Cell culturing device and cell culturing method

Abstract

The purpose of the present invention is to provide a cell culturing device and a cell culturing method that enable simultaneous removal of carbon dioxide in a liquid phase and a gas phase, sufficient removal of carbon dioxide in the liquid phase, preferential degassing of carbon dioxide in the gas phase over oxygen to facilitate retention of oxygen in a system, and that have high culture efficiency. The present invention provides a cell culturing device comprising: a culture vessel in which a liquid phase, which is a culture solution containing cells, and a gas phase are encapsulated; and at least one gas separation membrane module disposed in the culture vessel. The gas separation membrane module includes a gas separation membrane capable of separating carbon dioxide, and a housing case to which the gas separation membrane is fixed. The gas separation membrane in the culture vessel is installed such that a part of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase.

Description

Cell culture device and cell culture method

[0001] The present invention relates to a cell culture device and a cell culture method. This application claims priority based on Japanese Patent Application No. 2024-227242 filed in Japan on December 24, 2024, and incorporates its content herein by reference.

[0002] In the initial production steps of biopharmaceuticals and cultured meat using animal cells, a large number of cells are cultured. In cell culture, it is necessary to continuously supply oxygen throughout the culture period and remove carbon dioxide accumulated during culture. For example, Patent Document 1 proposes discharging dissolved carbon dioxide in the culture solution to the outside of the system using a gas separation membrane module. Also known is a method of culturing by degassing carbon dioxide in the gas phase in a sweep manner while rocking a culture bag filled with a culture solution (Patent Document 2).

[0003] Japanese Patent Application Laid-Open No. 2023-073597 Japanese Patent Application Laid-Open No. 2016-063816

[0004] An equilibrium state is formed between carbon dioxide dissolved in the culture solution (liquid phase) and carbon dioxide gas in the gas phase. The culture device and method described in Patent Document 1 mainly aim to remove dissolved carbon dioxide in the liquid phase. On the other hand, the sweep method described in Patent Document 2 mainly aims to remove carbon dioxide gas in the gas phase, and dissolved carbon dioxide in the liquid phase needs to be converted into carbon dioxide gas in the gas phase in order to be removed. Also, in the sweep method, oxygen (O ２ ) and carbon dioxide (CO ２ ) are simultaneously discharged, so the utilization efficiency of oxygen (O ２ ) required during cell culture may decrease, and there is a risk of increased energy costs.

[0005] The main object of the present invention is to provide a cell culture device and a cell culture method that can simultaneously remove carbon dioxide in the liquid phase and the gas phase, sufficiently remove carbon dioxide in the liquid phase, preferentially degas carbon dioxide in the gas phase over oxygen, and easily retain oxygen in the system, with high culture efficiency.

[0006] In view of the above problems, the inventors of the present invention have solved the above problems by adopting a configuration in which a part of the gas separation membrane is placed in the gas phase and a part of it is placed in the liquid phase, and have completed the present invention. That is, the present invention includes the following embodiments: [1] A cell culture apparatus comprising: a culture vessel containing a liquid phase which is a culture medium containing cells and a gas phase; and one or more gas separation membrane modules placed in the culture vessel, wherein the gas separation membrane module has a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed, and the gas separation membrane in the culture vessel is installed such that a part of the gas separation membrane is exposed in the gas phase and the remainder is immersed in the liquid phase. [2] The cell culture apparatus according to [1], wherein the specific gravity of the material of the gas separation membrane is less than the specific gravity of the culture medium. [3] The cell culture apparatus according to [1] or [2], further comprising a rocking means for rocking the culture vessel. [4] The cell culture apparatus according to any one of [1] to [3], further comprising a depressurization means for depressurizing the secondary side of the gas separation membrane. [5] The cell culture apparatus according to any one of [1] to [4], wherein the culture vessel is a culture bag. [6] The cell culture apparatus according to any one of [1] to [5], wherein the gas separation membrane consists of a plurality of hollow fiber membranes. [7] The cell culture apparatus according to [6], wherein the plurality of hollow fiber membranes of the gas separation membrane module have free ends that are not fixed to the housing case by a potting material. [8] The cell culture apparatus according to any one of [1] to [7], further comprising an adjustment member for adjusting the position of the gas separation membrane module. [9] The cell culture apparatus according to [4], further comprising a pH sensor for measuring the pH of the culture medium.

[10] The cell culture apparatus according to [9], further comprising a control device for controlling the amount of dissolved carbon dioxide in the culture medium released from the system by adjusting the output of the decompression means based on the measurement value of the pH sensor.

[11] CO ２ The cell culture apparatus according to [4], further comprising a sensor.

[12] The CO ２

[11] A cell culture apparatus further comprising a control device that controls the amount of dissolved carbon dioxide in the culture medium released from the system by adjusting the output of the depressurization means based on the measurement value of a sensor.

[13] A cell culture method using a cell culture apparatus comprising: a culture vessel containing a liquid phase which is a culture medium containing cells and a gas phase; and one or more gas separation membrane modules disposed in the culture vessel, wherein the gas separation membrane module has a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed, and when culturing the cells, the gas separation membrane module in the culture vessel is in a state in which a part of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase.

[14] A cell culture method according to

[13] , wherein the specific gravity of the material of the gas separation membrane is less than the specific gravity of the culture medium.

[15] A cell culture method according to

[13] or

[14] , wherein the culture vessel is shaken when culturing the cells.

[16] The cell culture method according to any one of

[13] to

[15] , wherein the cell culture apparatus further comprises a depressurization means for reducing the pressure on the secondary side of the gas separation membrane, and when culturing the cells, the pressure on the secondary side of the gas separation membrane is reduced to separate and remove dissolved carbon dioxide in the culture medium and carbon dioxide gas in the gas phase.

[17] The cell culture method according to any one of

[13] to

[16] , wherein the gas separation membrane consists of a plurality of hollow fiber membranes, and the plurality of hollow fiber membranes have free ends that are not fixed to the housing case by a potting material.

[18] The cell culture method according to

[16] , wherein the cell culture apparatus further comprises a pH sensor for measuring the pH of the culture medium, and the output of the depressurization means is adjusted based on the measurement value of the pH sensor to control the amount of dissolved carbon dioxide in the culture medium released from the system.

[19] The cell culture apparatus comprises a CO2 sensor for measuring the dissolved carbon dioxide concentration of the culture medium. ２ The sensor is further equipped with the CO ２ A cell culture method according to

[16] , wherein the output of the depressurization means is adjusted based on the measurement value of the sensor to control the amount of dissolved carbon dioxide in the culture medium released from the system.

[20] The overall mass transfer coefficient (k) of dissolved carbon dioxide in the culture medium. Ｌ aＣＯ２ A cell culture method according to any one of

[13] to

[19] , having a simulation step of simulating optimal degassing conditions for the separation and removal of the dissolved carbon dioxide by using (1).

[0007] According to the present invention, it is possible to simultaneously remove carbon dioxide in the liquid phase and the gas phase, and while sufficiently removing carbon dioxide in the liquid phase, carbon dioxide in the gas phase is preferentially degassed over oxygen, making it easy to retain oxygen in the system, and a cell culture apparatus and a cell culture method with high culture efficiency are provided.

[0008] It is a front view schematically showing an example of a cell culture apparatus according to an embodiment. It is a plan view schematically showing an example of a cell culture apparatus according to an embodiment. It is a partial cross-sectional view schematically showing an example of a connection structure from the housing case of the gas separation membrane module to the gas discharge pipe. It is a partial cross-sectional view schematically showing an example of a connection structure from the housing case of the gas separation membrane module to the gas discharge pipe. It is a plan view schematically showing a state of adjusting the orientation of the separation membrane module in the cell culture apparatus according to the embodiment. It is a side view schematically showing an example of a cell culture apparatus according to the embodiment. It is a side view schematically showing a state where the rocking means of the cell culture apparatus of FIG. 5 is rocking. The overall mass transfer coefficient (k Ｌ a ＣＯ２ ) is a schematic diagram showing an example of an acquisition method. It is a diagram showing the time change of the dissolved carbon dioxide concentration and pH of the simulated solution during culture in Experimental Example 1. ln[DCO ２ ０ - [DCO ２ ｔThis figure shows the change over time. The first graph shows the pH of culture medium A in the culture bags in Example 1 and Comparative Example 1, plotted with culture time on the x-axis and pH on the y-axis. The second graph shows the dissolved carbon dioxide concentration of culture medium A in the culture bags in Example 1 and Comparative Example 1, plotted with culture time on the x-axis and dissolved carbon dioxide concentration on the y-axis. The third graph shows the change in viability from day 3 onwards, plotted with culture time on the x-axis and VCD retention rate on the y-axis. The fourth graph shows the change in viability from day 3 onwards, plotted with culture time on the x-axis and viability retention rate on the y-axis.

[0009] The cell culture method and cell culture apparatus of the present invention will be described below with reference to the drawings, with an example provided. Note that the dimensions and other specifications shown in the following description are examples only, and the present invention is not necessarily limited to them. It can be implemented with appropriate modifications without altering the essence of the invention.

[0010] [Cell Culture Apparatus] The cell culture apparatus according to the embodiment comprises a culture vessel containing a liquid phase which is a culture medium containing cells, and a gas phase, and one or more gas separation membrane modules disposed within the culture vessel. The gas separation membrane module has a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed. The gas separation membrane in the culture vessel is installed such that a part of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase.

[0011] Any culture vessel that can enclose the culture medium (liquid phase) and gas phase while ensuring liquid-tightness and airtightness is acceptable, such as glass or stainless steel culture tanks, or vinyl culture bags. When using a culture tank, the ratio of the surface area of ​​the culture medium to the liquid depth tends to be small. On the other hand, when using a culture bag, the ratio of the surface area of ​​the culture medium to the liquid depth tends to be large, making it easier to obtain a wider liquid surface. Furthermore, in order to uniformly supply oxygen and degass carbon dioxide in cell culture, it is preferable to have at least one of a stirring means for stirring the culture medium and an agitating means for agitating the culture vessel itself. From the viewpoint of suppressing physical damage to cells, it is more desirable to use an agitating means that does not directly contact the cells than a stirring means that directly contacts the cells.

[0012] Figure 1 is a schematic front view of a cell culture apparatus 1 according to one embodiment. Figure 2 is a top view of the cell culture apparatus 1, and Figure 5 is a side view of the cell culture apparatus 1. The cell culture apparatus 1 shown in Figures 1, 2, and 5 is an example of a cell culture apparatus that employs a culture bag as a culture container and is equipped with a rocking mechanism. For convenience in the following description, the left-right direction when viewing the cell culture apparatus 1 from the front will be the X direction, the direction parallel to the horizontal direction and perpendicular to the X direction (direction from the front to the back) will be the Y direction, and the height direction will be the Z direction.

[0013] The cell culture apparatus 1 comprises a culture bag 10 containing a liquid phase and a gas phase, which are culture medium A containing cells; a gas separation membrane module 12 having a gas separation membrane 18, positioned inside the culture bag 10; a rocking means 14 for rocking the culture bag 10; and a depressurization means 16 positioned outside the culture bag 10. As shown in Figure 1, inside the culture bag 10, the gas separation membrane module 12 is installed such that a portion of the gas separation membrane 18 is exposed to the gas phase and the remainder is immersed in the liquid phase. The cell culture apparatus 1 may also include a gas supply means for supplying an oxygen-containing gas, typically air, to the culture medium A.

[0014] In the cell culture apparatus 1, the culture bag 10 is placed on the rocking mechanism 14, and cells are cultured while the culture bag 10 is rocked by the rocking mechanism 14. During cell culture, the dissolved carbon dioxide in the culture medium A (liquid phase) and the carbon dioxide gas in the gas phase can be separated and removed through the gas separation membrane module 12 by reducing the pressure using the decompression mechanism 16. Details of each part will be described later.

[0015] The culture bag 10 is a bag for cell culture in which a culture medium containing cells is sealed. The culture bag 10 is not particularly limited as long as it can seal the culture medium (liquid phase) and gas phase while ensuring liquid-tightness and airtightness, and a resin bag commonly used for cell culture can be used, for example, a single-use resin bag.

[0016] The shape of the culture bag 10 can be set as appropriate. In the example shown in Figure 2, the plan view shape of the culture bag 10 is a square with rounded corners, but it is not limited to this shape, and may be rectangular, circular, elliptical, etc. The dimensions of the culture bag 10 are not particularly limited and can be designed as appropriate depending on the application. Also, when using a culture bag, the ratio of the surface area of ​​the culture solution to the liquid depth tends to be large. Under such conditions, an equilibrium state is formed between the dissolved carbon dioxide in the liquid phase and the carbon dioxide gas in the gas phase, which greatly affects the degassing effect. Therefore, the configuration of this embodiment exhibits the optimal effect under such conditions.

[0017] When the culture apparatus is stationary, the surface area S (cm²) of the culture medium is... ２ The ratio S / D, expressed as the ratio of the liquid depth D (cm) to the liquid depth, is preferably 1 cm or more, more preferably 10 cm or more, and most preferably 100 cm or more. There is no particular upper limit to S / D.

[0018] A gas outlet 24 is formed on the surface of the culture bag 10 for discharging carbon dioxide gas separated from the culture medium A by the gas separation membrane module 12 to the outside of the system. Since the contact efficiency between the culture medium A and the gas separation membrane module 12 is high inside the culture bag 10 which is being rocked by the rocking means 14, the position of the gas outlet 24 on the culture bag 10 is preferably at the corner of the culture bag 10, as shown in the example in Figure 2. However, the position of the gas outlet 24 on the culture bag 10 is not limited to the embodiment in this example and should be designed according to the shape, size, etc. of the culture bag. For example, the gas outlet 24 may be provided in the center of the culture bag 10.

[0019] The opening shape of the gas outlet 24 is typically circular, but is not limited to that. The size of the gas outlet 24 can be set as appropriate. Typically, the gas outlet 24 is shaped so that only the gas outlet piping is inserted, but it may also be shaped so that the gas separation membrane module 12 can be inserted into and removed from the culture bag 10 through the gas outlet 24. In this example, a detachable cap 30 is attached to the gas outlet 24, but is not limited to that. Details of the cap 30 will be described later. Alternatively, the gas outlet 24 may be configured without a cap 30, so that the edge of the gas outlet 24 in the culture bag 10 and the connecting piping between the gas separation membrane module 12 and external equipment are sealed when the gas separation membrane module 12 is placed inside the culture bag 10.

[0020] The culture bag 10 may be provided with connection parts for connecting various sensors other than the gas separation membrane module 12 housed inside the culture bag 10 to various devices placed outside the culture bag 10.

[0021] The gas separation membrane module 12 is positioned inside the culture bag 10 such that a portion of the gas separation membrane 18 is exposed to the gas phase and the remainder is immersed in the culture medium A. In this invention, as in this example, by positioning the gas separation membrane module inside the culture vessel such that a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase, it is possible to simultaneously remove carbon dioxide that accumulates as the culture concentration increases from both the liquid and gas phases. Furthermore, by sufficiently removing carbon dioxide from the liquid phase and preferentially degassing carbon dioxide from the gas phase over oxygen, it is possible to make it easier to retain oxygen in the system.

[0022] One example of a method in which a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase is to use a gas separation membrane made of a material with a lower specific gravity than the culture medium, i.e., a gas separation membrane that is lighter than the culture medium. This allows the gas separation membrane to always remain suspended on the surface of the culture medium, with a portion exposed, even when stirring or agitating during cell culture. The difference between the specific gravity d1 of the culture medium and the specific gravity d2 of the gas separation membrane material (d1-d2) is preferably 0.1 or greater, but 0.01 to 0.05 is also sufficient.

[0023] Another example of a method in which a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase is to fix the gas separation membrane module 12 in a position that is in contact with the culture medium surface within the culture bag 10, thereby maintaining a state in which a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase. This makes it easier to maintain a state in which the gas separation membrane always floats on the surface of the culture medium, even if there is stirring or agitation during cell culture.

[0024] The form of the gas separation membrane 18 is not particularly limited, but hollow fiber membranes and flat membranes are examples. From the viewpoint of specific surface area height, hollow fiber membranes are preferred. An example gas separation membrane module 12 shown in Figures 1 and 2 has a gas separation membrane 18 made up of a plurality of hollow fiber membranes 18a. More specifically, the gas separation membrane module 12 comprises a gas separation membrane 18 made up of a plurality of hollow fiber membranes 18a for separating dissolved carbon dioxide in culture medium A and carbon dioxide gas in the gas phase, and a housing case 20 into which one end of the plurality of hollow fiber membranes 18a is inserted and fixed.

[0025] In this example of a gas separation membrane module 12, each of the multiple hollow fiber membranes 18a is folded back in a U-shape, and one end, formed by bringing together both open ends of each folded hollow fiber membrane 18a, is fixed to the housing case 20 by a potting material. Both ends of each hollow fiber membrane 18a in the longitudinal direction are fixed together with the potting material and are open on the inside of the housing case. The U-shaped folded portion of each hollow fiber membrane 18a is not fixed to the housing case 20 by the potting material and is a free end 18b. Because the multiple hollow fiber membranes 18a of the gas separation membrane module 12 have free ends 18b, each hollow fiber membrane 18a can easily spread out in the culture medium A due to shaking during cell culture. As a result, the contact efficiency between the culture medium A and each hollow fiber membrane 18a is increased, and the separation efficiency of dissolved carbon dioxide is improved. Furthermore, since cultured cells are less likely to clog between each hollow fiber membrane 18a, it is easier to suppress the decrease in the separation efficiency of dissolved carbon dioxide, and the recovery of cultured cells also becomes easier.

[0026] The form of the gas separation membrane module 12 is not limited to the form shown in this example. For example, it may be a form in which multiple hollow fiber membranes are bundled together in a sheet, and both ends of these multiple hollow fiber membranes are inserted into a first housing case and a second housing case, respectively, and fixed in place.

[0027] The hollow fiber membrane 18a is not particularly limited as long as it can separate carbon dioxide gas from the culture medium A and the gas phase by allowing dissolved carbon dioxide gas in the culture medium A and carbon dioxide gas in the gas phase to permeate into the membrane. The hollow fiber membrane 18a may be a membrane composed only of a homogeneous layer, or a membrane made of a porous layer may be used to increase the efficiency of gas permeability. A composite hollow fiber membrane comprising a thin homogeneous layer with gas permeability and a porous support layer supporting the homogeneous layer is particularly preferred as the hollow fiber membrane 18a because it has excellent strength and can efficiently separate and remove dissolved carbon dioxide gas and carbon dioxide gas in the gas phase. The layer configuration of the composite hollow fiber membrane 18a is not particularly limited, and for example, it may be a two-layer configuration in which a porous support layer is provided inside the homogeneous layer, or a three-layer configuration in which porous support layers are provided both inside and outside the homogeneous layer.

[0028] The material used to form the homogeneous layer of the hollow fiber membrane 18a is not particularly limited, and known materials can be used. Examples include silicone rubber resins, polyolefin resins, fluorine-containing resins, cellulose resins, polyphenylene oxide, poly-4-vinylpyridine, and urethane resins. These materials may be used individually or in combination of two or more.

[0029] The homogeneous layer of the hollow fiber membrane 18a has a flux ratio of carbon dioxide to oxygen (CO2). ２ / O ２ It is preferable that the flux ratio (CO) of the homogeneous layer be 2.0 or higher. This is because it becomes easier to separate and remove dissolved carbon dioxide in culture medium A and carbon dioxide gas in the gas phase while maintaining a good culture state. ２ / O ２ ) is more preferably 3.0 or higher. Flux ratio (CO ２ / O ２ The upper limit of the flux ratio (CO) is not particularly limited and can be, for example, 10 or less. ２ / O ２ The lower and upper limits of ) can be any combination, for example, 2.0 to 10.0 or 3.0 to 10. Flux ratio of homogeneous layer (CO ２ / O ２This value is calculated based on the flow rate of each gas when pure gas is supplied to the membrane at a constant pressure.

[0030] The material used to form the porous support layer of the hollow fiber membrane 18a is not particularly limited, and known materials can be used. For example, polyolefin resins with low specific gravity are preferred, and fluorine-containing resins, cellulose resins, polyphenylene oxide, poly-4-vinylpyridine, urethane resins, polystyrene, polyetheretherketone, etc. can also be used depending on the shape. These materials may be used individually or in combination of two or more.

[0031] The average thickness of the hollow fiber membrane 18a is preferably 20 μm or more and 150 μm or less, and more preferably 30 μm or more and 70 μm or less. If the average thickness of the hollow fiber membrane 18a is above the lower limit, the durability of the hollow fiber membrane 18a is excellent. If the average thickness of the hollow fiber membrane 18a is below the upper limit, it is easier to maintain good carbon dioxide separation and removal performance, and the processability is also excellent. The average thickness of the hollow fiber membrane is calculated by measuring the thickness of the hollow fiber membrane at multiple locations (five or more locations) in the circumferential direction and calculating the average value.

[0032] As shown in Figure 1, a gas outlet 20a is formed in the housing case 20, and a connecting pipe 22 is connected to the gas outlet 20a. The other end of the connecting pipe 22 is connected to a gas outlet 24 formed on the surface of the culture bag 10. In this way, within the culture bag 10, the housing case 20 of the gas separation membrane module 12 is connected to the gas outlet 24 by the connecting pipe 22. As another example of the connecting pipe 22, a flexible continuous pipe may be used.

[0033] Outside the gas outlet 24, an adjustment member 26 connected to a connecting pipe 22 is positioned. One end of the gas outlet pipe 28 is connected to the adjustment member 26, and the other end of the gas outlet pipe 28 is connected to the depressurization means 16. In this way, in the cell culture apparatus 1, the gas separation membrane module 12 inside the culture bag 10 and the depressurization means 16 outside the culture bag 10 are connected by a connecting pipe 22 that connects the housing case 20 of the gas separation membrane module 12 inside the culture bag 10 to the gas outlet 24, an adjustment member 26 positioned outside the gas outlet 24 and connected to the connecting pipe 22, and a gas outlet pipe 28 that connects the adjustment member 26 to the depressurization means 16.

[0034] The connecting pipe 22 is not particularly limited and may include, for example, a combination of an L-shaped elbow made of resin, two resin tubes, etc. The gas discharge pipe 28 is not particularly limited and may include, for example, a resin tube or a metal pipe.

[0035] The adjustment member 26 is a member for adjusting the position of the gas separation membrane module 12. It is preferable that the cell culture apparatus, as in this example, is equipped with an adjustment member for adjusting the position of the gas separation membrane module in the culture vessel. The adjustment member 26 in this example is a cylindrical member having a flow path inside that connects a connecting pipe 22 connected to one end and a gas discharge pipe 28 connected to the other end, and by rotating it around its central axis, the orientation of the gas separation membrane module 12 in the culture bag 10 can be changed and its position adjusted.

[0036] One example of how the orientation of the gas separation membrane module 12 inside the culture bag 10 can be changed by the adjustment member 26 is shown in Figure 3A. In this example, the connecting pipe 22 includes a first tube 22a, one end of which is connected to the gas outlet 20a of the housing case 20 of the gas separation membrane module 12; an elbow 22b, the other end of which is connected to the first tube 22a; and a second tube 22c, the other end of which is connected to the elbow 22b. The other end of the second tube 22c extends to a cap 30 that is detachably attached to the gas outlet 24 of the culture bag 10.

[0037] The cap 30 has a threaded portion on its inner wall surface, which is screwed onto the gas outlet 24. The cap 30 has a connecting portion 32 in the center, which is a through hole for connecting the connecting pipe 22 and the adjustment member 26. The material of the cap 30 is not particularly limited, and it can be made of resin, for example.

[0038] The adjustment member 26 comprises a main body 26a having a flow path inside, and a nut 26b that is screwed onto the side of the main body 26a to which the gas discharge pipe 28 is connected. The side of the main body 26a to which the gas discharge pipe 28 is connected is designed to receive the gas discharge pipe 28 into the flow path. Furthermore, the flow path of the main body 26a is provided with a step that protrudes inward, and the surface of this step on the gas discharge pipe 28 side is a contact surface 26c that the tip surface of the received gas discharge pipe 28 abuts against. The side of the main body 26a on which the connecting pipe 22 is connected is also designed to receive the connecting pipe 22 into the flow path. For example, a bore-through connector can be used as the adjustment member 26.

[0039] In the example shown in Figure 3A, the main body portion 26a of the adjustment member 26 is inserted into and fixed to the connection portion 32 of the cap 30. At the connection portion 32 of the cap 30, the end of the second tube 22c of the connecting pipe 22 opposite to the elbow 22b is connected to the main body portion 26a of the adjustment member 26. In addition, one end of the gas discharge pipe 28 is inserted into the main body portion 26a of the adjustment member 26 on the side opposite to the side to which the connecting pipe 22 is connected until it contacts the contact surface 26c. The first tube 22a of the connecting pipe 22 on the side opposite to the adjustment member 26 is connected to the housing case 20 of the gas separation membrane module 12. It is more preferable that the second tube 22c and the gas discharge pipe 28 are a single tube, as shown in Figure 3B.

[0040] The gas separation membrane module 12 is inserted into the culture bag 10 through the gas outlet 24, and the cap 30 is attached to the gas outlet 24 by screwing it on. When the nut 26b of the adjustment member 26 is not tightened, as shown in Figures 2 and 4, the position of each hollow fiber membrane 18a of the gas separation membrane module 12 in the culture bag 10 can be adjusted by rotating the adjustment member 26 around its central axis. In this example, in a plan view from the direction of the central axis of the adjustment member 26, the housing case 20 of the gas separation membrane module 12 rotates around the central axis of the adjustment member 26 together with the connecting pipe 22 as the adjustment member 26 rotates around the central axis. Subsequently, by tightening the nut 26b of the adjustment member 26, the gas separation membrane module 12 can be inserted into the desired position in the culture bag 10 while suppressing disturbance of the hollow fiber membrane 18a. In this way, the position of the gas separation membrane module 12 in the culture bag 10 can be freely adjusted using the adjustment member 26.

[0041] The agitation means 14 is not particularly limited as long as it can agitate the culture bag 10 containing the culture medium A during cell culture, and known agitation means can be used. By agitating the culture bag 10 with the agitation means 14 during cell culture, the culture medium A inside the culture bag 10 is stirred, increasing the contact efficiency between each hollow fiber membrane 18a of the gas separation membrane module 12 and the culture medium A, thereby improving the culture efficiency. Furthermore, compared to the case where a stirring blade is inserted into the culture bag 10 to stir the culture medium A, the agitation means 14 does not subject the cultured cells in the culture medium A to physical impact from the stirring blade, thus reducing damage to the cultured cells.

[0042] An example of the rocking mechanism 14 shown in Figures 1 and 5 comprises a base 42 and a flat rocking platform 46 supported on the base 42 via a support portion 44. As shown in Figure 6, the rocking platform 46 is designed to rock so that, in a side view from the X direction, both ends in the Y direction alternately move up and down. By placing a culture bag 10 containing culture medium A on the rocking platform 46 and rocking the rocking platform 46, the culture bag 10 can be rocked. The rocking speed and angle of the rocking platform 46 in the rocking mechanism 14 can be appropriately set depending on the type of cell being cultured.

[0043] The depressurization means 16 is a means for reducing the pressure on the secondary side of the gas separation membrane 18. However, the "primary side" of the gas separation membrane means the side of the gas separation membrane where the culture medium is present, and the "secondary side" of the gas separation membrane means the side opposite to the primary side of the gas separation membrane, that is, the side opposite to the side of the gas separation membrane where the culture medium is present. When the gas separation membrane consists of multiple hollow fiber membranes, the outside of each hollow fiber membrane is the primary side, and the inside of each hollow fiber membrane is the secondary side. Dissolved carbon dioxide in the culture medium and carbon dioxide gas in the gas phase are separated by permeation from the primary side to the secondary side of the gas separation membrane.

[0044] The depressurization means 16 is not particularly limited and includes, for example, a vacuum pump and an aspirator. By driving the depressurization means 16, the inside of each hollow fiber membrane 18a of the gas separation membrane module 12 can be depressurized through the gas discharge pipe 28, the adjustment member 26, and the connecting pipe 22. As a result, dissolved carbon dioxide in the culture medium A and carbon dioxide in the gas phase permeate through each hollow fiber membrane 18a and are separated, and discharged outside the system through the connecting pipe 22, the adjustment member 26, and the gas discharge pipe 28.

[0045] As shown in the example in Figure 2, the cell culture apparatus 1 according to this embodiment preferably further includes a pH sensor 52 for measuring the pH of the culture medium A in the culture bag 10. The pH sensor 52 is not particularly limited as long as it can measure the pH of the culture medium, and any known pH sensor can be used.

[0046] Furthermore, it is preferable that the cell culture apparatus 1 further includes a control device 60 that controls the output of the decompression means 16 based on the measurement value of the pH sensor 52. In this case, the pH of the culture medium A during cell culture is monitored by the pH sensor 52, and the measurement value is transmitted to the control device 60. The control device 60 then controls the amount of dissolved carbon dioxide released from the culture medium A to the outside of the system by adjusting the output of the decompression means 16 based on the pH measurement value from the pH sensor 52.

[0047] The cell culture apparatus 1 measures the dissolved carbon dioxide concentration of the culture medium A in the culture bag 10. ２ A sensor 54 may be provided. ２ The sensor 54 is not particularly limited as long as it is capable of measuring the dissolved carbon dioxide concentration in the culture medium, and any known dissolved carbon dioxide sensor can be used.

[0048] Cell culture device 1 is CO ２ When equipped with a sensor 54 and a control device 60, CO ２ The output of the depressurization means 16 may be controlled by the control device 60 based on the measurement value of the sensor 54. For example, if the dissolved carbon dioxide concentration of the culture medium A during cell culture is CO ２ The sensor 54 monitors the CO2 readings, and the measured values ​​are transmitted to the control device 60. The control device 60 then processes the CO2 readings. ２ Based on the dissolved carbon dioxide concentration measured by the sensor 54, the output of the decompression means 16 is adjusted to control the amount of dissolved carbon dioxide released from the culture medium A to the outside of the system.

[0049] Before performing the culture, the overall mass transfer coefficient (k) of the dissolved carbon dioxide in the simulated solution should be measured. Ｌ a ＣＯ２ It is preferable to conduct chemical engineering tests to confirm the carbon dioxide removal rate and optimal degassing conditions using the following: Here, the overall mass transfer coefficient (k) of dissolved carbon dioxide. Ｌ a ＣＯ２ The overall mass transfer coefficient (k) of dissolved carbon dioxide is an indicator of how efficiently carbon dioxide is transported (emitted). Generally, the overall mass transfer coefficient (k) of dissolved carbon dioxide is used. Ｌ a ＣＯ２ ) is 0.25h －１It is said that the above is sufficient. The components of the simulated solution mimic the viscosity, pH, dissolved carbon dioxide, and other elements of the actual culture medium, but do not contain cells.

[0050] The control by the control device 60 controls the overall mass transfer coefficient (k) of dissolved carbon dioxide in the culture medium. Ｌ a ＣＯ２ ) may be used to simulate the optimal degassing conditions for the separation and removal of dissolved carbon dioxide using the following steps 1 to 4. Step 1: Confirm the highest dissolved carbon dioxide concentration when cells are cultured under conditions in which dissolved carbon dioxide is not degassed from the culture medium. Step 2: Set up several different degassing conditions corresponding to the dissolved carbon dioxide concentration value obtained in Step 1. Step 3: Using the multiple degassing conditions set up in Step 2, set up a simulation step to simulate the overall mass transfer coefficient (k) of multiple dissolved carbon dioxide. Ｌ a ＣＯ２ ) calculate the multiple overall mass transfer coefficients (k) obtained in step 3. Step 4: Ｌ a ＣＯ２ Among these, the overall mass transfer coefficient (k) that does not damage the cells and obtains the optimal degassing effect. Ｌ a ＣＯ２ Select the corresponding degassing conditions.

[0051] To obtain the highest dissolved carbon dioxide concentration when culturing cells in Step 1, you may use the values ​​from the paper or measure it yourself. The degassing conditions in Step 2 are at least one of the effective membrane area or number of gas separation membrane modules of the gas separation membrane module, the vacuum level of the vacuum pump, the placement of one or more gas separation membrane modules in the culture bag 10, and the shaking speed of the shaking means. The overall mass transfer coefficient (k) in Step 3. Ｌ a ＣＯ２ The calculation of ) will utilize a known calculation method.

[0052] For example, when the control device 60 controls the output of the depressurization means 16, the overall mass transfer coefficient (k) obtained in the simulation process described above is used. Ｌ a ＣＯ２ The upper limit (target DCO) of the dissolved carbon dioxide concentration corresponding to ) ２Let the value be CO2. The actual dissolved carbon dioxide concentration of culture medium A during cell culture is CO2. ２ The sensor 54 monitors the CO2 level, and the measured values ​​are transmitted to the control device 60. In the early stages of cell culture, CO2 ２ Because the measurement value from sensor 54 is low, the control device 60 operates in energy-saving mode by not activating the decompression means 16 or by adjusting the output of the decompression means 16 to a lower level. As cells proliferate, the dissolved carbon dioxide concentration gradually increases. ２ The measurement value from sensor 54 is the target DCO ２ If the value is higher, the control device 60 controls the amount of dissolved carbon dioxide released from the culture medium A to the outside of the system by increasing the output of the depressurization means 16. ２ The measurement value from sensor 54 is the target DCO ２ If the value falls below the specified level, the control device 60 adjusts the output of the depressurization means 16 back to its original value and operates in energy-saving mode. This control can be repeated during cell culture.

[0053] [Cell Culture Method] The cell culture method according to the embodiment is a method using the cell culture apparatus according to the embodiment described above. More specifically, the cell culture method uses a cell culture apparatus comprising a culture vessel containing a liquid phase which is a culture medium containing cells, and a gas phase, and one or more gas separation membrane modules disposed within the culture vessel. The gas separation membrane module has a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed. When culturing cells, the gas separation membrane module in the culture vessel is in a state in which a part of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase. In the cell culture method according to the embodiment, it is preferable to shake the culture vessel during cell culture in order to enable more uniform oxygen supply and carbon dioxide degassing during cell culture.

[0054] Below, an example of a cell culture method according to the embodiment will be described, specifically a method using the cell culture apparatus 1.

[0055] The gas separation membrane module 12 is inserted into the culture bag 10 through the gas outlet 24, the cap 30 is attached to the gas outlet 24 by screwing it on, and the gas separation membrane module 12 is inserted into the desired position in the culture bag 10 by rotating the adjustment member 26. After tightening the nut 26b of the adjustment member 26, the culture medium A is supplied into the culture bag 10 from a separately provided culture medium supply port, so that a part of the gas separation membrane 18 is exposed to the gas phase and the rest is immersed in the liquid phase. Then, as shown in Figures 1 and 5, the culture bag 10 containing the culture medium A with cells is placed on the rocking platform 46 of the rocking means 14, and cell culture is performed while rocking the culture bag 10, as shown in Figure 6.

[0056] In this embodiment, by changing the orientation and adjusting the position of each hollow fiber membrane 18a of the gas separation membrane module 12 using the adjustment member 26, entanglement of each hollow fiber membrane 18a due to shaking during cell culture can be suppressed, the contact efficiency between the culture medium A and each hollow fiber membrane 18a can be increased, and the culture efficiency can be improved. From the viewpoint of suppressing entanglement of each hollow fiber membrane 18a due to shaking and improving culture efficiency, it is preferable that, when viewed from above the shaking means 14, the length direction of each hollow fiber membrane 18a of the gas separation membrane module 12 before shaking is perpendicular to the direction of shaking. In this example, as shown in Figure 6, the direction of shaking of the shaking means 14 is the Y direction, and as shown in Figure 2, the length direction of each hollow fiber membrane 18a of the gas separation membrane module 12 before shaking is in the X direction. Note that the position and orientation of the gas separation membrane module 12 are not limited to this embodiment.

[0057] The cells to be cultured are not particularly limited; any cells from which the desired substance, such as antibodies, enzymes, or viral vectors, can be obtained. Examples of cells to be cultured include animal cells (such as mammalian cells), plant cells, insect cells, bacteria, yeast, and fungi.

[0058] The culture medium A is not particularly limited as long as it is suitable for the cells being cultured. The temperature of the culture medium A during culture can be set appropriately according to the cells being cultured, and is preferably 30 to 40°C, and more preferably 35 to 38°C.

[0059] The pH of culture medium A is preferably set to a range suitable for the cells being cultured. Culture medium A may contain a carbonate with pH buffering capacity. Examples of carbonates include sodium bicarbonate, sodium carbonate, potassium carbonate, potassium bicarbonate, and magnesium carbonate.

[0060] As the culture progresses, when the dissolved carbon dioxide concentration in culture medium A increases, the depressurization means 16 is driven to reduce the pressure inside each hollow fiber membrane 18a of the gas separation membrane module 12 through the gas discharge pipe 28, adjustment member 26, and connecting pipe 22, thereby separating and removing dissolved carbon dioxide in culture medium A and carbon dioxide gas in the gas phase. The separation and removal of dissolved carbon dioxide and carbon dioxide gas in the gas phase by the gas separation membrane module 12 may be performed continuously or intermittently.

[0061] When the dissolved carbon dioxide concentration in culture medium A increases, the pH of culture medium A decreases. In a preferred example, a pH sensor 52 is provided in the cell culture apparatus 1, and the control device 60 controls the amount of dissolved carbon dioxide released from culture medium A to the system by adjusting the output of the depressurization means 16 based on the measured pH of culture medium A during cultivation. For example, when the measured pH of culture medium A falls below a predetermined value, the depressurization means 16 is driven or its output is increased to increase the amount of dissolved carbon dioxide released from culture medium A to the system. This makes it possible to increase the culture efficiency.

[0062] Furthermore, under multiple degassing conditions, the overall mass transfer coefficient (k) of dissolved carbon dioxide can be determined from the change in the dissolved carbon dioxide concentration of the simulated liquid over time. Ｌ a ＣＯ２ The calculation is performed, and the calculated value is the threshold (0.25h). －１ If the value is above this, it is preferable to use the degassing conditions corresponding to the calculated value as the cell culture conditions. When culturing cells, the target DCO of the culture medium ２ The value should preferably be set appropriately depending on the cell type. Generally, from the viewpoint of damage to cells due to dissolved carbon dioxide, it is preferable to set it to 20% or less, more preferably 10% or less. Target DCO ２It is also preferable to adjust the output of the depressurization means 16 so as not to exceed the value, thereby controlling the amount of dissolved carbon dioxide in the culture medium A released from the system. ２ It is more preferable to provide a sensor 54 and control the amount of dissolved carbon dioxide released from the culture medium A to the system by adjusting the output of the depressurization means 16 based on the measured value of the dissolved carbon dioxide concentration using a control device 60. While it is preferable, the output control of the depressurization means 16 is controlled by the control device 60, it is not limited to this method. The dissolved carbon dioxide concentration of the culture medium A can be measured, for example, by periodically taking a sample of the culture medium A and using a dissolved carbon dioxide sensor, but it is not limited to this method.

[0063] Furthermore, the overall mass transfer coefficient (k) of dissolved carbon dioxide in culture medium A in the embodiment Ｌ a ＣＯ２ ) is obtained as shown in Figure 7. The abbreviations in Figure 7 have the following meanings: [DCO ２ ] ０ : Dissolved carbon dioxide concentration at the start of operation (t=0) [DCO ２ ] ｓ Dissolved carbon dioxide concentration in steady state after continued operation [DCO ２ ] ｔ : Dissolved carbon dioxide concentration after t time elapsed t: Operating time (hours)

[0064] For example, when controlling the output of the depressurization means 16 based on the dissolved carbon dioxide concentration, the cell culture method according to the embodiment uses the overall mass transfer coefficient (k) of dissolved carbon dioxide in the culture medium A as described above. Ｌ a ＣＯ２ Preferably, the system includes a simulation step that uses () to simulate the optimal degassing conditions for the separation and removal of dissolved carbon dioxide.

[0065] In the cell culture method according to this embodiment, the dissolved carbon dioxide concentration of culture medium A is preferably adjusted to be between 5% and 15%, and more preferably between 5% and 10%. This makes it easier to culture active cells and further increases the recovery rate of cultured cells.

[0066] As described above, in this invention, by installing a gas separation membrane module in the culture vessel such that a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase, it is possible to simultaneously remove carbon dioxide from both the liquid and gas phases. Furthermore, by sufficiently removing carbon dioxide from the liquid phase and preferentially degassing carbon dioxide from the gas phase over oxygen, it is possible to retain oxygen in the system. In addition, by agitating the culture bag with an agitator during cell culture, damage to the cultured cells can be reduced compared to agitation using a stirring blade, further increasing the culture efficiency. Moreover, by adjusting the position of the gas separation membrane module in the culture bag with an adjustment member, entanglement of each hollow fiber membrane due to agitation during culture can be suppressed, thereby increasing the contact efficiency between each hollow fiber membrane and the culture medium, and further increasing the culture efficiency.

[0067] The cell culture apparatus and cell culture method of the present invention are not limited to the embodiments described above. The number of gas separation membrane modules is not limited to one, but may be two or more. In the case of two or more modules, it is preferable to disperse the separation membrane modules in the culture bag from the viewpoint of increasing the contact efficiency between each hollow fiber membrane and the culture medium. Each hollow fiber membrane in the gas separation membrane module may have both ends fixed and may not have the free end 18b described above. The cell culture apparatus according to the embodiment may not be equipped with either a pH sensor or a dissolved oxygen sensor, or both. The cell culture apparatus according to the embodiment may not be equipped with a rocking means or a control device.

[0068] Furthermore, without departing from the spirit of the present invention, the components in the above embodiments may be replaced with well-known components as appropriate, and the above-described modifications may be combined as appropriate.

[0069] The present invention will be specifically described below with reference to experimental examples (chemical engineering tests using simulated solutions), examples, and comparative examples, but the present invention is not limited to the following description.

[0070] [Experimental Example 1] Using the cell culture apparatus 1 and simulated solution illustrated in Figures 1-6, a situation with a high dissolved carbon dioxide concentration was simulated by continuously supplying carbon dioxide, and the overall mass transfer coefficient (k) was calculated.Ｌ a ＣＯ２ A test was conducted to determine the following. Using the cell culture apparatus 1 (WAVE Bioreacoer: ABLE-Biott SWING) illustrated in Figure 1, a Cellbag™ (Cytiva) 20L was used as the culture bag, and the gas separation membrane module was inserted using the screw cap at the corner. As the gas separation membrane module, a membrane module with a three-layer composite hollow fiber membrane (MHF300EPE manufactured by Mitsubishi Chemical Corporation) was used. The effective membrane area of ​​this membrane module was 0.4 m². ２ The effective length is 40 cm. When viewed from above the rocking mechanism, the gas separation membrane module in its pre-rocking state was adjusted so that the length direction of each hollow fiber membrane was perpendicular to the rocking direction. As a simulated solution, a solution was prepared that mimicked the viscosity, pH, and dissolved carbon dioxide concentration of the culture medium, and did not contain cells. Specifically, 10 L of Poloxamer 188 0.33 g / L was prepared, and the temperature of the simulated solution was set to 37°C. In order to make the dissolved carbon dioxide concentration in the simulated solution in the culture bag high, air was continuously supplied at 100 mL / min and carbon dioxide at 100 mL / min, and the culture bag was rocked by the rocking mechanism. When the dissolved carbon dioxide concentration in the simulated solution reached equilibrium, the inside of each hollow fiber membrane of the gas separation membrane module was depressurized by the depressurization mechanism, and the dissolved carbon dioxide concentration over time was measured. The specific gravity of the hollow fiber membrane material is lower than that of the culture medium, and during cell culture, the gas separation membrane, consisting of multiple hollow fiber membranes, constantly floated on the surface of the culture medium, with a portion exposed and the remainder immersed. The time changes in dissolved carbon dioxide concentration and pH of the simulated solution are shown in Figure 8, ln[DCO ２ ] ０ - [DCO ２ ] ｔ The time evolution is shown in Figure 9.

[0071] As shown in Figure 8, the dissolved carbon dioxide concentration in the simulated liquid decreased from 32.6% at the start of the separation and removal of dissolved carbon dioxide by the gas separation membrane module to 13.6% in one hour. Also, the pH of the simulated liquid increased from 4.97 at the start of the separation and removal of dissolved carbon dioxide to 5.41 in one hour. As shown in Figure 9, the overall mass transfer coefficient (k) of dissolved carbon dioxide was calculated from the measured dissolved carbon dioxide concentration. Ｌa ＣＯ２ ), the calculated value was 0.8104 h －１ which is higher than the threshold value of 0.25 h －１ It was found that the dissolved carbon dioxide in the simulated liquid could be efficiently removed. Therefore, it was confirmed that cell culture could be carried out using the degassing conditions (membrane area, number of membrane modules, degree of vacuum, etc.) corresponding to the k Ｌ a ＣＯ２ corresponding to.

[0072] Hereinafter, in Example 1 and Comparative Example 1, a comparison was made between the case where the dissolved carbon dioxide was removed by the gas separation membrane module 12 and the case where it was not removed.

[0073] [Example 1] Using the cell culture device 1 (WAVE Bioreactor: ABLE - Biott SWING) illustrated in FIG. 1, Cellbag ＴＭ (Cytiva) 20 L was used as the culture bag, and the gas separation membrane module was inserted using the screw cap at the corner, and the seed culture (N - 1 culture) of suspension HEK293 cells (VP002 cells) was performed. As the gas separation membrane module, a membrane module (MHF300EPE manufactured by Mitsubishi Chemical Corporation) having a three - layer composite hollow fiber membrane was used. The effective membrane area of the membrane module is 0.4 m ２ , and the effective length is 40 cm. As the culture medium A, 10 L of EX - CELL CD HEK293 Viral Vector Medium + 6 mM L - Glutamine + 1% P / S was prepared, the seeding concentration was 3.5×10 ５ cells / mL, the temperature of the culture medium A was 37°C, and the culture was carried out while supplying air at 200 mL / min. The specific gravity of the material of the hollow fiber membrane is smaller than the specific gravity of the culture medium, and during cell culture, the gas separation membrane composed of a plurality of hollow fiber membranes always floats on the surface of the culture medium, a part of it is exposed, and the remaining part is maintained in a state of being immersed. After the 3rd day of culture, the carbon dioxide concentration of the gas ventilated into the gas phase above the liquid level of the culture medium A was increased to the target DCO ２The aeration conditions (high carbon dioxide concentration aeration conditions) were changed to achieve a 30% concentration. In this way, by setting the dissolved carbon dioxide concentration to a high level after the cell density increased, a scaled-down model was created that simulated the high carbon dioxide conditions during large-scale production culture. After the change in aeration conditions on the third day of culture, a vacuum pump was operated after the start of aeration of the carbon dioxide mixed gas to remove dissolved carbon dioxide in the culture medium and carbon dioxide gas in the gas phase using a gas separation membrane module. The vacuum level for vacuuming with the vacuum pump was set to -90 kPa.

[0074] Figure 10 shows the change in pH of culture medium A in the culture bag. Figure 11 shows a graph plotting the dissolved carbon dioxide concentration of culture medium A in the culture bag with culture time on the x-axis and dissolved carbon dioxide concentration on the y-axis. Figure 12 shows a graph plotting the change in viable cell density (VCD) in culture medium A in the culture bag when high carbon dioxide concentration aeration conditions were applied from the third day of culture onwards, with culture time on the x-axis and VCD retention rate on the y-axis. Furthermore, Figure 13 shows a graph plotting the change in viability in culture medium A in the culture bag when high carbon dioxide concentration aeration conditions were applied from the third day of culture onwards, with culture time on the x-axis and viability retention rate on the y-axis. Viable cell density (VCD) and viability were measured using Vi-CELL BLU (Beckman Coulter). The VCD retention rate or viability retention rate was calculated as a ratio based on the VCD or viability immediately after changing to high carbon dioxide concentration aeration conditions from the third day of culture onward.

[0075] [Comparative Example 1] Cell culture was carried out for 6 days in the same manner as in Example 1, except that the removal of dissolved carbon dioxide in the culture medium and carbon dioxide gas in the gas phase using a gas separation membrane module was not performed from the third day of culture onwards. Figure 10 shows the change in pH of culture medium A in the culture bag. Figure 11 shows a graph plotting the dissolved carbon dioxide concentration of culture medium A in the culture bag with culture time on the x-axis and dissolved carbon dioxide concentration on the y-axis. Figure 12 shows a graph plotting the change in VCD when high carbon dioxide concentration aeration conditions were applied from the third day of culture onwards, with culture time on the x-axis and VCD retention rate on the y-axis. Furthermore, Figure 13 shows a graph plotting the change in Viability when high carbon dioxide concentration aeration conditions were applied from the third day of culture onwards, with culture time on the x-axis and Viability retention rate on the y-axis.

[0076] As shown in Figure 10, in Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were removed from the culture medium using a gas separation membrane module, the pH of the culture medium was maintained closer to that at the start of culture, even when high carbon dioxide concentration aeration conditions were applied from the third day of culture onward, compared to Comparative Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were not removed. As shown in Figure 11, in Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were removed from the culture medium, the dissolved carbon dioxide concentration in the culture medium from the third day of culture onward was reduced by 40% to 50% compared to Comparative Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were not removed, and was stably maintained at 20% or less even under high carbon dioxide concentration conditions simulating production. As shown in Figure 12, in Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were removed from the culture medium using a gas separation membrane module, the reduction in viable cell density under high carbon dioxide concentration aeration conditions from the third day of culture onward was mitigated compared to Comparative Example 1, where dissolved carbon dioxide and gaseous carbon dioxide gas were not removed. As shown in Figure 13, in Example 1, where dissolved carbon dioxide and carbon dioxide gas in the gas phase were removed from the culture medium using a gas separation membrane module, the decrease in the viable cell rate was suppressed even under high carbon dioxide concentration aeration conditions from the third day of culture onward, compared to Comparative Example 1, where dissolved carbon dioxide and carbon dioxide gas were not removed. Furthermore, compared to Comparative Example 1, where almost all cells died by the sixth day of culture, viable cells were maintained in the culture medium.

[0077] 1 Cell culture apparatus 10 Culture bag 12 Gas separation membrane module 14 Oscillating means 16 Depressurization means 18 Gas separation membrane 18a Hollow fiber membrane 20 Housing case 22 Connecting piping 24 Gas outlet 26 Adjustment member 28 Gas outlet piping 30 Cap 42 Base 44 Support part 46 Oscillating platform 52 pH sensor 54 CO ２ Sensor 60 Control device

Claims

1. A cell culture apparatus comprising: a culture vessel containing a liquid phase which is a culture medium containing cells, and a gas phase; and one or more gas separation membrane modules disposed within the culture vessel, wherein the gas separation membrane module has a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed; and the gas separation membrane in the culture vessel is installed such that a portion of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase.

2. The cell culture apparatus according to claim 1, wherein the specific gravity of the material of the gas separation membrane is less than the specific gravity of the culture medium.

3. The cell culture apparatus according to claim 1 or 2, further comprising a rocking means for rocking the culture vessel.

4. The cell culture apparatus according to claim 1 or 2, further comprising a vacuum means for reducing the pressure on the secondary side of the gas separation membrane.

5. The cell culture apparatus according to claim 1 or 2, wherein the culture vessel is a culture bag.

6. The cell culture apparatus according to claim 1 or 2, wherein the gas separation membrane consists of a plurality of hollow fiber membranes.

7. The cell culture apparatus according to claim 6, wherein the plurality of hollow fiber membranes of the gas separation membrane module have free ends that are not fixed to the housing case by a potting material.

8. The cell culture apparatus according to claim 1 or 2, further comprising an adjustment member for adjusting the position of the gas separation membrane module.

9. The cell culture apparatus according to claim 4, further comprising a pH sensor for measuring the pH of the culture medium.

10. The cell culture apparatus according to claim 9, further comprising a control device that controls the amount of dissolved carbon dioxide in the culture medium released from the system by adjusting the output of the depressurization means based on the measurement value of the pH sensor.

11. Measuring the dissolved carbon dioxide concentration of the culture medium CO ２ The cell culture apparatus according to claim 4, further comprising a sensor.

12. The aforementioned CO ２ The cell culture apparatus according to claim 11, further comprising a control device that controls the amount of dissolved carbon dioxide in the culture medium released from the system by adjusting the output of the depressurization means based on the measurement value of the sensor.

13. A cell culture method using a cell culture apparatus comprising: a culture vessel containing a liquid phase which is a culture medium containing cells, and a gas phase; and one or more gas separation membrane modules disposed within the culture vessel, wherein the gas separation membrane module comprises a gas separation membrane capable of separating carbon dioxide and a housing case to which the gas separation membrane is fixed, and when culturing the cells, the gas separation membrane module in the culture vessel is in a state in which a part of the gas separation membrane is exposed to the gas phase and the remainder is immersed in the liquid phase.

14. The cell culture method according to claim 13, wherein the specific gravity of the material of the gas separation membrane is less than the specific gravity of the culture medium.

15. The cell culture method according to claim 13 or 14, wherein the culture vessel is shaken when culturing the cells.

16. The cell culture method according to claim 13 or 14, wherein the cell culture apparatus further comprises a depressurization means for reducing the pressure on the secondary side of the gas separation membrane, and when culturing the cells, the pressure on the secondary side of the gas separation membrane is reduced to separate and remove dissolved carbon dioxide in the culture medium and carbon dioxide gas in the gas phase.

17. The cell culture method according to claim 13 or 14, wherein the gas separation membrane consists of a plurality of hollow fiber membranes, and the plurality of hollow fiber membranes have free ends that are not fixed to the housing case by a potting material.

18. The cell culture method according to claim 16, wherein the cell culture apparatus further comprises a pH sensor for measuring the pH of the culture medium, and controls the amount of dissolved carbon dioxide in the culture medium released from the system by adjusting the output of the decompression means based on the measurement value of the pH sensor.

19. The cell culture apparatus measures the dissolved carbon dioxide concentration of the culture medium. ２ The sensor is further equipped with the CO ２ The cell culture method according to claim 16, wherein the output of the decompression means is adjusted based on the measurement value of the sensor to control the amount of dissolved carbon dioxide in the culture medium released from the system.

20. The overall mass transfer coefficient (k) of dissolved carbon dioxide in the culture medium. Ｌ a ＣＯ２ The cell culture method according to claim 13 or 14, further comprising a simulation step of simulating the optimal degassing conditions for the separation and removal of dissolved carbon dioxide using ).

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