JP2022016884A - Shaking culture device and shaking culture method - Google Patents

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

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

By culturing microorganisms, insect cells, plant cells, animal cells, etc., various substances useful as pharmaceuticals, foods, cosmetics, and raw materials thereof are produced.
Patent Document 1 describes a single-use cell in which a baffle made of a curved convex protrusion is provided on a part of an abutting surface that abuts on the inner surface of a housing portion that houses a flexible culture bag. The incubator is described.
Patent Document 2 describes a culture vessel having a protruding baffle extending in the horizontal direction inside a columnar stirring vessel for containing a liquid content.
In Patent Document 3, the outer surface of the inner bottom surface of the culture vessel is circular, and as a concave annular path provided concentrically with respect to the bottom surface, a flow path capable of determining the direction of the flow of the culture solution during shaking culture. The culture vessel provided with is described.

Japanese Unexamined Patent Publication No. 2017-35009 Japanese Unexamined Patent Publication No. 2019-198850 Japanese Unexamined Patent Publication No. 9-65876

According to Patent Document 1, when a part of the culture solution collides with the baffle, the direction of the swirling flow is changed to generate a vertical flow, and the culture solution is efficiently mixed so as to be uniform. There is a statement that it can be done.
Patent Document 2 describes that even if the mixture is mildly stirred, it can be uniformly mixed with high stirring efficiency in order to suppress the share acting on the contents.
According to Patent Document 3, animal cells floating in a culture medium move smoothly in a predetermined flow direction along the flow of the culture medium, so that the collision between animal cells is greatly reduced, and the animal cells are greatly reduced. There is a description that it can prevent the animal from being hurt.

When culturing a large number of cells, such as stem cells used in regenerative medicine, the cells are subjected to shear stress by stirring or shaking, which causes growth inhibition. Simply reducing the flow rate for the purpose of reducing shear stress causes insufficient mixing of the contents and thus inhibits growth.

The present invention has been made in view of the above circumstances, and provides a shaking culture apparatus and a shaking culture method capable of reducing shear stress acting on a culture and suppressing growth inhibition. Make it an issue.

In order to solve the above-mentioned problems, the present invention has a curved surface on the bottom surface of the culture vessel, and the height of the curved surface is high in the central portion of the bottom surface and low in the peripheral portion of the bottom surface. Provided is a shaking culture apparatus characterized in that a surface is formed.

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 rotationally symmetric shape 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 comprises a flexible inner bag, a curved member having the curved surface, and an outer shell for accommodating the inner bag, and the curved member comprises a bottom surface of the outer shell and the inner bag. It may be installed in between.

The present invention also provides a shaking culture method characterized by 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 in the central portion of the bottom surface and low in the peripheral portion of the bottom surface, so that the center of the bottom surface on which the power of shaking is difficult to act is difficult. The depth of the culture solution in the portion is shallow, and the depth of the culture solution in the peripheral portion of the bottom surface where the power of shaking is easily applied becomes deep. As a result, the speed gradient between the central portion and the peripheral portion of the bottom surface can be reduced, and the shear stress can be reduced by shaking with a smaller power.

It is a perspective view which showed the appearance of the culture container in the shaking culture apparatus of 1st Embodiment. It is sectional drawing which showed the inside of the culture container in the shaking culture apparatus of 2nd Embodiment. It is sectional drawing which showed the inside of the culture container in the shaking culture apparatus of 3rd Embodiment. It is sectional drawing which showed the inside of the culture container in the shaking culture apparatus of 4th Embodiment.

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

FIG. 1 shows the appearance of the culture vessel in the shaking culture apparatus of the first embodiment. The culture vessel 10 is columnar and has a curved surface 14 on the bottom surface 13. The height of the curved surface 14 is high at the central portion of the bottom surface 13 and low at the peripheral portion of the bottom surface 13. The bottom surface 13 of the culture vessel 10 is circular. Further, the culture vessel 10 has a cylindrical side surface 11 on the bottom surface 13. At the upper part of the side surface 11, the upper surface 12 of the culture vessel 10 may be closed, or the upper surface 12 may be open.

When shaking culture of a culture using a shaking culture device provided with a culture container 10, the liquid level S of a liquid L such as a culture liquid is above the curved surface 14 in a stationary state. Gas G is present on the liquid surface S. According to the culture vessel 10, the depth D1 of the liquid L in the central portion of the bottom surface 13 on which the power of shaking is hard to act is shallow, and the depth D2 of the liquid L in the peripheral portion of the bottom surface 13 on which the power of shaking is easy to act. Becomes deeper. As a result, the speed gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shear stress can be reduced by shaking with a smaller power. The liquid L may contain, for example, water as a main component.

When the bottom surface 13 is flat without providing the curved surface 14, the flow velocity of the liquid L tends to be small because the shaking power is difficult to act on the central portion of the bottom surface 13. Further, since the power of shaking is likely to act on the peripheral portion of the bottom surface 13, the flow velocity of the liquid L tends to increase. When the curved surface 14 is provided, since the depth D1 of the liquid L is shallow at the center of the bottom surface 13, an appropriate flow velocity can be obtained even if the liquid L is shaken with a smaller power. Therefore, the velocity of the peripheral portion of the bottom surface 13 tends to be lower than that of the conventional one, the velocity gradient in the radial direction is reduced, and milder stirring becomes possible. As a result, even if 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 as the content liquid can be promoted and growth inhibition can be suppressed.

FIG. 2 shows the inside of the culture vessel 10A in the shaking culture apparatus of the second embodiment. The culture vessel 10A has a cylindrical side surface 11, a circular upper surface 12, and a bottom surface 13 having a circular outer circumference. A curved surface 14 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 in the central portion of the bottom surface 13 of the culture vessel 10A is shallow, and the depth D2 of the liquid L in the peripheral portion of the bottom surface 13 is deep. As a result, the speed gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shear stress can be reduced by shaking with a smaller power.

FIG. 3 shows the inside of the culture vessel 10B in the shaking culture apparatus of the third embodiment. The 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 having a circular outer circumference, and a curved surface 14. Similar to the culture vessel 10 of the first embodiment, the depth D1 of the liquid L in the central portion of the bottom surface 13 of the culture vessel 10B is shallow, and the depth D2 of the liquid L in the peripheral portion of the bottom surface 13 is deep. As a result, the speed gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shear stress can be reduced by shaking with a smaller power.

FIG. 4 shows the inside of the culture vessel 10C in the shaking culture apparatus of the fourth embodiment. The culture container 10C includes a flexible inner bag 16, a curved member 17 having a curved surface 14, and an outer shell 18 for accommodating 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. Similar to the culture vessel 10 of the first embodiment, the depth D1 of the liquid L in the central portion of the bottom surface 13 of the culture vessel 10C is shallow, and the depth D2 of the liquid L in the peripheral portion of the bottom surface 13 is deep. As a result, the speed gradient between the central portion and the peripheral portion of the bottom surface 13 can be reduced, and the shear stress can be reduced by shaking with a smaller power.

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

In the outer shells 15 and 18 of the culture container 10, 10A or the culture container 10B, the side surface 11, the upper surface 12 and the bottom surface 13 may be integrally configured, and the side surface is in the middle of the side surface 11 and between the side surface 11 and the upper surface 12. A seam may be provided between the 11 and the bottom surface 13. The portions on both sides of the seam may be joined to each other. Each part on both sides of the seam may be separable. For example, the side surface 11 and the bottom surface 13 may be an integral container body, and the top surface 12 may be a lid portion that can be opened and closed with respect to the container body.

When the liquid L is housed 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 be projected upward from the upper end of the side surface 11. The inner bag 16 may be superposed on the inside of the curved surface 14 and may be 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 touch the outer shells 15 and 18. The shape of the inner bag 16 is not particularly limited, and examples thereof include polygonal columns such as square columnar, hexagonal columnar, and octagonal columnar columns, or columnar columns. It is preferable that the inner bag is a gusset bag because it can be easily folded outside the outer shells 15 and 18. It is preferable that the inner bag 16 is a multiple packaging bag such as a double bag or a triple bag because the liquid L does not easily leak from the inner bag 16.

The shaking culture device can be provided with a shaking device (not shown) for shaking the culture containers 10, 10A, 10B, 10C in addition to the above-mentioned culture containers 10, 10A, 10B, 10C. It is preferable that the shaking device shakes while rotating the center of the bottom surface 13 in a horizontal direction on a circular orbit. As a result, the liquid surface S rises to the left and right during stirring, and the central portion becomes concave. The height of the liquid level S vibrates up and down in the peripheral portion near the side surface 11 of the bottom surface 13.

The material of the culture containers 10 and 10A or the materials of the outer shells 15 and 18 of the culture containers 10B and 10C are not particularly limited, but are preferably having sufficient hardness (rigidity), for example, metal such as stainless steel, resin, and wood. , Glulam, composite materials such as fiber reinforced plastics. The material of the film constituting the inner bag 16 is not particularly limited, and 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).

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

The inner bag 16 and the curved member 17 may be provided in a state of being installed at predetermined positions of the outer shells 15 and 18, or may be provided as accessories of the outer shells 15 and 18, with the outer shells 15 and 18. May be provided separately. Culture containers 10, 10A that can be used without the inner bag 16 may be used as the outer shells 15, 18 and may be combined with the inner bag 16.

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, and 200 L or more. The volume may be 1000 L, 3000 L, 5000 L or the like. The larger volume is suitable for a large amount of stirring. When the liquid L is contained in the inner bag 16, the volume of the inner bag 16 preferably matches the volumes of the culture vessels 10B and 10C. The contents of the inner bag 16 housed in the outer shells 15 and 18 preferably include a liquid L and a gas G.

The inner bag 16 has a tube, a hose, a spout, an inlet, a cock, and a cap for the purpose of adding and taking out contents such as a medium and atmospheric gas, controlling the pH of the contents, and adding additives. , One or more spouts and the like may be provided. The tube may be provided with a filter, a flow monitor, a flow meter, a valve, a pump, or the like. The inner bag 16 and its accessories are preferably sterilized before use, if necessary for purposes such as culture and hygiene. The sterilization means can be appropriately selected according to the purpose of use and the like, and specific examples thereof include heating with radiation such as γ-rays, gas such as ethylene oxide, and steam.

The above-mentioned culture vessels 10, 10A, 10B, and 10C can be applied to any of batch culture (batch culture), fed-batch culture (fed-batch method), and perfusion culture (perfusion method) as a shaking type culture container. can. The culture of microorganisms, cells, etc. to be cultivated in the culturing step is not particularly limited, but is limited to fungi, bacteria, viruses, yeast, algae, insect cells, plant cells, animal cells, and CHO (Chinese Hamster) for producing biopharmaceuticals. Ovari) cells, HeLa cells, COS cells, iPS cells for regenerative medicine, stem cells such as mesenchymal stem cells, animal cells such as differentiated tissue cells, and the like. Further, in the culture step, not only when a culture such as a microorganism or a cell is obtained as a target product, 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 the liquid L can be adjusted by using the temperature adjusting function of the heater or the like. The temperature at the time of culturing depends on the type of the culture, but in the case of animal cells, for example, 4 to 40 ° C. is preferable, and 25 to 37 ° C. is particularly preferable. When the culture is not cultured and is used as a stirring device for a medium or the like, it may be cooled. Examples of the cooling temperature include 4 to 20 ° C. For temperature control, feedback control can be performed by using the measured value of the thermometer. The thermometer may be a contact type thermometer, but a non-contact type thermometer such as a stationary type or a mounting type is preferable. The viscosity (viscosity) of the liquid L may be about 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 a shape that is rotationally symmetric around the central portion of the bottom surface 13. Examples of the rotationally symmetric shape that can form the curved surface 14 include curved surfaces such as a spherical surface, a spheroidal surface, a rotating paraboloid, and a rotating hyperboloid. As shown in FIGS. 2 to 4, examples of the shape of the curved surface 14 in the cross section including the central axis of the bottom surface 13 include curved shapes such as arcs, elliptical arcs, parabolas, and hyperbolas. The shape may be such that the inclination in the peripheral portion is large and the inclination in the central portion is small from the peripheral portion to the central portion of the bottom surface 13. The entire bottom surface 13 may be a curved surface 14. The shape of the portion of the bottom surface 13 other than the curved surface 14 is not particularly limited, but may be, for example, a horizontal surface, an inclined surface, or the like.

The curved surface 14 is not limited to a smooth curved surface having no ridge line, and may have, for example, a shape in which a plurality of surfaces are connected by radial or concentric ridge lines. A ridge is a line that constitutes a boundary between a plurality of different faces. The ridge line included in the curved surface 14 may be a straight line or a curved line. The change in height of the curved surface 14 from the peripheral portion to the central portion of the bottom surface 13 may be continuous or gradual. In the cross section including the central axis of the bottom surface 13, the curved surface 14 may be curved in a stepped manner. Examples of the stepped curved surface 14 include a shape in which the stepped curved surface 14 is laminated so that 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 central portion 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 forming between 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 a plurality of steps. It is also possible to form a stepped curved member 17 by combining the step members divided for each step. The shape of the step member forming each step of the stepped curved surface 14 may be, for example, a columnar shape or a truncated cone shape having different diameters. The upper surface or the 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 a plurality of members in which the curved surface 14 is divided into a radial shape, a concentric shape, or the like. Curved members 17 having different shapes of the curved surface 14 may be installed on the outer shells 15 and 18. The curved member 17 may be installed on the bottom surface 13 having the curved surface 14. Another curved member 17 may be installed on the curved member 17 having the curved surface 14. By attaching / detaching or replacing the curved member 17, an appropriate curved surface 14 according to the conditions may be made usable.

The height of the curved surface 14 can be appropriately designed so that the ratio of the depth D1 of the liquid L in the central portion of the bottom surface 13 and the depth D2 of the liquid L in the peripheral portion of the bottom surface 13 is appropriate. be. The ratio of the depth D1 in the central portion to the depth D2 in the peripheral portion is not particularly limited, and examples of the value of the depth ratio D1 / D2 include 0.01 to 0.99. Specific examples of the value of the depth ratio D1 / D2 are 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 about 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, for example, the maximum diameter such as a major axis or a diagonal length can be used 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 are 0.1, 0.3, 0.5, 0.7, 0.8, 0.9, 0.95, 0.99, 1.0 or These intermediate values are mentioned.

Although the present invention has been described above based on a preferred embodiment, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention. Modifications include addition, replacement, omission, and other changes of components in each embodiment. It is also possible to appropriately combine the components used in the two or more embodiments.

When the curved member 17 having a curved surface 14 and the outer shell 18 having no curved surface on the bottom surface 13 are used in combination, the inner bag 16 is omitted and the outer shell 18 to which the curved member 17 is fixed is used as a culture container. It can also be used. If there is no problem such that the curved member 17 is securely fixed to the bottom surface 13 of the outer shell 18, the liquid L is directly applied to the inside of the outer shell 18 to which the curved member 17 is fixed without going through the inner bag 16. Can be accommodated.

A ... Curved surface diameter, B ... Bottom diameter, D1 ... Culture solution depth in the center of the bottom surface, D2 ... Culture solution depth in the periphery of the bottom surface, G ... Gas, L ... Liquid, S ... Liquid Surfaces, 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 … An outer shell that does not have a curved surface.

Claims (6)

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 in the central portion of the bottom surface and low in the peripheral portion of the bottom surface. Shaking culture device. 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. The shaking culture apparatus according to claim 1 or 2, wherein the curved surface has a rotationally symmetric shape around a central portion of the bottom surface. The shake according to any one of claims 1 to 3, wherein the culture vessel includes an outer shell having the curved surface and a flexible inner bag housed in the outer shell. Incubator. The culture vessel includes a flexible inner bag, a curved member having the curved surface, and an outer shell for accommodating the inner bag, and the curved member comprises a bottom surface of the outer shell and the inner bag. The shaking culture apparatus according to any one of claims 1 to 3, wherein the shaking culture apparatus is installed between the two. A method for shaking culturing a culture using the shaking culturing apparatus according to any one of claims 1 to 5.

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