showing the external appearance of the culture vessel in the shaking culture apparatus of the first embodiment. [Figure 2] This is a cross-sectional view showing the inside of the culture vessel in the shaking culture apparatus of the second embodiment. [Figure 3] This is a cross-sectional view showing the inside of the culture vessel in the shaking culture apparatus of the third embodiment. [Figure 4] This is a cross-sectional view showing the inside of the culture vessel in the shaking culture apparatus of the fourth embodiment. [Modes for carrying out the invention]

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

[0013] Figure 1 shows the external appearance of the culture vessel in the shaking culture apparatus of 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 in the center of the bottom surface 13 and lower in 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. The top surface 12 of the culture vessel 10 may be closed or open at the top of the side surface 11.

[0014] When performing shaking culture of a culture using a shaking culture apparatus equipped with a culture vessel 10, in a stationary state, the liquid surface S of the liquid L such as a culture solution is above the curved surface 14. A gas G exists on the liquid surface S. According to the culture vessel 10, the depth D1 of the liquid L at the central portion of the bottom surface 13 where the shaking power is difficult to act is shallow, and the depth D2 of the liquid L at the peripheral portion of the bottom surface 13 where the shaking power is easy to act is deep. Thereby, the velocity gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and shaking can be performed with a smaller power to reduce the shear stress. The liquid L may be mainly composed of, for example, water.

[0015] When the bottom surface 13 is flat without providing the curved surface 14, at the central portion of the bottom surface 13, since the shaking power is difficult to act, the flow velocity of the liquid L tends to be small. Also, at the peripheral portion of the bottom surface 13, since the shaking power is easy to act, the flow velocity of the liquid L tends to be large. When the curved surface 14 is provided, at the central portion of the bottom surface 13, since the depth D1 of the liquid L is shallow, an appropriate flow velocity can be obtained even when shaking is performed with a smaller power. Therefore, the velocity at the peripheral portion of the bottom surface 13 tends to be lower in flow velocity than in the conventional case, the velocity gradient in the radial direction decreases, and milder stirring becomes possible. Thereby, even when the distribution of the culture is widely dispersed from the central portion to the peripheral portion of the bottom surface 13, mixing of the liquid L which is the content liquid can be promoted to suppress growth inhibition.

[0016] Fig. 2 shows the inside of the culture vessel 10A in the shaking culture apparatus of the second embodiment. This culture vessel 10A has a cylindrical side surface 11, a circular upper surface 12, and a bottom surface 13 with a circular outer periphery. A curved surface \alpha is formed integrally with the bottom surface 13. Similar to the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the central portion of the bottom surface 13 of the culture vessel 10A is shallow, and the depth D2 of the liquid L at the peripheral portion of the bottom surface 13 is deep. Thereby, the velocity gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and shaking can be performed with a smaller power to reduce the shear stress.

[0017] Figure 3 shows the inside of the culture vessel 10B in the shaking culture apparatus of the 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 upper surface 12, a bottom surface 13 with a circular outer periphery, and a curved surface 14. Similar to the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the central portion of the bottom surface 13 of the culture vessel 10B is shallow, and the depth D2 of the liquid L at the peripheral portion of the bottom surface 13 is deep. Thereby, the velocity gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shaking can be performed with a smaller power to reduce the shear stress.

[0018] Figure 4 shows the inside of the culture vessel 10C in the shaking culture apparatus 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 the curved surface 14 on the bottom surface 13, the bottom surface 13 may be flat. Similar to the culture vessel 10 of the first embodiment, the depth D1 of the liquid L at the central portion 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. Thereby, the velocity gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shaking can be performed with a smaller power to reduce the shear stress.

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

[0020] The outer shells 15 and 18 of the culture vessel 10, 10A or the culture vessel 10B may be integrally formed with the side surface 11, the upper surface 12, and the bottom surface 13, or may have joints in the middle of the side surface 11, between the side surface ǁ and the upper surface 12, between the side surface 11 and the bottom surface 13, etc. Each part on both sides of the joint may be coupled to each other. Each part on both sides of the joint may be separable. For example, the side surface 11 and the bottom surface 13 may be an integral container body, and the upper surface 12 may be a lid portion that can be opened and closed with respect to the container 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 part of the inner bag 16 may protrude above the upper end of the side surface 11. The inner bag 16 may be superimposed 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 and 18. The shape of the inner bag 16 is not particularly limited, but examples include polygonal prisms such as rectangular prisms, hexagonal prisms, octagonal prisms, or cylindrical shapes. It is preferable that the inner bag be a gusset bag because it is easy to fold outside the outer shells 15 and 18. It is preferable that the inner bag 16 be a multi-layered packaging bag such as a double bag or a triple bag because it is less likely for the liquid L to leak from the inner bag 16.

[0022] The shaking culture apparatus may include, in addition to the culture vessels 10, 10A, 10B, and 10C described above, a shaking device (not shown) for shaking the culture vessels 10, 10A, 10B, and 10C. It is preferable that the shaking device shakes the vessels while causing the center of the bottom surface 13 to rotate horizontally on a circular orbit. As a result, the liquid surface S rises from side to side during stirring, and the central part becomes concave. In the peripheral area near the side surface 11 of the bottom surface 13, the height of the liquid surface S vibrates up and down.

[0023] The material of the culture containers 10, 10A or the outer shells 15, 18 of the culture containers 10B, 10C is not particularly limited, but it is preferable that it has sufficient hardness (rigidity). Examples include metals such as stainless steel, resins, wood, laminated wood, fiber-reinforced plastics, and other composite materials. The material of the film constituting the inner bag 16 is not particularly limited, but examples 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. Curved convex projections as described in Patent Document 1 may be provided on at least a portion of the side surface 11, bottom surface 13, or curved surface 14. The shape, number, arrangement, etc., of the curved convex projections are not particularly limited. A horizontally extending baffle as described in Patent Document 2 may be provided on the side surface 11. The shape, number, arrangement, etc., of the baffle is not particularly limited.

[0025] The inner bag 16 and the curved member 17 may be provided already installed in the outer shells 15 and 18, as accessories to the outer shells 15 and 18, or separately from the outer shells 15 and 18. Culture containers 10 and 10A that can be used without the inner bag 16 may be used as the outer shells 15 and 18, and the inner bag 16 may be combined with them.

[0026] The volume of the culture vessels 10, 10A, 10B, and 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 also be 1000 L, 3000 L, 5000 L, etc. A larger volume is more suitable for large-scale stirring. When the liquid L is contained in the inner bag 16, the capacity of the inner bag 16 is preferably matched to the volume of the culture vessels 10B and 10C. The contents of the inner bag 16 contained in the outer shells 15 and 18 preferably include the liquid L and the gas G.

[0027] The inner bag 16 may be equipped with one or more tubes, hoses, spouts, inlets, cocks, caps, or spouts for purposes such as adding or removing contents like culture media or atmospheric gas, controlling the pH of the contents, or adding additives. The tubes may also be equipped with filters, flow monitors, flow meters, valves, pumps, etc. The inner bag 16 and its accessories are preferably sterilized before use, as necessary for cultivation and hygiene purposes. Sterilization methods can be appropriately selected depending on the purpose of use, and specific examples include radiation such as gamma rays, heating with gases such as ethylene oxide or steam.

[0028] The culture vessels 10, 10A, 10B, and 10C described above can be used as shaking-type culture vessels for batch culture, fed-batch culture, and perfusion culture. The cultured products, such as microorganisms and cells, that are cultured in the culture process are not particularly limited, but include fungi, bacteria, viruses, yeasts, 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 differentiated tissue cells. Furthermore, in the culture process, it is not limited to obtaining cultured products such as microorganisms and cells as the target product; 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 of the liquid L can be controlled using a temperature control function such as a heater. The temperature for culturing 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 used as a stirring device for culture media etc. without culturing cultures, cooling may be performed. Examples of cooling temperatures include 4 to 20°C. Temperature control can be performed using feedback control by utilizing the measurement value of a thermometer. The thermometer may be a contact type, but a non-contact thermometer such as a stationary type or a mounted type is preferred. The viscosity (viscosity) of the liquid L may be about the same as water (about 1 mPa·s), or it may be more viscous than water.

[0030] The shape of the curved surface 14 may be rotationally symmetric around the center of the base surface 13. Examples of rotationally symmetric shapes that can constitute the curved surface 14 include spheres, ellipsoids of revolution, parabolas of revolution, and hyperbolas of revolution. As shown in Figures 2 to 4, examples of curved shapes for the curved surface 14 in a cross-section including the central axis of the base surface 13 include circular arcs, elliptical arcs, parabolas, and hyperbolas. The shape may have a steeper incline at the periphery and a shallower incline at the center of the base surface 13. The entire base surface 13 may be the curved surface 14. The shape of the part of the base surface 13 other than the curved surface 14 is not particularly limited, but may be a horizontal surface, an inclined surface, etc.

[0031] The curved surface 14 is not limited to a smooth curved surface without edges, but may also be a shape in which multiple surfaces are connected by radial or concentric edges, for example. An edge is a line that constitutes a boundary line between multiple different surfaces. The edges included in the curved surface 14 may be straight or curved. The change in height of the curved surface 14 from the periphery to the center of the base surface 13 may be continuous or stepped. In a cross-section including the central axis of the base surface 13, the curved surface 14 may be curved in a step-like manner. An example of a step-like curved surface 14 is a shape in which layers are stacked such that the diameter of each step decreases as the height from the base surface 13 increases.

[0032] The shape of the stepped curved surface 14 may be rotationally symmetrical around the center of the base surface 13. Each step constituting the stepped curved surface 14 may be formed concentrically. In the stepped curved surface 14, the surfaces constituting the space between the concentric edges may be selected from horizontal surfaces, inclined surfaces, vertical surfaces, conical surfaces, cylindrical surfaces, etc. The stepped curved surface 14 may be integrated with the base surface 13, or it may be composed of a curved member 17 that includes multiple steps. The stepped curved member 17 can also be composed of stepped members divided into steps. The shape of the stepped members forming each step of the stepped curved surface 14 may be, for example, cylindrical or frustoconical shapes of different diameters. The upper or lower surface of the stepped member is not limited to a curved surface 14, but may be a flat surface.

[0033] The curved member 17 may be composed of multiple members obtained by dividing the curved surface 14 radially, concentrically, or in other ways. Curved members 17 with different shapes of curved surfaces 14 may be installed on the outer shells 15 and 18. The curved member 17 may be installed on the bottom surface 13 having a curved surface 14. Another curved member 17 may be installed 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 used depending on the conditions.

[0034] 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 depth ratio D1 / D2 can be 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 ​​therebetween.

[0035] As shown in Figure 1, when the diameter of the curved surface 14 is A and the diameter of the base surface 13 is B, the diameter A of the curved surface 14 may be approximately the same as the diameter B of the base surface 13. The diameter A of the curved surface 14 may also be smaller than the diameter B of the base surface 13. If the planar shape of the base surface 13 or the curved surface 14 is not circular, the diameter can be the largest diameter, such as the major axis or diagonal length. The value of the diameter ratio A / B can be, 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 ​​therebetween.

[0036] Although the present invention has been described above based on preferred embodiments, the present invention is not limited to the embodiments described above, and various modifications are possible without departing from the spirit of the invention. Modifications include adding, substituting, omitting, or otherwise changing the components in each embodiment. It is also possible to combine components used in two or more embodiments as appropriate.

[0037] When using a curved member 17 having a curved surface 14 in combination with an outer shell 18 that does not have a curved surface on its 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 container. If there are no problems, such as the curved member 17 being 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. [Explanation of symbols]

[0038] A...Diameter of the curved surface, B...Diameter of the bottom surface, D1...Depth of the culture medium at the center of the bottom surface, D2...Depth of the culture medium at the periphery of the bottom surface, G...Gas, L...Liquid, S...Liquid level, 10, 10A, 10B, 10C...Culture container, 11...Side surface, 12...Top surface, 13...Bottom surface, 14...Curved surface, 15...Outer shell with a curved surface, 16...Inner bag, 17...Curved member, 18...Outer shell without a curved surface.

Claims

1. A shaking culture apparatus characterized in that the bottom surface of the culture vessel has a curved surface, and the height of the curved surface is higher in the center of the bottom surface and lower in the peripheral part of the bottom surface.

2. The shaking culture apparatus according to claim 1, characterized in that the bottom surface is circular and the culture vessel has cylindrical sides on the bottom surface.

3. The shaking culture apparatus according to claim 1 or 2, characterized in that the curved surface has a shape that is rotationally symmetrical around the center of the bottom surface.

4. The shaking culture apparatus according to any one of claims 1 to 3, characterized in that the culture vessel comprises an outer shell having the curved surface and a flexible inner bag housed in the outer shell.

5. The shaking culture apparatus according to any one of claims 1 to 3, wherein the culture vessel comprises a flexible inner bag, a curved member having a curved surface, and an 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.

6. A shaking culture method characterized by performing shaking culture of a culture using a shaking culture apparatus according to any one of claims 1 to 5.

Images