Culture device and culture method

Abstract

Provided is a culture device and the like that achieve improvements both in oxygen supplying performance and carbon dioxide removal performance in a liquid phase within a culture tank. A culture device (1) according to the present invention comprises an oxygen supply device (3) for supplying an oxygen gas into a liquid phase (LP) obtained by supplying the inside of a culture tank (2) with a culture medium (CM), an air supply device (4) for supplying air into the liquid phase (LP), a liquid surface ventilation device (5) that controls ventilation of air in a gas phase (GP) formed above the liquid phase (LP) on the inside of the culture tank (2), a first sensor unit (6) that detects an indicator regarding dissolved carbon dioxide of the liquid phase (LP), and a control device (7). The control device (7) executes a second operation for improving carbon dioxide removal performance from the liquid phase (LP) on the basis of the information from the first sensor unit (6). When the control device (7) executes the second operation, the ventilation quantity via the air supply device (4) and the ventilation quantity via the liquid surface ventilation device (5) are determined on the basis of at least the information from the first sensor unit (6).

Description

Culture apparatus and culture method

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

[0002] There is a technique called suspension culture in which animal cells, microorganisms, or those attached to beads (microcarriers) (hereinafter referred to as cells) are added to the culture medium in a culture tank, and the cells are cultured by mechanically agitating the mixture while supplying pure oxygen or air into the liquid (submerged aeration). In this way, the cells themselves are produced, or valuable substances secreted by the cells are produced.

[0003] Animal cells and microorganisms respire during the growth and production processes, requiring the supply of oxygen (hereinafter sometimes referred to as O2) and the removal of carbon dioxide (hereinafter sometimes referred to as CO2). Therefore, from the perspective of growth rate, productivity, and ensuring product quality, there is a desire to improve the oxygen supply and carbon dioxide removal performance within the culture vessel. Those skilled in the art can determine the behavior of dissolved oxygen (hereinafter sometimes referred to as DO) and dissolved carbon dioxide (hereinafter sometimes referred to as DCO2) concentrations within the culture vessel, the supply rate of oxygen (sometimes air) and carbon dioxide degassing gas, the gas bubble diameter, the liquid surface area / liquid volume (or aspect ratio), the liquid surface permeability in the gas phase at the top of the culture vessel, and the CO2 partial pressure (p ＣＯ２ We are striving to achieve the above requirements by appropriately selecting operating control conditions such as the above.

[0004] Improving the oxygen supply performance within the culture vessel can be achieved relatively easily, for example, by using pure oxygen or increasing the amount of pure oxygen supplied. On the other hand, improving the CO2 removal performance within the culture vessel is difficult because the partial pressure of CO2 in the bubbles rising in the culture medium and the DCO2 concentration in the culture medium reach equilibrium relatively quickly, and thereafter DCO2 in the culture medium no longer transfers to the bubbles. As a result, the CO2 generation rate (Carbon dioxide (CO2) Evolution Rate; CER) stops increasing, making it difficult to improve CO2 removal performance.

[0005] In recent years, intensive research has been conducted on CO2 removal in culture tanks, and numerous findings have been reported. For example, Non-Patent Document 1 states that "carbon dioxide has high solubility in water, and the partial pressure of carbon dioxide in bubbles easily reaches equilibrium with the dissolved carbon dioxide in the culture medium. Therefore, measures such as improving liquid surface gas exchange and large-diameter bubble aeration are necessary for the removal of dissolved carbon dioxide." Non-Patent Document 1 also states that "VVM is the most important indicator of gas exchange." Furthermore, Non-Patent Document 1 states that "even if VVM is constant, the residence time of aeration bubbles increases with increasing liquid depth due to scale-up, and the overall oxygen transfer capacity coefficient (KLa) improves. Therefore, comparison and evaluation including not only VVM but also aeration tower velocity is necessary."

[0006] Furthermore, for example, Non-Patent Document 2 states that "the gas transport capacity coefficient KLa is equal to the gas tower velocity U ｇ Since it is proportional to U ｇ It states that scaling up at a constant level is sufficient. However, in culture tanks, scaling up often involves keeping the vvm constant, taking into account not only oxygen supply but also the emission of carbon dioxide generated by respiration. Non-patent document 2 also describes an example of scaling up with VVM = 0.5 during koji mold cultivation.

[0007] Satoshi Murakami, "Cognitive Biases and Innovation in Animal Cell Culture," Journal of the Japan Society for Biotechnology, 2024, Vol. 102, No. 9, pp. 463-466. Tomohiro Ogawa, Hirotoshi Handa, Chiho Kato, and Noriyuki Yamagami, "Study on the Application of Swingstar (Registered Trademark) to Culture Vessels," Kobe Steel Environmental Solutions Technical Report, Vol. 13, No. 2 (2017 / 3), pp. 27-31.

[0008] Oxygen supply and carbon dioxide removal are achieved by aeration of oxygen gas and air into the liquid and aeration of the liquid surface. Although there is knowledge from non-patent documents such as 1 and 2, oxygen supply and carbon dioxide removal are determined by various factors, such as the conditions of the equipment and environment related to cell culture. Furthermore, some of these conditions affect both oxygen supply performance and carbon dioxide removal performance, or there is a trade-off relationship between them. As a result, operations to improve one of the performances, oxygen supply performance or carbon dioxide removal performance, sometimes deteriorated the performance of the other. In this technical field, it is desirable to be able to achieve both improved oxygen supply performance and improved carbon dioxide removal performance in the liquid phase (culture medium) of the culture vessel.

[0009] The present invention has been made in view of the above circumstances. The object of the present invention is to provide a culture apparatus and culture method that achieve both improved oxygen supply performance and carbon dioxide removal performance in the liquid phase of the culture vessel.

[0010] The culture apparatus according to the present invention, which solves the aforementioned problems, comprises a culture tank for culturing cells in a culture medium, an oxygen supply device for supplying oxygen gas into the liquid phase formed by supplying the culture medium inside the culture tank, an air supply device for supplying air into the liquid phase, a liquid level ventilator connected to the gas phase formed above the liquid phase inside the culture tank and controlling the airflow in the gas phase, a first sensor unit provided so as to be in contact with at least the liquid phase and detecting an indicator of dissolved carbon dioxide in the liquid phase, and the oxygen supply device, the air supply device and the liquid level ventilator The system includes a control device that controls the following: The control device performs a first operation to improve the oxygen supply performance to the liquid phase and a second operation to improve the carbon dioxide removal performance from the liquid phase based on information from the first sensor unit, wherein the second operation is at least one of an operation to increase the amount of air permeation by the air supply device and an operation to increase the amount of air permeation by the liquid surface ventilator, and when the control device performs the second operation, it determines the amount of air permeation by the air supply device and the amount of air permeation by the liquid surface ventilator based on information from at least the first sensor unit.

[0011] According to the present invention, it is possible to provide a culture apparatus and culture method that achieve both improved oxygen supply performance and carbon dioxide removal performance in the liquid phase of the culture vessel. Problems, configurations, and effects other than those described above will be clarified by the following description of embodiments. Further features related to the present invention will be evident from the description herein and the accompanying drawings.

[0012] This is a schematic diagram illustrating one example of the configuration of the culture apparatus 1 according to this embodiment. This is a table summarizing the relationships between each parameter in the culture apparatus 1. This is a flowchart explaining the content of the culture method according to this embodiment. This is a block diagram of the computer that constitutes the control device in the culture apparatus 1 of Figure 1.

[0013] To achieve the aforementioned objectives, a culture apparatus and culture method according to one embodiment of the present invention have the following configuration. The object, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description herein, and those skilled in the art will be able to easily reproduce the present invention from the description herein. The specific embodiments of the invention described below are examples of preferred embodiments of the present invention and are provided for illustrative or explanatory purposes only, and do not limit the present invention to them. It will be apparent to those skilled in the art that various modifications and modifications can be made based on the description herein, within the intent and scope of the present invention as disclosed herein.

[0014] Furthermore, the embodiments described below are illustrative examples for illustrating the present invention, and have been omitted and simplified as appropriate for clarity of explanation. The present invention can be implemented in various other forms. Unless otherwise specified, each component may be singular or plural. The positions, sizes, shapes, and ranges of each component shown in the drawings may not represent the actual positions, sizes, shapes, and ranges in order to facilitate understanding of the invention. For this reason, the present invention is not necessarily limited to the positions, sizes, shapes, and ranges disclosed in the drawings.

[0015] When there are multiple components with the same or similar function, they may be described using the same symbol but with different subscripts. Furthermore, if it is not necessary to distinguish between these multiple components, the subscript may be omitted. Duplication of explanations for components with the same or similar function may be omitted.

[0016] In embodiments, processing performed by executing a program may be described. Here, the computer executes the program using a processor (e.g., a central processing unit (CPU), a graphics processing unit (GPU)) and performs processing defined by the program using memory resources (e.g., memory) and interface devices (e.g., communication ports). Therefore, the main entity performing the processing by executing the program may be the processor. Similarly, the main entity performing the processing by executing the program may be a controller, device, system, computer, or node having a processor. The main entity performing the processing by executing the program may be an arithmetic unit, and may include a dedicated circuit that performs a specific processing. Here, a dedicated circuit is, for example, an FPGA (Field Programmable Gate Array), an ASIC (Application Specific Integrated Circuit), or a CPLD (Complex Programmable Logic Device).

[0017] The program may be installed on a computer from a program source. The program source may be, for example, a program distribution server or a computer-readable storage medium. If the program source is a program distribution server, the program distribution server includes a processor and storage resources for storing the program to be distributed, and the processor of the program distribution server may distribute the program to other computers. In addition, in some embodiments, two or more programs may be implemented as a single program, or one program may be implemented as two or more programs.

[0018] [Culture Apparatus] Figure 1 is a schematic diagram illustrating one example of the configuration of the culture apparatus 1 according to this embodiment. As shown in Figure 1, the culture apparatus 1 comprises a culture tank 2, an oxygen supply device 3, an air supply device 4, a liquid level aeration device 5, a first sensor unit 6, and a control device 7.

[0019] The culture vessel 2 is used to culture cells in the culture medium CM and is a sealed container in which external access is restricted, at least during cell culture. The culture vessel 2 is equipped with various devices and sensors, such as an oxygen supply device 3 used for cell culture. The culture vessel 2 can be made of any metal material or single-use plastic material, but is not limited to these.

[0020] The oxygen supply device 3 supplies oxygen gas into the liquid phase LP, which is formed by supplying culture medium CM inside the culture tank 2. To supply oxygen gas into the liquid phase LP, the oxygen supply device 3 is connected to an oxygen supply sparger 31 located at the bottom of the culture tank 2, and supplies oxygen gas into the liquid phase LP from the sparger 31. The oxygen gas supply source for the oxygen supply device 3 can be an oxygen cylinder filled with pure oxygen. The oxygen supply device 3 has a control valve 33 in the piping 32 leading to the culture tank 2. This control valve 33 is connected to a control device 7 by wire or wireless, and controls the amount of oxygen gas supplied based on a control signal from the control device 7.

[0021] The air supply device 4 supplies air to the liquid phase LP to remove carbon dioxide from the culture medium CM. The air supply device 4 is connected to an air supply sparger 41 located at the bottom of the culture tank 2 in order to remove carbon dioxide from the culture medium CM in the liquid phase LP, and supplies air to the liquid phase LP from the sparger 41. An air compressor or the like can be used as the air supply source for the air supply device 4. The air supply device 4 has a control valve 43 in the piping 42 from the air supply source to the culture tank 2. This control valve 43 is connected to the control device 7 by wire or wireless, and controls the amount of air that flows through based on a control signal from the control device 7.

[0022] The liquid level aeration device 5 is connected to the gas phase GP formed above the liquid phase LP inside the culture tank 2, and controls the aeration of air in the gas phase GP. The liquid level aeration device 5 has an air inlet 51 in the upper part of the culture tank 2 to supply air to the gas phase GP. The air inlet 51 is connected to an air supply source by piping 52. The air supply source in the liquid level aeration device 5 can be an air compressor, compressed air gas cylinder, nitrogen gas cylinder, etc. The liquid level aeration device 5 has a control valve 53 in the piping 52 from the air supply source to the air inlet 51. This control valve 53 is connected to a control device 7 by wire or wireless, and controls the amount of air aeration based on a control signal from the control device 7.

[0023] Furthermore, a pipe 55 connected to a CO2 gas supply device 54 may be connected to the piping 52 leading to the air introduction section 51 of the liquid surface aeration device 5. In this case, the connection point of the pipe 55 connected to the CO2 gas supply device 54 can be between the liquid surface aeration device 5 and the control valve 53 of the liquid surface aeration device 5. The CO2 gas supply device 54 has a control valve 56 in its piping 55. This control valve 56 is connected to the control device 7 by wire or wireless, and controls the amount of CO2 gas supplied (aeration amount) from the CO2 gas supply device 54 based on a control signal from the control device 7. In this way, it is possible to supply CO2 gas to the gas phase GP of the culture tank 2 as needed.

[0024] Furthermore, the culture tank 2 has an air discharge section 57 at its upper part for discharging air from inside the culture tank 2. The air discharge section 57 is connected to a pipe 59 equipped with a control valve 58. This control valve 58 is connected to a control device 7 by wire or wireless, and based on control signals from the control device 7, it is opened and closed and the valve opening is adjusted to release air from inside the culture tank 2 to the outside or adjust the pressure inside the culture tank 2. In Figure 1, a pressurizing / depressurizing device 60 is shown installed downstream of this control valve 58. The pressurizing / depressurizing device 60 can be an exhaust pump or the like. The pressurizing / depressurizing device 60 is also connected to the control device 7 by wire or wireless, and the exhaust output can be adjusted based on control signals from the control device 7. The pressurizing / depressurizing device 60 may also be configured to control the pressure inside the culture tank 2, thereby pressurizing or depressurizing the inside of the culture tank 2. This allows the culture apparatus 1 to more actively and accurately control the exhaust of air from inside the culture tank 2 and the adjustment of the pressure inside the culture tank 2.

[0025] Furthermore, the culture tank 2 may be equipped with a culture medium extraction device 10. The culture medium extraction device 10 is also connected to the control device 7 by wire or wireless connection, and can extract the culture medium CM from the culture tank 2 based on a control signal from the control device 7. A pump or the like can be used as the culture medium extraction device 10.

[0026] The first sensor unit 6 is provided so as to be in contact with at least the liquid phase LP and detects an indicator related to dissolved carbon dioxide in the liquid phase LP (for example, dissolved carbon dioxide concentration). The first sensor unit 6 is connected to the control device 7 by wire or wireless and transmits the detected indicator related to dissolved carbon dioxide to the control device 7. The first sensor unit 6 can be a commercially available dissolved carbon dioxide meter.

[0027] The control device 7 controls the oxygen supply device 3, the air supply device 4, and the liquid level aeration device 5. The control device 7 also performs various controls necessary for cell culture. For example, the control device 7 can control the rotational speed of the stirring blades 11a, inclined stirring blades 11b, and stirring blades 11c, which are located inside the culture tank 2. The rotational speed of the stirring blades 11a, inclined stirring blades 11b, and stirring blades 11c is controlled by a motor 11 and a rotation control device 12, which are connected to the control device 7 by wire or wireless. Furthermore, for example, the control device 7 can control temperature control devices (not shown), such as heaters and coolers, located outside the culture tank 2. Such a control device 7 is implemented using a central processing unit (CPU), storage media such as hard disk drives or solid-state drives, random access memory (RAM), and read-only memory (ROM) built into the culture apparatus 1, but it may also be implemented using a computer connected to the culture apparatus 1 by wire or wireless. This computer also includes a CPU, storage media, RAM, ROM, etc.

[0028] In addition to the above, the culture tank 2 may also have a baffle 13 for generating turbulence in the agitated culture medium CM to improve agitation efficiency, a liquid volume meter 14 for measuring the liquid volume of the culture medium CM, and a live cell density meter 15 for measuring the density of live cells. The culture tank 2 may also be equipped with a culture medium tank 16 for storing the liquid culture medium supplied to the culture tank 2. The culture medium tank 16 and the culture tank 2 are connected by a pipe 161. A culture medium supply device and liquid level control device 17 is provided on the pipe 161, and by operating this device, liquid culture medium can be supplied from the culture medium tank 16 to the culture tank 2 (and the height of the liquid phase LP in the culture tank 2 can be controlled). Any pump can be used for the culture medium supply device and liquid level control device 17. Furthermore, the culture tank 2 may also have a thermometer (not shown) for measuring the temperature of the culture medium CM and a pH meter (not shown) for measuring the pH of the culture medium CM. By providing these configurations and measuring instruments, the culture apparatus 1 can perform cell culture more appropriately.

[0029] (Operation of the control device 7) In the culture apparatus 1 equipped with the above-described components, the control device 7 operates as follows: The control device 7 performs a first operation to improve the oxygen supply performance to the liquid phase LP, and a second operation to improve the carbon dioxide removal performance from the liquid phase LP based on the information from the first sensor unit 6. Of these, the first operation can be performed by increasing the amount of oxygen gas supplied from the oxygen supply device 3. The second operation can be performed by increasing the amount of air supplied by the air supply device 4 and increasing the amount of air supplied by the liquid surface air supply device 5, at least one of these.

[0030] Then, when the control device 7 performs the second operation, it determines the amount of air supplied by the air supply device 4 and the amount of air supplied by the liquid level air supply device 5 based on at least the information from the first sensor unit 6. In this case, the control device 7 may decide to perform, for example, the following controls <control 1> to <control 4>.

[0031] <Control 1> The control device 7 can decide to increase the amount of air supplied by the air supply device 4 until the partial pressure of CO2 in the bubbles rising in the liquid phase LP and the DCO2 concentration in the liquid phase LP reach equilibrium, but not increase the amount of air supplied by the liquid surface ventilator 5 or set it to zero. In other words, when the control device 7 performs the second operation, it can decide to increase only one of the air supply device 4 and the liquid surface ventilator 5 (only the air supply device 4). The embodiment of <Control 1> allows for a reliable and rapid reduction of the DCO2 concentration in the liquid phase by preferentially using the air supply device 4, which has superior CO2 removal performance.

[0032] <Control 2> After the partial pressure of CO2 in the bubbles rising in the liquid phase LP and the DCO2 concentration in the liquid phase LP reach equilibrium, the control device 7 can decide to maintain the amount of air supplied by the air supply device 4 as is, and in addition, increase the amount of air supplied by the liquid surface aeration device 5. In other words, when the control device 7 performs the second operation, it can decide to increase both the air supply device 4 and the liquid surface aeration device 5. The <Control 2> mode can achieve the highest CO2 removal performance for the liquid phase LP. Therefore, the <Control 2> mode is suitable when the cell density is high and the DCO2 concentration in the liquid phase LP approaches the upper limit that cells can tolerate.

[0033] <Control 3> After the CO2 partial pressure in the bubbles rising in the liquid phase LP and the DCO2 concentration in the liquid phase LP reach equilibrium, the control device 7 can decide to reduce and maintain the amount of air supplied by the air supply device 4, while also increasing the amount of air supplied by the liquid surface aeration device 5. In other words, when the control device 7 performs the second operation, it can decide to increase both the air supply device 4 and the liquid surface aeration device 5. The embodiment of <Control 3> can reduce the DCO2 concentration in the liquid phase LP while reducing the bubbles supplied by the air supply device 4, thereby further preventing cell death and further reducing damage to cells.

[0034] <Control 4> The control device 7 can decide to stop the aeration rate by the air supply device 4 and increase the aeration rate by the liquid surface aeration device 5 after the CO2 partial pressure in the bubbles rising in the liquid phase LP and the DCO2 concentration in the liquid phase LP have reached equilibrium. In other words, when the control device 7 performs the second operation, it can decide to increase only one of the air supply device 4 and the liquid surface aeration device 5 (only the liquid surface aeration device 5). Depending on the size of the culture tank 2, the amount of culture medium CM, the state of the cultured cells, etc., it may be appropriate to adopt this <Control 4> configuration, in which case it is preferable to adopt the <Control 4> configuration.

[0035] The culture apparatus 1 according to the present embodiment has the above-described configuration, and when the control device 7 executes the above-described second operation, the control device 7 determines the aeration amount by the air supply device 4 and the aeration amount by the liquid surface aeration device 5 based at least on the information from the first sensor unit 6. Therefore, the culture apparatus 1 can achieve both improvement of the oxygen supply performance and the carbon dioxide removal performance in the liquid phase LP in the culture tank 2.

[0036] Next, description will be given on that the culture apparatus 1 can achieve both improvement of the oxygen supply performance and the carbon dioxide removal performance in the liquid phase LP in the culture tank 2.

[0037] (Regarding improvement of oxygen supply performance) In the culture apparatus 1, by increasing the oxygen concentration (up to 100%) of the submerged aeration in the oxygen supply device 3 (note that "%" in the present specification means "% by volume"), the oxygen concentration difference between gas and liquid (ΔC Ｏ２ ) can be set to a large value (= 9·DO). Combined with the increased solubility due to the reduction of bubble diameter, the oxygen supply performance can be improved relatively easily. OTR ｂ = kLa Ｏ２ · ΔC Ｏ２ Note that OTR ｂ : Oxygen permeability related to bubbles kLa Ｏ２ : Overall mass transfer capacity coefficient (in this case, the oxygen transfer capacity coefficient) ΔC Ｏ２ = DO ｓａｔ - DO = (Saturated concentration with pure oxygen) - (e.g., 50% of the air saturated concentration) = 5 × (Air saturated concentration) - 0.5 × (Air saturated concentration) = 9 · DO

[0038] (Regarding improvement of carbon dioxide removal performance) The submerged aeration in the air supply device 4 for CO2 degassing uses air that contains (substantially) no CO2 at the beginning of aeration (CO2 partial pressure = 0.04 ≈ 0), and the saturated solubility DCO2 of the liquid phase LP corresponding to the partial pressure of 0 ｓａｔ = 0. On the other hand, CO2 is dissolved in the liquid phase LP due to cell respiration. Since the CO2 in the liquid phase LP is in a supersaturated state relative to the CO2 partial pressure in the bubbles, CO2 moves from the liquid phase LP to the bubbles. The CO2 concentration (DCO2) in the liquid phase LP cannot be set to about 20% or higher, which is the level that cells can tolerate, and ΔC ＣＯ２It is difficult to take a large value. Therefore, the CO2 concentration difference between gas and liquid (-ΔC) ＣＯ２ It is not possible to take a large amount of (DCO2). CER ｂ =kLa ＣＯ２ ΔC ＣＯ２ Furthermore, CER ｂ CO2 generation rate related to bubbles (kLa) ＣＯ２ : Overall mass transfer capacity coefficient (in this case, carbon dioxide transfer capacity coefficient) - ΔC ＣＯ２ =DCO2-DCO2 ｓａｔ，ｂ =DCO2-0 =DCO2

[0039] Therefore, as the bubbles rise through the liquid phase LP, the partial pressure of CO2 in the bubbles and DCO2 reach equilibrium, and thereafter DCO2 does not transfer to the bubbles, so the CER does not rise. For this reason, the DCO2 removal performance cannot be solved by improving the kLa of the gas permeation through the liquid, and ΔC ＣＯ２ To maintain this, there was a challenge in keeping the partial pressure of CO2 in the bubbles as low as possible. This was due to the kLa of the ventilator velocity. ＣＯ２ This means that instead of evaluating the CO2 volume directly, it is important to evaluate the number of moles of air, oxygen, etc. that can be blown in per minute using VVM (gas volume per liquid volume per minute), which represents how many times the volume of liquid is blown in with air.

[0040] (How the idea for the present invention was arrived at) Based on the behavior of oxygen transition from the liquid phase LP to the air bubbles in the liquid due to air permeation in liquid and air permeation at the liquid surface, and the behavior of DCO2 transitioning from the liquid phase LP to the air bubbles in the liquid and to the gas phase at the liquid surface (gas phase GP), the present invention will be explained, while showing the content and problems of the prior art.

[0041] (Oxygen Transfer Behavior) The oxygen transfer behavior will be explained. The oxygen transfer behavior is as follows: Volume of liquid in the tank: V (m ３ ), liquid depth: V / S (m), stirring power per unit volume of liquid: (N ３ ・Di ５ / V), liquid level air permeability q (m ３(m / s), velocity of oxygen supply gas in a hollow tower: Us (m / s), difference between the DO saturation value corresponding to the initial oxygen concentration in the oxygen supply gas bubble and the actual DO: Δp(O2) ｂ，ｉｎ (mM), the difference between the DO saturation value corresponding to the oxygen concentration when the oxygen supply gas bubble reaches the liquid surface and the actual DO: Δp(O2) ｂ，ｏｕｔ (mM), difference between the DO saturation value at the liquid surface and the actual DO: Δp(O2) ｓ It depends on (mM). Here, stirring speed: N (1 / s), impeller diameter: Di (m), and liquid surface area in the tank: S (m ２ ) Note that there are two types of kLa: the value between the gas bubbles in the liquid and the liquid phase LP, and the value between the gas phase GP above the liquid surface and the liquid phase LP. The former (kLa ｂ ) depends on the stirring power per unit volume of liquid and the gas velocity in the empty column, the latter (kLa ｓ ) depends on the volume of liquid in the tank and the stirring power per unit volume of liquid.

[0042] In oxygen supply, the Henry constant H for oxygen relative to water. Ｏ２ It is large and difficult to dissolve. Ｏ２ = H Ｏ２ ・(18 / 1000)・DO ｓａｔ Note H Ｏ２ = H0・e Ｘ X = A * (1 - 298 / T) + B * ln (T / 298) + C * (T / 298 - 1) H0 = 4420 A = 29.339 B = -24.453 C = 0 T: Absolute temperature (K) Therefore, for example, at T = 313K (40°C), which is close to the culture temperature, H Ｏ２ This is approximately 5400 MPa / M (Molar (mol / L)).

[0043] Therefore, the oxygen supply capacity (OTR; Oxygen Transfer Rate) is determined by the partial pressure of the bubbles p. Ｏ２，ｂ and the transition rate from gas phase GP to liquid phase LP (kLa Ｏ２，ｂ kLa Ｏ２，ｓ The size of the ) is important. For the DO of the liquid phase LP, for example, we assume it to be about 50% of the concentration when the air is saturated. OTR = kLa Ｏ２，ｂ ・(ΔC) Ｏ２，ｂ，ｉｎ -ΔC Ｏ２，ｂ，ｏｕｔ ) / ln(ΔC Ｏ２，ｂ，ｉｎ / ΔCＯ２，ｂ，ｏｕｔ ) + kLa Ｏ２，ｓ ΔC Ｏ２，ｓ Note that ΔC Ｏ２，ｂ，ｏｕｔ =ΔC Ｏ２，ｂ，ｉｎ - exp(-kLa Ｏ２，ｂ ・V / S / Us) kLa Ｏ２，ｂ ∝ (N ３ ・Di ５ / V) Ａ１ Us Ａ２ kLa Ｏ２，ｓ ∝ (N ３ ・Di ５ / V) Ａ３ ・V Ａ４ A1: For example, this is a value that can be determined experimentally, using as a reference the value "0.4" described in Reference 1 below or the value "0.4 to 0.7" described in Reference 2 below. A2: For example, this is a value that can be determined experimentally, using as a reference the value "0.5" described in Reference 1 below or the value "0.4 to 0.6" described in Reference 2 below. A3: For example, this is a value that can be determined experimentally, using as a reference the value "0.8 to 1.0" described in Reference 3 below. A4: For example, this is a value that can be determined experimentally, using as a reference the value "around -0.5" described in Reference 4 below. Reference 1: PH Calderbank and MB Moo-Young, Chem. Engng Sci.16: 39-54, 1961) Reference 2: J.-C. Gabelle et al. The Canadian Journal of Chemical Engineering, 89,5, 1139-1153, 2011 Reference 3: Imanaka, Kobe Steel Pantech Technical Report, Vol. 71, No. 2, March 1998, pp. 37-46 Reference 4: Mimura, J. Ferment. Technol., Vol. 48, No. 7, pp. 449-460, 1970 (Deciphered from Figure 6)

[0044] partial pressure of air bubbles p Ｏ２，ｂ , partial pressure p at the liquid level Ｏ２，ｓ Regarding this, it is as follows: ΔC Ｏ２，ｂ，ｉｎ =DO ｓａｔ，ｂ -DO = (p Ｏ２，ｂ / H Ｏ２ -0.5 p Ｏ２，ｓ / HＯ２ )・(1000 / 18) ΔC Ｏ２，ｓ =DO ｓａｔ，ｓ -DO =(p Ｏ２，ｓ / H Ｏ２ -0.5・p Ｏ２，ｓ / H Ｏ２ )・(1000 / 18) =0.5・p Ｏ２，ｓ / H Ｏ２ ・(1000 / 18) Here, when bubbles are supplied as 0.1 MPa pure oxygen, the partial pressure is about 5 times that of air (p Ｏ２，ｂ =5・p Ｏ２，ｓ ), so ΔC Ｏ２，ｂ，ｉｎ =4.5・p Ｏ２，ｓ / H Ｏ２ ・(1000 / 18) =4.5・0.1 / 5400・(1000 / 18) =0.46 mM.

[0045] ΔC Ｏ２，ｂ，ｏｕｔ (the oxygen concentration difference between gas and liquid corresponding to the oxygen concentration in the oxygen supply gas bubbles when reaching the liquid surface) does not significantly decrease from ΔC Ｏ２，ｂ，ｉｎ (the oxygen concentration difference between gas and liquid corresponding to the initial oxygen concentration in the oxygen supply gas bubbles), and a sufficient concentration difference for oxygen transfer is maintained even at the stage where the bubbles reach the liquid surface. Regarding kLa Ｏ２，ｂ , aeration with small-diameter bubbles increases the gas-liquid specific surface area (increases the a of kLa). Note that kL is the liquid-phase side mass transfer coefficient (m / s), and a is the gas-liquid interface area per unit dispersion phase volume (m ２ / m ３ ). In addition, kLa increases with the superficial velocity Us related to aeration (=volume flow rate / liquid surface cross-sectional area, m / s).

[0046] (Conventional ideas related to improving oxygen supply performance) Conventionally, to improve oxygen supply performance, as in the aforementioned Non-Patent Document 1 and Document 5 (Japanese Unexamined Patent Publication No. 1-86867), the oxygen aeration amount increases with scale-up, and pure oxygen gas (p Ｏ２The aeration of small bubbles was carried out at a high empty-stack velocity. When small-diameter bubbles are aerated at a high empty-stack velocity, kLa increases. In addition, because the O2 concentration in the bubbles can be increased by aerating with pure oxygen gas, a large concentration difference between the O2 in the bubbles and the DO in the liquid can be maintained until the final stage when the bubbles are released from the liquid surface. However, when increasing the cell density and the viscosity of the culture medium CM to improve productivity, further improvement of oxygen supply was necessary.

[0047] (Challenges of conventional ideas regarding improving oxygen supply performance) When the partial pressure of various gases is increased, equilibrium is reached between the gas phase GP and the liquid phase LP, after which the saturation solubility of each gas component in the liquid phase LP increases. Air contains oxygen, and for oxygen supply performance, increasing the total pressure also increases the partial pressure of O2, so the saturation value of DO increases slightly, and the amount of oxygen dissolved from the bubbles increases slightly. For example, by pressurizing the culture tank 2 with the pressurizing / depressurizing device 60 and increasing the total pressure, the oxygen supply performance to the liquid phase LP is improved. However, if the contribution of oxygen gas aeration in the liquid is large, the above effect will be small. In that case, from the viewpoint of CO2 degassing and removal capacity, since the CO2 concentration in the air is extremely low, CO2 generation in the liquid phase LP and its release from there become the source (because Henry's law shows a proportional relationship between partial pressure and saturation solubility (mole fraction)). Therefore, even if the total pressure is increased by performing liquid surface aeration into the gas phase GP and discharging CO2 from the gas phase GP to the outside of the system, the partial pressure of CO2 does not increase and there is no effect on DCO2. However, when discharging gas from the pressurized culture tank 2 to the outside of the system, it is necessary to do so via a vacuum pump (pressure / depressurization device 60), and it is necessary to consider the applicability and the amount of increase in total pressure, taking into account the upper limit of the flow rate that can be performed per vacuum pump and the constraints on the number of vacuum pumps that can be installed in the culture tank 2.

[0048] From the above, depending on the apparatus configuration, operating conditions, and operating status of the culture tank 2, as another example of the first operation for improving the oxygen supply performance, an operation of increasing the internal tank pressure by the pressurization and depressurization device 60 may be performed. Then, when performing the operation of increasing the internal tank pressure as the first operation, control may be performed such that the operation of increasing the aeration rate by the liquid surface aeration device 5 is executed as the second operation. As a result, the oxygen supply performance may be improved and the decrease in CO₂ removal capability may be compensated, and in that case, this contributes to achieving both the improvement of the oxygen supply performance in the liquid phase LP (culture medium CM) in the culture tank 2 and the improvement of the carbon dioxide removal performance.

[0049] (CO₂ Transition Behavior) The CO₂ transition behavior will be described. The CO₂ transition behavior depends on the liquid volume in the tank: V (m ３ ), liquid depth: V / S (m), stirring power per unit liquid volume: (N ３ ·Di ５ / V), liquid surface aeration rate q (m ３ / s), superficial liquid velocity of the degassing gas (usually air): Uc (m / s), the difference between the DCO₂ saturation value corresponding to the initial CO₂ concentration in the degassing gas bubbles and the actual DCO₂: Δp(CO ２ ) ｂ，ｉｎ (mM), the difference between the DCO₂ saturation value at the liquid surface and the actual DCO₂: Δp(CO ２ ) ｓ (mM), the difference between the DCO₂ saturation value at the time of reaching the liquid surface in the CO₂ supply gas bubbles and the actual DCO₂: Δp(CO ２ ) ｂ，ｏｕｔ (mM). Here, stirring speed: N (1 / s), stirring blade diameter: Di (m), liquid surface area in the tank: S (m ２ ), the aeration rate in liquid of the CO₂ degassing air: Qc (m ３ / s = 4π·(d / 2) ３ ·n / 3), bubble diameter: d (m), number of bubbles: n ( / s).

[0050] In DCO₂ removal, the Henry's constant H of CO₂ in water ２ is small, and CO₂ is easily dissolved. p ＣＯ２ ＣＯ２ = H ＣＯ２ ·(18 / 1000)·DCO₂ ｓａｔ Note that H​ＣＯ２ = H0・e Ｘ X = A * (1 - 298 / T) + B * ln (T / 298) + C * (T / 298 - 1) H0 = 165.8 A = 29.319 B = -21.669 C = 0.3287 T: Absolute temperature (K) Therefore, for example, at T = 313K (40℃), which is close to the culture temperature, H ＣＯ２ It is approximately 240 MPa / M (Molar).

[0051] The equation relating to the transfer behavior of CO2 between gas and liquid is the same as for oxygen, as follows: CER = kLa ＣＯ２，ｂ ・(ΔC) ＣＯ２，ｂ，ｉｎ -ΔC ＣＯ２，ｂ，ｏｕｔ ) / ln(ΔC ＣＯ２，ｂ，ｉｎ / ΔC ＣＯ２，ｂ，ｏｕｔ ) + kLa ＣＯ２，ｓ ΔC ＣＯ２，ｓ Note that ΔC ＣＯ２，ｂ，ｏｕｔ =ΔC ＣＯ２，ｂ，ｉｎ - exp(-kLa ＣＯ２，ｂ ・V / S / Uc) kLa ＣＯ２，ｂ ∝ (N ３ ・Di ５ / V) Ａ５ ・Uc Ａ６ kLa ＣＯ２，ｓ ∝ (N ３ ・Di ５ / V) Ａ７ ・V Ａ８ A5: For example, it is a value that can be determined experimentally, using as a reference the value "0.4" described in the above-mentioned document 1 and the value "0.4 to 0.7" described in the above-mentioned document 2. A6: For example, it is a value that can be determined experimentally, using as a reference the value "0.5" described in the above-mentioned document 1 and the value "0.4 to 0.6" described in the above-mentioned document 2. A7: For example, it is a value that can be determined experimentally, using as a reference the value "0.8 to 1.0" described in the above-mentioned document 3. A8: For example, it is a value that can be determined experimentally, using as a reference the value "around -0.5" described in the above-mentioned document 4.

[0052] The vent bubbles for CO2 degassing (vent bubbles from the air supply device 4) and the liquid surface venting gas are air that does not contain CO2. The initial CO2 partial pressure p in the bubbles. ＣＯ２，ｂ and the partial pressure of CO2 in the liquid surface aeration gas p ＣＯ２，ｓSince both are 0, the saturation solubility for them is DCO2 ｓａｔ = p ＣＯ２，ｂ / H ＣＯ２ ・(1000 / 18) =p ＣＯ２，ｓ / H ＣＯ２ • (1000 / 18) = 0. On the other hand, in the culture medium CM, CO2 dissolves into the water due to cellular respiration, creating a supersaturated state with a dissolved CO2 concentration (DCO2). Therefore, the absolute value of the concentration difference is as follows, with the maximum value DCO2 immediately after aeration with bubbles or immediately after supplying liquid surface aeration gas. -ΔC ＣＯ２，ｂ，ｉｎ =DCO2-DCO2 ｓａｔ，ｂ =DCO2-0 =DCO2-ΔC ＣＯ２，ｓ =DCO2-DCO2 ｓａｔ，ｓ =DCO2-0 =DCO2

[0053] Furthermore, the DCO2 during culture is approximately 1 mM, assuming a CO2 gas concentration similar to that in vivo (5%). DCO2 = p ＣＯ２ / H ＣＯ２ ・(1000 / 18) =0.05・0.1 / 240・(1000 / 18) =1.17mM

[0054] However, regarding the partial pressure of the vented bubbles, H ＣＯ２ The CO2 concentration cannot exceed (18 / 1000) DCO2 (5% in this example, 0.05 MPa), and as the bubbles rise through the liquid, the partial pressure of CO2 in the bubbles and DCO2 reach equilibrium. After that, no more dissolved CO2 transfers to the bubbles, and the CER does not increase. This is in contrast to the improvement achieved by aeration with 100% pure oxygen supply, where CO2 removal only increases the CO2 concentration in the bubbles from about 5% to at most about 20% (a case where cell survival is difficult). This presents a challenge for improving the CER.

[0055] (Conventional ideas regarding improving CO2 removal performance) Considering that the partial pressure of CO2 in the bubbles of liquid aeration and the DCO2 in the liquid phase LP reach equilibrium, it is necessary to experimentally verify the volume of liquid aeration (VVM) corresponding to the amount of CO2 that needs to be removed per unit volume of liquid, and to maintain it during cultivation. Here, conventional ideas regarding improving CO2 removal performance include, for example, those described in Non-Patent Document 1 and Non-Patent Document 2, as well as References 6 and 7. Non-Patent Documents 1 and 2 contain the same information as described in the background art section. Reference 6 (by Satoshi Murakami, Susumu Harada, and Shuichi Yamamoto, "Scale-up Methods for Culture Vessels," Journal of the Japan Society for Food Engineering, Vol. 2, No. 2, pp. 53-61, June, 2001) states that "the ratio of height H to diameter D of a culture vessel (aspect ratio) is usually 1.6 to 1.8," and that "it is known that as the aspect ratio increases, the aeration velocity increases even if the aeration rate per unit volume of culture medium remains constant, resulting in increased gas hold-up." Furthermore, Reference 7 (by Imanaka, Kobe Steel Pantech Technical Report, Vol. 412, No. 2, pp. 37-46) states that "elongated culture vessels may have excessive dissolved carbon dioxide due to insufficient gas exchange at the liquid surface or poor top-to-bottom mixing. Therefore, an aspect ratio of 1 to 1.2 is preferable for cell culture vessels."

[0056] If there are two culture tanks 2 with the same volume and aeration rate, Uc = Qc / S ∝ kLa and VVM = Qc / V, then if you select the larger Uc, kLa will be Ｏ２，ｂ This increases, which is advantageous for oxygen supply. On the other hand, since VVM is equivalent if V and Qc are the same, the CO2 removal performance of liquid aeration will also be the same regardless of the tank shape. However, in this case, the amount of gas exchange at the liquid surface increases, and kLa ＣＯ２，ｂ To prevent an increase in the amount of gas, additional measures are needed, such as the need for large-diameter air bubbles to be aerated (i.e., reducing the 'a' in kLa), separate from the aeration for oxygen supply. The latter is important in scaling up culture tank 2 to avoid the CO2 partial pressure and DCO2 reaching equilibrium in the bubbles used for aeration in the liquid. Furthermore, large-diameter bubbles have the advantage of relatively little foam layer formation and less cell damage due to the pressure difference inside and outside the bubble caused by surface tension.

[0057] (Challenges of conventional ideas regarding CO2 removal performance) Therefore, if Uc cannot be increased, the only thing that can be done in reality is to keep the airflow rate Qc constant (d ３ Under the condition that n is constant, the only thing that needs to be selected is the bubble diameter d and the number of bubbles n. Also, both the empty column velocity in liquid and VVM have upper limits because if they are too high, gas-liquid separation will occur and increased shear force on cells will cause them to die. In particular, in animal cell culture such as CHO cells, excessively high VVM values ​​are undesirable, and it is especially desirable for them to be less than 0.5 VVM.

[0058] (How the idea of ​​the present invention was arrived at) (Increasing the amount of air permeation at the liquid surface in the gas phase GP formed at the top of the culture tank 2) From the viewpoint of oxygen supply performance, since the permeation of pure oxygen gas into the liquid is more effective than the permeation of air at the liquid surface, the effect of increasing the amount of air permeation at the liquid surface is small. On the other hand, from the viewpoint of CO2 removal performance, the difference in concentration between dissolved CO2, aeration bubbles, and CO2 in the liquid surface gas phase is ΔC ＣＯ２，ｂ，ｉｎ =ΔC ＣＯ２，ｓ = -1 mM, and ΔC ＣＯ２，ｂ，ｏｕｔ It gradually decreases from a large negative value to 0 (ΔC ＣＯ２，ｂ，ｏｕｔ →-0) Therefore, Abs{(ΔC ＣＯ２，ｂ，ｉｎ -ΔC ＣＯ２，ｂ，ｏｕｔ ) / ln(ΔC ＣＯ２，ｂ，ｉｎ / ΔC ＣＯ２，ｂ，ｏｕｔ )} << Abs(ΔC ＣＯ２，ｓ ) And when the liquid level permeability is large, CER is kLa ＣＯ２，ｓ Δp(CO2) ｓ The contribution of [this factor] becomes dominant. From this, it was considered desirable that the liquid surface airflow rate be large in order to reduce the CO2 concentration in the gas phase GP from the viewpoint of CO2 removal performance.

[0059] Here, the volume of the liquid-level gas phase in the culture tank 2 is V'(m ３ ), pressure: P (MPa), liquid level airflow rate: q (m ３ / s), CO2 concentration in gas phase GP: m (mol / m ３If the CO2 release rate from the liquid phase LP to the gas phase GP is M (mol / s), the gas constant is R (J / (mol・K)), and the absolute temperature is T (K), then if q is large enough that the partial pressure of CO2 in the space above the liquid surface (gas phase GP) and the DCO2 in the liquid phase LP do not reach equilibrium, then from V'・dm / dt = M - q・m = 0, we get m = M / q. → Partial pressure of CO2: p ＣＯ２ =P・m / (P / RT) =(8.31・T)・(M / q) H ＣＯ２ : Assuming the CO2 Henry constant (MPa), according to Henry's Law, when T = 313 (K), H ＣＯ２ ・(DCO2) / (10 ３ / 18) = p ＣＯ２ DCO2=8.31×10 ３ / 18・T・M / (H ＣＯ２ q) = 610 M / q. Therefore, in this case, it can be seen that increasing the liquid surface aeration rate (∝ VVM) reduces the DCO2 in the liquid phase LP. In other words, increasing the liquid surface aeration rate (∝ VVM) suppresses CO2 saturation in the gas phase GP of culture tank 2, thus improving CO2 removal efficiency. Note that the amount of liquid aeration for CO2 degassing is q. ｂ As such, the required amount is: 610 * (0.2 / 0.0224) * (q ｂ / q) ≤ 1.0 × 10 －３ Therefore, q ≥ 5.45 * q ｂ This shows that surface aeration is more efficient than submerged aeration in reducing DCO2 in the liquid phase LP.

[0060] Based on the research and studies described above, we concluded that the carbon dioxide removal performance from the liquid phase LP can be improved by actively using not only submerged aeration but also surface aeration when performing the second operation. Specifically, we concluded that the culture apparatus 1 should be controlled by the control device 7 to determine the amount of aeration by the air supply device 4 and the amount of aeration by the surface aeration device 5 when performing the second operation. More specifically, we concluded that it would be beneficial to perform the controls described above, such as Control 1 to Control 4.

[0061] Figure 2 is a table summarizing the relationships between each parameter in the culture apparatus 1. As indicated in the direction of operation in Figure 2, separate spargers are provided for oxygen supply and CO2 removal air. Therefore, the control device 7 can independently control oxygen supply and CO2 removal. For oxygen supply, it is desirable to make the bubble diameter small, and for CO2 removal air, it is desirable to make it large. The bubble diameter cannot be operated in either case. For oxygen supply, it is desirable to make the empty column velocity large, and for CO2 removal air, it is desirable to make it small. The empty column velocity can be operated in either case. For oxygen supply, vvm can be made small in the case of pure oxygen, and it is desirable to do so. On the other hand, for CO2 removal air, it is desirable to make vvm large. vvm can be operated in either case. For oxygen supply, it is desirable to make it small, and it is desirable to make it large. The liquid surface area cannot be operated in either case. It is preferable to design the liquid surface area to a value that balances these. Since the liquid level ventilation rate is not intended to supply oxygen, there is no particular preference for oxygen supply, but it is desirable to increase the rate for CO2 removal air. The liquid level ventilation rate can be used to operate the CO2 removal air. The liquid level ventilation rate can be controlled separately from the amount of CO2 removal air supplied from the air supply device 4, allowing for independent control of CO2 removal.

[0062] (Preferred Embodiments in Culture Apparatus 1) Next, preferred embodiments of the culture apparatus 1, (a) to (g2), will be described. (a) When the control device 7 performs the second operation, it is preferable to decide whether to perform either an operation to increase the amount of air supplied by the air supply device 4 or an operation to increase the amount of air supplied by the liquid level aeration device 5, or both, based on the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4. In this way, since the control device 7 controls based on the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4, it is possible to avoid situations in which the pressure in the culture tank 2 becomes excessively high or these amounts of air supply become insufficient.

[0063] (b) Preferably, the control device 7 decides to increase the amount of air supplied by the liquid level aeration device 5 when the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4 exceeds a threshold. When the sum exceeds the threshold, it is possible to consider that the CO2 removed from the liquid phase LP by the oxygen supply device 3 and the air supply device 4 is contained in the gas phase GP at a predetermined rate. In response to such a state, the control device 7 can reduce the concentration of CO2 contained in the gas phase GP by causing the culture apparatus 1 to perform an operation to increase the amount of air supplied by the liquid level aeration device 5. As the concentration of CO2 in the gas phase GP of the culture tank 2 is reduced, the removal of CO2 contained in the liquid phase LP progresses, and the DCO2 of the liquid phase LP can be reduced. In this embodiment, the threshold can be appropriately set according to the culture tank 2 used. Note that the control in this embodiment can also be performed by providing a measuring device (not shown) in the culture tank 2 to measure the carbon dioxide concentration in the gas phase GP and transmitting the measured value to the control device 7. The method of measuring the carbon dioxide concentration in the gas phase GP of the culture tank 2 has the advantage of enabling more accurate control, while the method of managing the threshold for the amount of aeration described above has the advantage of simplifying the configuration and reducing costs because it does not require a measuring device to measure the carbon dioxide concentration in the gas phase GP.

[0064] (c) Preferably, the culture apparatus 1 is provided so as to be in contact with at least the liquid phase LP and further comprises a second sensor unit 18 that detects an indicator related to dissolved oxygen in the liquid phase LP (for example, dissolved oxygen concentration). This second sensor unit 18 is connected to the control device 7 by wire or wireless and transmits the detected indicator related to dissolved oxygen to the control device 7. A commercially available dissolved oxygen meter can be used for the second sensor unit 18. In this way, the control device 7 can accurately know the dissolved oxygen concentration of the liquid phase LP and thus control the amount of air supplied by the oxygen supply device 3 more accurately. In particular, when performing the second operation described above, the control device 7 can also control the increase or decrease of the amount of air supplied by the oxygen supply device 3 when increasing the amount of air supplied by the air supply device 4 or increasing the amount of air supplied by the liquid level air supply device 5.

[0065] (d) In the culture apparatus 1, it is preferable that the hole diameter of the sparger 31 connected to the oxygen supply device 3 is smaller than the hole diameter of the sparger 41 connected to the air supply device 4. This increases the solubility of oxygen. Therefore, the culture apparatus 1 can improve or at least maintain its oxygen supply performance.

[0066] (e) In addition, in the culture apparatus 1, it is preferable that the air supply device 4 controls the amount of air supplied by the air supply device 4 so that the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4 is less than 0.5 vvm relative to the amount of liquid inside the culture tank 2. In this way, foam layer formation is relatively small, and cell damage due to the pressure difference inside and outside the bubbles caused by surface tension can also be reduced.

[0067] (f) In the culture apparatus 1, it is preferable to increase the amount of air supplied by the liquid surface aeration device 5 and to increase the empty column velocity of oxygen supplied by the oxygen supply device 3. This improves the CO2 removal performance of the liquid surface aeration device 5 and compensates for the decrease in oxygen supply performance by the oxygen supply device 3.

[0068] In the operation of this embodiment, the pressure inside the culture tank 2 tends to increase. If the pressure inside the culture tank 2 increases too much, it may cause malfunctions in the various devices attached to the culture tank 2, damage to cells, inhibit the production of valuable substances secreted by cells, or adversely affect the quality of valuable substances. For this reason, when performing the operation of this embodiment, it is preferable to provide a vacuum pump (the aforementioned pressurizing and depressurizing device 60) that can discharge gas from the pressurized culture tank 2 to the outside of the system. It is preferable to set the upper limit of the pressure inside the culture tank 2 taking into account the upper limit of the flow rate that can be performed per vacuum pump and the constraints on the number of vacuum pumps that can be installed in the culture tank 2. (g1) When the pressure inside the culture tank 2 increases after performing the first operation described above, it is preferable for the control device 7 to control the system to perform at least one of the operations described above as the second operation, which is to increase the flow rate of air supplied by the air supply device 4 and to increase the amount of air supplied by the liquid level aeration device 5. (g2) Furthermore, if the control device 7 increases the pressure inside the culture tank 2 after performing the first operation described above, it is preferable to control the control device 7 to perform the second operation described above, which is to increase the amount of air permeation by the liquid level aeration device 5. In either case of (g1) or (g2) above, the decrease in CO2 removal performance when performing the first operation described above can be compensated for.

[0069] [Culturing Method] Next, the culturing method according to this embodiment will be described. Figure 3 is a flowchart illustrating the contents of the culturing method according to this embodiment. The culturing method according to this embodiment uses the culture apparatus 1 described above. The configuration of the culture apparatus 1 has already been described in detail, so a detailed explanation will be omitted.

[0070] As shown in Figure 3, the culture method according to this embodiment includes an acquisition step S1 and a determination step S2. In the acquisition step S1, the control device 7 acquires information from the first sensor unit 6. In the determination step S2, when the control device 7 performs a second operation, it determines the amount of air supplied by the air supply device 4 and the amount of air supplied by the liquid level air supply device 5 based on at least the acquired information from the first sensor unit 6. In this determination step S2, based on the information from the first sensor unit 6, for example, the controls described above (control 1) to (control 4) can be performed. Since the culture method according to this embodiment includes an acquisition step S1 and a determination step S2, it is possible to improve both the oxygen supply performance and the carbon dioxide removal performance in the liquid phase LP in the culture tank 2.

[0071] (Preferred Embodiments of the Culture Method) Next, preferred embodiments of the culture method, (a') to (g2'), will be described. The preferred embodiments of the culture method, (a') to (g2'), described below correspond to the preferred embodiments (a) to (g2) of the culture apparatus 1 described above. The detailed configuration and effects of the preferred embodiments (a') to (g2') described below are the same as those described in the preferred embodiments (a) to (g2) described above, so we will refer to those descriptions and omit detailed descriptions of the configuration and effects in the following description.

[0072] (a') In decision step S2, when the control device 7 performs the second operation, it is preferable to decide whether to perform either an operation to increase the amount of air supplied by the air supply device 4 or an operation to increase the amount of air supplied by the liquid level ventilation device 5, or both, based on the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4.

[0073] (b') In decision step S2, it is preferable that the control device 7 decides to increase the amount of air supplied by the liquid level aeration device 5 when the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4 exceeds a threshold.

[0074] (c') In the culture method, it is preferable that the culture apparatus 1 is provided so as to be in contact with at least the liquid phase LP and further comprises a second sensor unit 18 that detects an indicator of dissolved oxygen in the liquid phase LP. (d') In the culture method, it is preferable that the hole diameter of the sparger 31 connected to the oxygen supply device 3 is smaller than the hole diameter of the sparger 41 connected to the air supply device 4.

[0075] (e') In the determination step S2, it is preferable that the air supply device 4 controls the amount of air supplied by the air supply device 4 so that the sum of the amount of air supplied by the oxygen supply device 3 and the amount of air supplied by the air supply device 4 is less than 0.5 vvm relative to the amount of liquid inside the culture tank 2. (f') In the determination step S2, it is preferable that the control device 7 increases the amount of air supplied by the liquid level aeration device 5 and increases the empty velocity of oxygen supplied by the oxygen supply device 3.

[0076] In the operation of this embodiment, the pressure inside the culture tank 2 tends to increase. If the pressure inside the culture tank 2 increases too much, it may cause malfunctions in the various devices attached to the culture tank 2, damage to cells, inhibit the production of valuable substances secreted by cells, or adversely affect the quality of valuable substances. For this reason, when performing the operation of this embodiment, it is preferable to provide a vacuum pump (the aforementioned pressurizing and depressurizing device 60) that can discharge gas from the pressurized culture tank 2 to the outside of the system. It is preferable to set the upper limit of the pressure inside the culture tank 2 taking into account the upper limit of the flow rate that can be performed per vacuum pump and the constraints on the number of vacuum pumps that can be installed in the culture tank 2. (g1') When the pressure inside the culture tank 2 increases after performing the first operation described above, it is preferable that the control device 7 controls the system to perform at least one of the operations described above as the second operation, for example, in the decision step S2, which is to increase the flow rate of air supplied by the air supply device 4 and to increase the amount of air supplied by the liquid level aeration device 5. (g2') Furthermore, if the control device 7 performs the first operation described above and the pressure inside the culture tank 2 increases, it is preferable to control the control device 7 to perform, for example, the second operation described above in the decision step S2, which is to increase the amount of air permeation by the liquid level aeration device 5.

[0077] Figure 4 is a block diagram of the computer that constitutes the control device in the culture apparatus shown in Figure 1. The control device 7 shown in Figure 1 is equipped with one or more computers 980 as shown in Figure 4. In Figure 4, the computer 980 includes a CPU (Central Processing Unit) 981, a storage unit 982, a communication port 983, an input / output port 984, and a DSP (Digital Signal Processor) 985.

[0078] Here, the memory unit 982 includes RAM 982a and ROM 982b. The communication port 983 is connected to the communication circuit 986. The input / output port 984 is connected to the input / output device 987. ROM 982b stores control programs (programs that perform first operations, second operations, etc.) and various data executed by the CPU 981 and DSP 985. The CPU 981 and DSP 985 realize various functions by executing these control programs.

[0079] Although the culture apparatus 1 and culture method according to the present invention have been described in detail above with reference to embodiments, the present invention is not limited to the embodiments described above and includes various modifications. For example, the embodiments described above are described in detail for the purpose of explaining the present invention in an easy-to-understand manner and are not necessarily limited to those having all the described configurations. Furthermore, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. In addition, it is possible to add, delete, or replace parts of the configuration of each embodiment with other configurations.

[0080] 1. Culture apparatus 2. Culture tank 3. Oxygen supply device 4. Air supply device 5. Liquid level aeration device 6. First sensor unit 7. Control device 18. Second sensor unit

Claims

1. A culture tank for culturing cells in a culture medium; an oxygen supply device for supplying oxygen gas into a liquid phase formed by supplying the culture medium inside the culture tank; an air supply device for supplying air into the liquid phase; a liquid surface venting device connected to a portion of the gas phase formed above the liquid phase inside the culture tank and controlling the airflow in the gas phase; a first sensor unit provided so as to be in contact with at least the liquid phase and detecting an indicator of dissolved carbon dioxide in the liquid phase; and a control device for controlling the oxygen supply device, the air supply device and the liquid surface venting device, wherein the control device performs a first operation to improve the oxygen supply performance to the liquid phase and a second operation to improve the carbon dioxide removal performance from the liquid phase based on information from the first sensor unit, wherein the second operation is at least one of an operation to increase the amount of airflow by the air supply device and an operation to increase the amount of airflow by the liquid surface venting device. A culture apparatus characterized in that, when the control device performs the second operation, it determines the amount of air supplied by the air supply device and the amount of air supplied by the liquid level air supply device based on at least the information from the first sensor unit.

2. A culture apparatus according to claim 1, wherein the control device, when performing the second operation, determines whether to perform either an operation to increase the amount of air supplied by the air supply device or an operation to increase the amount of air supplied by the liquid level aeration device, or both, based on the sum of the amount of air supplied by the oxygen supply device and the amount of air supplied by the air supply device.

3. A culture apparatus according to claim 2, wherein the control device determines to increase the amount of air permeation by the liquid level aeration device when the sum of the amount of air permeation by the oxygen supply device and the amount of air permeation by the air supply device exceeds a threshold.

4. A culture apparatus according to claim 1, further comprising a second sensor unit provided to be in contact with at least the liquid phase and for detecting an indicator of dissolved oxygen in the liquid phase.

5. A culture apparatus according to claim 1, characterized in that the hole diameter of the sparger connected to the oxygen supply device is smaller than the hole diameter of the sparger connected to the air supply device.

6. A culture apparatus according to claim 1, characterized in that the air supply device controls the supply of air by the air supply device until the sum of the air supply amount by the oxygen supply device and the air supply amount by the air supply device is less than 0.5 vvm with respect to the liquid volume inside the culture tank.

7. A culture apparatus according to claim 1, wherein the control device increases the amount of air supplied by the liquid surface aeration device and increases the empty column velocity of oxygen supplied by the oxygen supply device.

8. A culture method using a culture apparatus comprising: a culture tank for culturing cells in a culture medium; an oxygen supply device for supplying oxygen gas into a liquid phase formed by supplying the culture medium inside the culture tank; an air supply device for supplying air into the liquid phase; a liquid level ventilator connected to a portion that becomes a gas phase formed above the liquid phase inside the culture tank and controls the airflow in the gas phase; a first sensor unit provided so as to be in contact with at least the liquid phase and detecting an indicator of dissolved carbon dioxide in the liquid phase; and a control device for controlling the oxygen supply device, the air supply device and the liquid level ventilator, wherein the control device performs a first operation to improve the oxygen supply performance to the liquid phase and a second operation to improve the carbon dioxide removal performance from the liquid phase based on information from the first sensor unit, wherein the second operation is at least one of an operation to increase the amount of airflow by the air supply device and an operation to increase the amount of airflow by the liquid level ventilator, and the control device acquires information from the first sensor unit. A culture method characterized by comprising: a determination step in which, when the control device performs the second operation, the control device determines the amount of air supplied by the air supply device and the amount of air supplied by the liquid level air supply device based on at least the information acquired from the first sensor unit.

9. A culture method according to claim 8, wherein in the determination step, the control device determines, when performing the second operation, whether to perform either an operation to increase the amount of air supplied by the air supply device or an operation to increase the amount of air supplied by the liquid level air supply device, or both, based on the sum of the amount of air supplied by the oxygen supply device and the amount of air supplied by the air supply device.

10. A culture method according to claim 9, characterized in that, in the determination step, the control device determines to perform an operation to increase the amount of air permeation by the liquid level air permeator when the sum of the amount of air permeation by the oxygen supply device and the amount of air permeation by the air supply device exceeds a threshold.

11. A culture method according to claim 8, further comprising a second sensor unit provided to be in contact with at least the liquid phase and for detecting an indicator of dissolved oxygen in the liquid phase.

12. A culture method according to claim 8, characterized in that the hole diameter of the sparger connected to the oxygen supply device is smaller than the hole diameter of the sparger connected to the air supply device.

13. A culture method according to claim 8, characterized in that, in the determination step, the air supply device controls the supply of air by the air supply device until the sum of the air supply amount by the oxygen supply device and the air supply amount by the air supply device is less than 0.5 vvm with respect to the liquid volume inside the culture tank.

14. A culture method according to claim 8, characterized in that, in the determination step, the control device increases the amount of air supplied by the liquid surface aeration device and increases the empty column velocity of oxygen supplied by the oxygen supply device.

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