Method and cell culture device for culturing cells while controlling amount of carbon dioxide dissolved in culture medium produced by metabolism of cells

Abstract

To provide a novel method for culturing cells while controlling the amount of carbon dioxide produced by the metabolism of cells and dissolved in a culture medium, and to provide a culture device therefor.SOLUTION: The present invention provides a method for using a cell culture device comprising a culture tank, and an impeller for stirring a culture solution, the impeller being installed inside the culture tank and rotating around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank, the impeller having a blade and a connecting portion for connecting with the blade, the blade having an upper blade portion and a lower blade portion extending from the upper blade portion in the water depth direction, and the method comprising a step of culturing cells while rotating the impeller at a predetermined rotation speed with the upper blade portion protruding into the gas phase above the interface of the culture solution for a desired time or period; and a culture device therefor.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for culturing cells while controlling the amount of carbon dioxide produced by the metabolism of cells and dissolved in a culture solution, and also to a cell culture device for use in said method. [Background technology]

[0002] In the fields of pharmaceutical manufacturing, regenerative medicine, and cultured meat production, it is necessary to culture large quantities of animal cells. Aeration and agitation culture is the most proven method for mass-cultivation of animal cells.

[0003] In aeration and agitation culture of animal cells, the animal cells are easily damaged by excessive shear stress caused by agitation. Therefore, oxygen and carbon dioxide (CO 2 ) there is a limit to how much gas exchange efficiency can be improved by increasing the stirring speed to make the aeration bubbles finer or by improving the fluidization state.

[0004] Even when aeration is performed using a sparger (also called an "aeration tube"), the flow rate is not absolutely greater than the oxygen consumption of cells compared to microorganisms such as E. coli, and cells are killed when bubbles generated by aeration break at the culture solution interface, and it is said that the generation of bubbles on the culture surface reduces the efficiency of gas exchange, so there is a limit to how much aeration flow rate can be increased. Therefore, in normal animal cell culture, pure oxygen, rather than air, is aerated through a sparger to suppress the aeration flow rate while still satisfying the oxygen demand of the cells.

[0005] As mentioned above, the CO produced by cellular respiration is limited due to the limitations of aeration and stirring conditions. 2 Dissolved CO tends to accumulate in the culture medium, especially in large-scale animal cell cultures. 2 This leads to the accumulation of dissolved CO 2 The concentration of dissolved CO in the culture medium increases. 2It has been reported that increased concentrations of this compound inhibit cell proliferation and protein productivity.

[0006] Therefore, in addition to the oxygen supply sparger, 2 A sparger for removal is installed, and nitrogen (N 2 However, this method requires deep aeration of nitrogen or air, which is greater than the amount of aeration required for oxygen supply from a sparger in an animal cell culture tank.

[0007] In addition, this method also has the disadvantage that the efficiency of gas exchange may decrease due to the breakage of bubbles generated by aeration, and the supply of oxygen and CO 2 Since the removal is restricted by the aeration flow rate using two spargers, it is difficult to set optimal culture conditions.

[0008] Dissolved CO from culture medium 2 When removing CO 2 The only way for air to escape is through the gas phase on the top surface of the culture medium, and in ordinary animal cell culture tanks, air is ventilated into the gas phase on the top surface of the culture medium (top ventilation) in the space above the culture medium for gas exchange and to maintain positive pressure to ensure sterility within the culture tank.

[0009] By increasing the top ventilation volume, CO 2 However, there is a limit to how much the aeration volume can be increased due to the relationship with the positive pressure inside the culture tank, and in ordinary animal cell culture tanks, the top is simply ventilated for gas exchange and to maintain positive pressure inside the culture tank, with no further improvements being made.

[0010] Patent Documents 1 and 2 disclose defoaming impellers for removing bubbles that occur on the upper surface of a culture solution. The defoaming impellers stir the interface of the culture solution, thereby defoaming bubbles that occur at the interface of the culture solution and cause cell death. However, the defoaming impellers disclosed in Patent Document 1 have a blade shape that stirs only the vicinity of the interface of the culture solution, and the liquid volume needs to be fixed so that the interface of the culture solution is located at the position of the defoaming impeller, and therefore cannot be used for culture involving changes in the liquid level, such as fed-batch. In addition, the defoaming impeller needs to be moved as the volume of the culture solution changes.

[0011] Patent Document 3 discloses an animal culture device that agitates the culture liquid while suppressing disturbance of the liquid surface and vortex formation, and continuously discharges oxygen-containing gas from a suction nozzle toward the liquid surface to form an inverted cone-shaped depression on the liquid surface. This culture device also aims to improve the efficiency of oxygen supply, and 2 The disclosure does not specifically disclose the removal of

[0012] Non-Patent Document 1 describes a culture method using microcarriers in which bubbles are generated by direct oxygen aeration through a sparger in the culture solution, which causes cells attached to the microcarriers to separate out as bubbles. Instead of direct aeration, the method uses an agitator blade to generate turbulence on the surface of the culture solution, and dissolves oxygen by the turbulence generated by the blade. However, this document also describes the 2 The disclosure does not specifically disclose the removal of [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Patent No. 3391455 [Patent Document 2] European Patent Application Publication No. 0257750 [Patent Document 3] International Publication No. 88 / 00965 [Non-patent literature]

[0014] [Non-Patent Document 1] WSHu, et al., Use of surface aerator improve oxygen transfer in cell culture, Biotechnol Bioeng. 1986 Jan;28(1):122-5. Summary of the Invention [Problem to be solved by the invention]

[0015] Conventional culture devices and culture methods using them (e.g., Patent Documents 1-3, Non-Patent Document 1) have been developed mainly for the purpose of increasing the amount of dissolved oxygen in the culture solution. When the amount of culture solution used is relatively small (e.g., less than 50 L), it is sufficient to use a means for increasing the amount of dissolved oxygen alone, but when the amount of culture solution used is large (e.g., 50 L or more), a problem occurs in that carbon dioxide produced by the metabolism of cells accumulates in the culture solution.

[0016] Therefore, an object of the present invention is to provide a new culture method and a culture apparatus for culturing cells while controlling the amount of carbon dioxide produced by the metabolism of cells and dissolved in the culture medium (e.g., 50 L or more of culture medium). [Means for solving the problem]

[0017] As a result of intensive research by the present inventors into the above-mentioned problems, they discovered that it is possible to control the amount of carbon dioxide dissolved in a culture solution (e.g., 50 L or more of culture solution) by culturing cells using an agitator impeller equipped with a blade having an upper blade portion and a lower blade portion extending from the upper blade portion in the water depth direction, with the upper blade portion protruding into the gas phase above the interface of the culture solution, while rotating the agitator impeller at a predetermined rotation speed, and thus completed the present invention.

[0018] That is, the present invention includes the following aspects.

[0019] [1] A method for culturing a cell while controlling the amount of carbon dioxide dissolved in the culture medium produced by the metabolism of the cell, comprising: A culture tank for storing the culture medium; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; A method for using a cell culture device comprising: The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has a wing upper portion and a wing lower portion extending in a water depth direction from the wing upper portion, Cultivating cells while rotating the impeller at a predetermined rotation speed with the upper portion of the blade protruding into the gas phase above the interface of the culture solution for a desired time or period; A method comprising: [2] The method according to item 1, further comprising measuring an amount of carbon dioxide dissolved in the culture solution, and adjusting the rotation speed of the stirring blade and / or the position of the interface depending on the amount of carbon dioxide dissolved in the culture solution. [3] The method according to item 1 or 2, wherein the stirring impeller is rotated so that the rotation speed is a blade tip speed of 0.5 m / sec to 1.5 m / sec. [4] The method according to any one of items 1 to 3, wherein the culture tank is adapted to accommodate 50 L to 100,000 L of culture medium. [5] The method according to any one of items 1 to 4, wherein the lower part of the wing contacts the culture medium at a depth of 50% or more of the distance from the interface to the deepest part of the bottom of the culture tank. [6] The method according to any one of items 1 to 5, wherein the wing portion has a predetermined distance between it and the rotation shaft. [7] The method according to any one of items 1 to 6, wherein the connecting portion of the agitating impeller is a blade bottom portion that is arranged approximately vertically with a gap provided from the bottom portion of the culture tank. [8] The method according to any one of items 1 to 7, further comprising an oxygen control mechanism for controlling the amount of oxygen dissolved in the culture medium. [9] The method of claim 8, wherein the oxygen control mechanism is a sparger.

[10] The method of claim 9, wherein the sparger is a sintered metal, an orifice, or an SPG membrane.

[11] The method according to any one of items 1 to 10, wherein the cell is an animal cell.

[12] The method according to any one of items 1 to 11, wherein the cells are cultured while adding fresh culture medium.

[13] The method according to any one of items 1 to 12, wherein the culture is performed while adding a fresh culture medium and discharging the culture medium from the culture tank.

[14] The method according to Item 13, wherein the cell-containing concentrated culture medium after removing or reducing any components from the discharged culture medium is added to the culture tank.

[0020]

[15] A cell culture device for use in the method according to any one of items 1 to 14, A culture tank for storing the culture medium; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; Equipped with The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has an upper portion protruding into the gas phase above the interface of the culture solution, and a lower portion extending in the water depth direction from the upper portion through the interface. Cell culture equipment. Effect of the Invention

[0021] The present invention uses CO generated by cell metabolism and dissolved in the culture medium. 2 This allows precise control of the amount of CO dissolved in the culture medium, which affects the cell culture state, at the desired time or period. 2 The amount of can be precisely and easily adjusted, which contributes to improving the cell culture environment. [Brief description of the drawings]

[0022] [Figure 1]FIG. 1 is a schematic diagram (longitudinal cross-sectional view) showing an example of the configuration of the cell culture device of the present invention. [Diagram 2] FIG. 2 is a schematic diagram (longitudinal cross-sectional view) showing an example of the configuration of the cell culture device of the present invention. [Diagram 3] FIG. 3 is a schematic diagram (longitudinal cross-sectional views ((A) and (B)) and a top view (C)) showing an example of the configuration of the cell culture device of the present invention. FIG. 3(B) shows the impeller of FIG. 3(A) rotated by 90 degrees. [Figure 4] FIG. 4 is a schematic diagram showing an example of a usage mode of the present invention. [Diagram 5] FIG. 5 is a schematic diagram showing an example of a usage mode of the present invention. [Figure 6] FIG. 6 is a schematic diagram showing the configuration of a cell culture device equipped with the MR210 blade used in Example 1 (A) or the two-paddle blade of the comparative example (B). [Figure 7] Figure 7 is a set of photographs showing the state of the liquid when the stirring speed (tip speed) and liquid volume (143 L or 156 L) were changed using the MR210 impeller implemented in Example 1. The numerical values ​​under each photograph indicate the overall carbon dioxide transfer coefficient (kLa(CO2)). [Figure 8] FIG. 8 is a graph showing the results of the overall carbon dioxide transfer coefficient (kLa(CO2)) measured using the MR210 impeller or the two-paddle impeller implemented in Example 1, with the stirring speed (impeller tip speed) and liquid volume (143 L or 156 L) as parameters. [Figure 9] 9 shows the results of a comparison of the overall oxygen transfer capacity coefficient (kLa) and the overall carbon dioxide transfer capacity coefficient (kLa(CO2)) when the MR210 impeller and the sparger were used in combination, as carried out in Example 2. (A) Overall oxygen transfer capacity coefficient (kLa) when the aeration rate (100 mL / min, 250 mL / min, 500 mL / min) and (B) the rotation speed of the impeller (25 rpm, 50 rpm, 75 rpm) were changed. (C) Overall carbon dioxide transfer capacity coefficient (kLa(CO2)) when the aeration rate was 500 mL / min and the rotation speed of the impeller (50 rpm, 75 rpm) was changed. [Figure 10]FIG. 10 is a graph showing the change in dissolved CO2 (%) in the cell culture experiment carried out in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Hereinafter, the embodiments for carrying out the present invention will be described, but the technical scope of the present invention is not limited to the following embodiments. The prior art documents cited in this specification are incorporated herein by reference. The present invention can be modified, for example, by adding, deleting, or substituting the constituent elements of the present invention, provided that the modifications do not depart from the spirit of the present invention.

[0024] In this specification, terms such as "first", "second", etc. are used to distinguish one element from another. For example, a first element may be expressed as a second element, and similarly, a second element may be expressed as a first element, without departing from the scope of the present invention.

[0025] Unless otherwise defined, all terms (technical and scientific) used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] In one embodiment, the present invention provides: A method for culturing a cell while controlling the amount of carbon dioxide dissolved in the culture solution produced by the metabolism of the cell, comprising the steps of: A culture tank for storing the culture medium; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; A method for using a cell culture device comprising: The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has a wing upper portion and a wing lower portion extending in a water depth direction from the wing upper portion, Cultivating cells while rotating the impeller at a predetermined rotation speed with the upper portion of the blade protruding into the gas phase above the interface of the culture solution for a desired time or period; The present invention provides a method comprising:

[0027] In another embodiment, the present invention provides a cell culture device for carrying out the method of the present invention, A culture tank for storing a culture solution; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; Equipped with The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has an upper portion protruding into the gas phase above the interface of the culture solution, and a lower portion extending in the water depth direction from the upper portion through the interface. A cell culture device is provided.

[0028] An example of the configuration of a cell culture device capable of carrying out the method of the present invention will be described with reference to FIGS.

[0029] 1 to 3 show examples of the configuration of a cell culture device (1, 1a, 1b, 1c, 1d, 1e) capable of carrying out the method of the present invention, but the configuration of the cell culture device applicable to the present invention is not limited to these configurations. Here, the components of the cell culture device 1 in FIG. 1 will be mainly described, but components that require further explanation as other aspects will be described separately. The components described here can be combined in any combination and can be applied to the present invention.

[0030] A cell culture device 1 to which the method of the present invention can be applied includes a culture tank 10 that stores a culture medium, and an agitator blade 11 for agitating the culture medium, which is installed inside the culture tank 10 and rotates around a rotation axis 12 that extends from the top of the culture tank 10 toward the bottom of the culture tank.

[0031] The culture tank 10 can be equipped with an agitator blade 11 and may be any commonly used culture tank, for example, cylindrical or rectangular, with the cylindrical type being preferred.

[0032] The agitator blade 11 has a blade portion 110 and a connecting portion 111 to the blade portion 110. The blade portion 110 has a blade portion upper portion 1100 that protrudes into the gas phase above the interface of the culture solution (corresponding to LS in FIG. 1), and a blade portion lower portion 1101 that extends from the blade portion upper portion 1100 through the interface in the water depth direction. In one embodiment, the connecting portion 111 may be disposed substantially vertically with a gap from the bottom surface portion of the culture tank 10 as shown in FIG. 1, forming a blade bottom portion (111) that agitates the liquid at the bottom of the culture tank. For example, the blade bottom portion (111) is formed to be 25% or less of the inner diameter of the culture tank.

[0033] The impeller exemplified in Fig. 1 may be, for example, the impeller disclosed in JP 2004-321967 A, and the MR210 impeller (Super-Mix MR210) sold by Satake Multimix Co., Ltd. (Saitama, Japan) can be used in the present invention. The impeller 11 may be one impeller, or a pair (two impellers) symmetrically arranged with respect to the rotating shaft 12 as shown in Fig. 1, or three or more impellers 11, for example, three, four, five, six or more impellers 11, may be provided.

[0034] In one embodiment, there may be a predetermined interval (gap) between the blade 110 of the impeller 11 and the rotating shaft 12 as in the cell culture device (1, 1a, 1b, 1d, 1e) shown in FIG. 1, FIG. 2(A), (B) or FIG. 2(D), or there may be almost no gap as in the cell culture device 1c of FIG. 2(c). Due to the gap provided between the impeller 11 and the rotating shaft 12 in the former case, a vortex is generated around the rotating shaft 12 as the impeller 11 rotates, and the CO in the culture medium is dispersed. 2 The effect of further promoting the removal of the fertilizer can be expected. The predetermined interval (gap) between the inner side edge of the blade portion 110 of the impeller 11 and the rotating shaft 12 may be such that a vortex is generated around the rotating shaft 12 when the impeller 11 rotates, and may be, for example, 10% to 90%, 15% to 80%, 15% to 60%, or 15% to 40% of the distance from any side surface of the rotating shaft 12 to the inner wall of the culture tank in the horizontal direction.

[0035] In one embodiment, the wing upper part 1100 and the wing lower part 1101 may have different shapes, like the cell culture device 1 (FIG. 1). For example, the wing upper part 1100 may protrude in a triangular mountain shape on the outer side when the rotation shaft 12 is the center. The wing lower part 1101 is a plate-like body that connects the lower end of the wing upper part 1100 and one end of the connecting part (wing bottom part) 111, and may have a width that increases from the upper part to the lower part, for example, as shown in FIG. 1.

[0036] In another embodiment, as shown in FIG. 2(C), the upper wing portion 1100c may have a wing shape having a width that widens from the top to the bottom, and the lower wing portion 1101c may have a wing shape having a width equal to the lower end of the upper wing portion 1100c.

[0037] In another embodiment, as shown in FIG. 2(D), upper wing portion 1100d and lower wing portion 1101d may have a helical ribbon-type wing that is integrally connected in a spiral shape.

[0038] In another embodiment, as illustrated in Figures 2(A) and (B), the upper wing portion (1100a, 1100b) and the lower wing portion (1101a, 1101b) may be formed as a single rectangular wing, as in the cell culture device (1a, 1b).

[0039] In one embodiment, the lower part of the wing part (1101, etc.) may be in contact with the culture solution at a depth of 50% or more, for example, 50% to 99%, of the distance from the interface to the deepest part of the bottom surface of the culture tank. The deeper the culture solution is in contact with the lower part of the wing part, the more the culture solution can be stirred in an integrated manner from the gas phase above the interface to the deep part of the liquid phase below the interface.

[0040] In another embodiment, as illustrated in Fig. 3, the impeller may have two or more impellers, one above the other, such as a first impeller 11e1 and a second impeller 11e2. In this case, the first impeller 11e1 located at the top has an upper blade portion protruding into the gas phase above the interface of the culture liquid, and a lower blade portion extending from the upper blade portion through the interface in the water depth direction, which is the effect of the present invention, i.e., the reduction in CO 2 Dissolved CO in the culture medium 2 It has the effect of controlling the amount of.

[0041] In one embodiment, there may be a certain gap between the stirring blades 11 and the inner wall of the culture tank 10 (see, for example, Fig. 1, Fig. 2 (A) and (C)). A sparger 15 may be provided in this gap. In addition, a baffle 14 may be provided on the inner wall of the culture tank 10, thereby enhancing the stirring effect of the culture solution.

[0042] By using the above cell culture device, it is possible to culture the cells while controlling the amount of carbon dioxide dissolved in the culture solution produced by the metabolism of the cells to a desired concentration. The inventors of the present application have used the cell culture device having the above-mentioned configuration to culture the cells while rotating the stirring blades with the upper part of the blade protruding into the gas phase above the interface of the culture solution, thereby controlling the amount of CO 2 For example, it has been found that the oxidizing agent removes carbon dioxide (CO ) dissolved in the culture medium at a desired time or period during the culture of cells (see, for example, FIG. 8 ). 2 ) and measure the amount of dissolved CO in the culture medium. 2 When the amount of dissolved CO in the culture medium exceeds a certain threshold (for example, 13%, which affects cell growth), the amount of culture medium is adjusted to adjust the interface of the culture medium so that the upper part of the blade protrudes into the gas phase above the interface of the culture medium. In this state, the impeller is rotated to measure the amount of dissolved CO in the culture medium. 2 Therefore, the amount of dissolved CO in the culture medium can be reduced. 2 If the amount does not exceed a predetermined threshold, the culture is continued with the upper part of the wing immersed in the culture medium, and if necessary, the upper part of the wing may be protruded into the gas phase above the interface of the culture medium.

[0043] Increasing the impeller rotation speed increases the amount of dissolved CO in the culture medium. 2 However, if the rotation speed is too fast, the shear stress becomes too high, which damages the cells. Therefore, the rotation speed may be adjusted within a range that does not damage the cells. The rotation speed of the impeller is, for example, 0.5 m / s to 1.5 m / s, preferably 1.0 m / s to 1.5 m / s, and the impeller tip speed is set to 0.5 m / s to 1.5 m / s, and the dissolved CO 2 The removal effect can be improved.

[0044] Due to the limitations of aeration and stirring conditions, CO produced by cell respiration 2 Dissolved CO tends to accumulate in the culture medium, especially in large-scale animal cell cultures. 2 This leads to the accumulation of dissolved CO 2 Therefore, the present invention can be particularly effective when a large amount of cell culture is performed, for example, when 50 L to 100,000 L, preferably 100 L to 100,000 L of culture solution is applied to a culture tank. Up until now, it has been known that agitation culture with the upper part of the wing protruding into the gas phase above the interface of the culture solution is effective in reducing the amount of CO dissolved in a large amount of culture solution (particularly 50 L or more of culture solution). 2 The present invention is an effective method for efficiently removing dissolved CO2, which leads to deterioration of the culture environment in large-scale culture. 2 This is a revolutionary method that allows for easy control of the above.

[0045] The cell culture device applied in the present invention may be equipped with an oxygen control mechanism for controlling the amount of oxygen dissolved in the culture solution, and a sparger is preferred. As the sparger, any commercially available sparger that can be used for culture can be used in the present invention, and for example, a sintered metal sparger or an orifice sparger (see, for example, Biotechnology and Bioengineering, Vol. 40, Pp 252-259 (1992)) or a sparger having an SPG membrane (see, for example, International Publication No. 2016 / 002492) can be used, and a sparger having an SPG membrane is preferred.

[0046] The sparger 15 can be, for example, a tubular porous body extending in the vertical direction, composed of a porous body (porous membrane) formed in an approximately cylindrical shape so that the internal region is hollow. For example, a sparger described in International Publication No. 2016 / 002492 can be used.

[0047] The shape of the sparger 15 may be a shape other than one that extends linearly in the vertical direction. In addition, when the sparger extends linearly in the vertical direction, the position where the sparger is installed in the culture vessel may be anywhere, but it is preferable to install the sparger 15 on the outside of the major diameter side of the stirring blade, since the sparger 15 does not affect the large circulation flow accompanied by the upstream and downstream and swirling flow of the culture liquid.

[0048] The upper end of the porous body is airtightly connected to the gas supply passage 16, and the lower end is sealed, for example, by a sealing member (not shown). A large number of minute pores having a pore diameter of, for example, 50 μm or less are uniformly formed over the entire surface of the porous body, and the internal region of the porous body 19 is configured to communicate with the outside of the sparger 15 at a large number of points via the pores.

[0049] The porous body is made of, for example, volcanic ash and lime (CaO or CaCO 3 ) and boric acid (H 3 BO 3The porous body 19 is obtained by mixing glass raw materials such as silica (SiO 2 ) and alumina (Al 2 O 3 ) and boron oxide (B 2 O 3 Since the first glass phase is separated extremely uniformly into a first glass phase mainly composed of calcium oxide (CaO) and a second glass phase mainly composed of calcium oxide (CaO), by adjusting the temperature and time of the heat treatment or the amount of the components added, a porous body with extremely fine pores formed uniformly can be obtained after the acid treatment. This porous body is called, for example, an SPG (Shirasu Porous Glass or Shirasu Porous Glass) membrane, and one manufactured by SPG Techno Co., Ltd. can be used.

[0050] The present invention relates to a method for producing a culture medium containing carbon dioxide (CO 2 ) and adjust the amount of dissolved CO 2 The rotation speed of the stirring blades may be adjusted so that the concentration of dissolved carbon dioxide (CO 2 ) to measure the amount of DCO 2 It may also include a sensor.

[0051] The present invention also relates to a method for producing a culture medium containing carbon dioxide (CO 2 ) and adjust the amount of dissolved CO 2 Alternatively, the adjustment of the rotation speed of the impeller and the adjustment of the position of the interface may be combined. For example, in a region with a relatively low rotation speed (e.g., a blade tip speed of 0.5 m / s to 1.0 m / s), the rotation speed of the impeller can be adjusted to adjust the dissolved CO concentration in the culture solution. 2 In a region where the rotation speed is relatively high (for example, impeller tip speed of 1.0 m / sec to 1.5 m / sec), adjustment of the rotation speed of the stirring impeller may be switched to adjustment of the interface position.

[0052] In addition, the present invention may involve culturing cells while adding fresh culture medium, and in another embodiment, culturing may involve culturing cells while adding fresh culture medium and discharging the culture medium from the culture tank.

[0053] In addition, the present invention may be such that the culture medium in which the cells are concentrated after any component has been removed or reduced from the discharged culture medium is added to the culture tank.

[0054] In one embodiment, the method of the present invention can be carried out by a batch culture method. In this case, for example, at the start of the culture, the amount of the culture solution may be adjusted so that the upper part of the impeller protrudes into the gas phase above the interface of the culture solution. In this case, the dissolved CO 2 can be controlled by setting the stirring rotation speed to a desired speed.

[0055] In another embodiment, the method of the present invention can be carried out in a fed-batch culture method as shown in FIG. 4. In this case, for example, the amount of culture solution (LS in FIG. 4) that is lower than the interface level of the culture solution in the batch culture method is used. low Cultivation is started at a temperature of 1000° C. (equivalent to 1000 mL of dissolved CO2), and concentrated supplemented culture solution is added successively as necessary. Cultivation is terminated at an amount sufficient to maintain the upper blade portion 1100 of the impeller 11 protruding into the gas phase above the interface of the culture solution. 2 can be controlled by setting the stirring rotation speed to a desired speed.

[0056] In another embodiment, the method of the present invention can be carried out in a perfusion culture method (concentration batch culture method) as shown in FIG. 5. In this case, for example, at the start of the culture, the upper blade portion 1100 of the agitator blade 11 is immersed in the culture solution (LS in FIG. 5). high The culture medium is dissolved in CO 2When the temperature rises, part of the culture solution is discharged from the culture tank, and the interface (liquid surface) level of the culture solution is lowered so that the upper blade portion 1100 of the impeller 11 protrudes into the gas phase above the interface of the culture solution (corresponding to LS in FIG. 5). In this state, the rotation speed of the impeller 11 is further controlled to reduce the amount of dissolved CO in the culture solution. 2 It is possible to control the amount of dissolved CO in the culture medium. 2 If the dissolved CO falls below a desired level, the amount of fresh culture medium added can be increased and / or the amount of culture medium drained can be decreased by immersing the upper blade portion 1100 of the impeller 11. 2 The decrease in the amount of

[0057] The amount of dissolved oxygen in the culture solution can be adjusted by adjusting the amount of oxygen supplied by the sparger 15. 2 can be controlled independently.

[0058] The type of cells that can be cultured by the method of the present invention is not particularly limited, and can be used to grow any cell. For example, the cells are selected from the group consisting of animal cells, insect cells, plant cells, yeast cells, and bacteria. Animal cells are broadly divided into cells derived from animals belonging to the phylum Vertebrate and cells derived from invertebrates (animals other than those belonging to the phylum Vertebrate). The origin of the animal cells in this specification is not particularly limited. Preferably, it means cells derived from animals belonging to the phylum Vertebrate. The phylum Vertebrate includes the classes Agnathostomata and Gnathostomata, and the classes Gnathostomata include the classes Mammalia, Aves, Amphibia, and Reptilia. Preferably, the cells are derived from animals belonging to the class Mammalia, which is generally referred to as mammals. Mammals are not particularly limited, but preferably include mice, rats, humans, monkeys, pigs, dogs, sheep, goats, and the like.

[0059] The culture medium used in the present invention is appropriately selected depending on the type of cells to be cultured, and is not particularly limited, and any of known culture media and culture media optimized for the cells can be used. EXAMPLES

[0060] The present invention will be described in more detail below with reference to examples, but these are not intended to limit the present invention.

[0061] <Example 1> The impeller used was an MR210 impeller (Super-Mix MR210, Satake Multimix Co., Ltd., Saitama, Japan) (corresponding to FIG. 1). As a comparative example, an experiment was conducted using a paddle impeller (Satake Multimix Co., Ltd.) (see FIG. 6) (hereinafter referred to as "two-paddle impeller") having two layers of impellers 30 with three blades each.

[0062] A brushless motor (BLM6400SHP-30S, Oriental Motor Co., Ltd., Tokyo, Japan) was used as the impeller rotation means, and the impeller connected to the impeller was installed in a cylindrical culture tank (CVi Incorporated, USA) (diameter 558.8 mm, height 885 mm).

[0063] The culture tank was charged with 156 L or 143 L of the simulated medium. The simulated medium is a solution having the following composition, and mimics the physical properties (viscosity, density, surface tension) of a culture solution for culturing animal cells. [Table 1]

[0064] In the case of 156L, the upper part of the impeller blade is completely immersed. On the other hand, when 143L is added, part of the upper part of the MR210 impeller blade is exposed to the gas phase. On the other hand, in the case of the two-paddle impeller, the upper part of the impeller blade is completely immersed in either the case where 156L or 143L of simulated medium is added.

[0065] The overall carbon dioxide transfer coefficient (k L a(CO 2) was calculated. This example was carried out without ventilation. For each impeller, the stirring experiment was carried out at a stirring speed of 25 rpm, 50 rpm or 75 rpm. However, since the turning radius of the impeller tip differs for each impeller, the comparison was carried out by converting it to the impeller tip speed (m / s). MR210 impeller: 25 rpm → 0.476 m / s, 50 rpm → 0.952 m / s, 75 rpm → 1.429 m / s. Two-paddle impeller: 50 rpm → 0.860 m / s, 75 rpm → 1.291 m / s.

[0066] In addition, the overall carbon dioxide transfer coefficient (k L a(CO 2 )) was determined by the following method.

[0067] Prepare 156L or 143L of simulated medium, set the liquid temperature to 37℃, and k L a(CO 2 ) measurement of oxygen k L The static method of Gassing Out, P.F.Stanbury and A. Whitaker, “Principles of Fermentation Technology”, 1984 Pergamon Press, p.174-175, is used to measure the amount of L a(CO 2 ) measurement. 2 Bubbling with 25% DCO 2 After that, start aeration from above (37.6L / min) and exhaust gas CO 2 When the concentration falls below 0.8%, start aeration (or no aeration) through the sparger and add 15% DCO 2 After the following time or after 1 hour, the aeration was stopped and the measurement was completed. 2 From the change in concentration of k L a(CO 2 ) was calculated. 2 DCO with sensor (Mettler Toledo Corporation) 2 Concentration of CO on the exhaust gas side of the vent 2 Concentration is exhaust gas CO 2 The measurements were performed using a meter (BioT Corporation).

[0068] As a result, the k of the MR210 wing L a(CO 2 ) was significantly higher than that of the two-paddle impeller, and the value for the 143L feed was higher than that of the 156L feed for the MR210 impeller (Figure 7).

[0069] This shows that the MR210 impeller produces less CO when mixed with the 143L impeller, which is at the culture medium interface and agitates the culture medium interface. 2 The fluctuation of the culture medium surface due to stirring and the entrainment of air from the culture medium interface were effective in removing CO 2 It was confirmed that the method was effective in removing the above-mentioned substances (Figure 8).

[0070] The two-paddle blade is 2 The removal effect is low, while the MR210 blade is CO 2 It became clear that this stirring impeller has a high removal effect.

[0071] When the mixing state was observed, it was confirmed that at speeds of 0.952 m / s or more with the MR210 impeller, the interface was moving up and down and air bubbles were being entrained.

[0072] <Example 2> Overall oxygen transfer coefficient (k L a) and the overall carbon dioxide transfer coefficient (k L a(CO 2 Comparison of

[0073] The impeller used was the MR210 impeller (Super-Mix MR210, Satake Multimix Co., Ltd., Saitama, Japan) (FIG. 1(A)), as in Example 1, and the other configurations of the culture apparatus were the same as in Example 1. The sparger was installed at the position shown in FIG. 6(A).

[0074] The spargers used were those equipped with a Shirasu Porous Glass (SPG) membrane (Kiyomoto Tech Ichi Co., Ltd.), sintered metal (Bio-T Corporation), or an orifice (Kiyomoto Tech Ichi Co., Ltd.). Each sparger was connected to a gas reservoir (oxygen cylinder) in which gas (oxygen) was stored via a gas supply line. The liquid volume was set to 156 L so that the upper wing of the MR210 blade was completely immersed.

[0075] For each sparger, the aeration rate (100 mL / min, 250 mL / min, 500 mL / min) and the rotation speed of the agitator (25 rpm, 50 rpm, 75 rpm) were changed to obtain the overall oxygen transfer coefficient (k L The overall carbon dioxide transfer coefficient (k a) was measured (Figures 9(A) and (B)). The aeration rate was set at 500 mL / min, and the rotation speed of the agitator blade (50 rpm, 75 rpm) was changed. L a(CO 2 ) was measured (Figure 9(C)).

[0076] As a result, by applying a sparger to a culture tank, the overall oxygen transfer coefficient (k L a) was able to be controlled (Figures 9(A) and (B)). On the other hand, when comparing the case with aeration by the sparger and the case without aeration, the CO 2 The removal effects were comparable (FIG. 9(C)). Considering this result and the result of Example 1, it is possible to reduce the amount of O2 removed from the culture medium by combining the presence or absence of aeration, the stirring speed, and the culture medium level (i.e., the presence or absence of exposure of the upper part of the impeller to the gas phase). 2 Supply and CO 2 It was found that the removal could be controlled independently.

[0077] <Example 3> The stirring blade used was the same as in Example 1, MR210 blade (Super-Mix MR210, Satake Multimix Co., Ltd., Saitama, Japan) (FIG. 6(A)), and the other configurations of the culture apparatus were the same as in Example 1. The sparger used was a Shirasu Porous Glass (SPG) membrane sparger, and was installed at the position shown in FIG. 6(A).

[0078] Culture medium (JX G017-MAB01 medium, Fujifilm Wako Pure Chemical Industries, Ltd.) was added under conditions where the upper part of the MR210 blade was above the liquid surface (143 L), and CHO-S cells (initial cell concentration: 6.1 × 10 5 The mixture was incubated at 37°C and agitated at 50 rpm (impeller tip speed: 0.952 m / s). After 48 hours of incubation, the agitation was increased to 60 rpm (impeller tip speed: 1.143 m / s). 2 The removal of urea was observed. In addition, the presence or absence of shear-induced inhibition of CHO-S cells was confirmed. The top aeration rate was 37.6 NL / min, and O 2 A constant amount of gas was manually aerated. The pH was adjusted to 7.2 (control range: pH 7.2 ± 0.2) by CO 2 Gas was vented.

[0079] Immediately after the start of incubation, dissolved carbon dioxide (DCO 2 ) exceeded 13%, the level at which cell growth inhibition occurs, but gradually decreased. After 48 hours of culture, the agitation speed was increased from 50 to 60 rpm, and DCO 2 The rate of decrease in the cell density increased (Figure 10). No damage to the cells due to shear stress was observed, demonstrating that cells can be cultured effectively. [Explanation of symbols]

[0080] 1, 1a, 1b, 1c, 1d, 1e Cell culture equipment 10 Culture tank 11, 11a, 11b, 11c, 11d Agitating blade 11e1 First stirring blade 11e2 Second mixing blade 110 Wings 1100, 1100a, 1100b, 1100c, 1100d, 1100e Upper part of wing 1101, 1101a, 1101b, 1101c, 1101d, 1101e Lower wing part 111, 111a, 111b, 111c, 111d connection part (bottom of wing) 12 Rotation axis 13 Agitator blade rotation means 14 Baffle 15 Sparger 16 Gas supply line 17 Gas storage section 18 Culture medium storage tank 180 Culture solution supply path 181 Agitator blade rotation means 182 Supply culture medium stirring blade 19 Culture medium drain channel 20 Cell separation device 21 Supply channel for culture medium containing concentrated cells 22 Drain for the culture medium containing concentrated cells 30 Agitator blade (Comparative example) L.S., L.S. high , L.S. low interface

Claims

1. A method for culturing a cell while controlling the amount of carbon dioxide dissolved in the culture solution produced by the metabolism of the cell, comprising the steps of: A culture tank for storing the culture medium; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; A method for using a cell culture device comprising: The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has a wing upper portion and a wing lower portion extending in a water depth direction from the wing upper portion, Cultivating cells while rotating the impeller at a predetermined rotation speed with the upper portion of the blade protruding into the gas phase above the interface of the culture solution for a desired time or period; A method comprising:

2. The method according to claim 1, further comprising measuring an amount of carbon dioxide dissolved in the culture solution, and adjusting the rotation speed of the stirring blade and / or the position of the interface in accordance with the amount of carbon dioxide dissolved in the culture solution.

3. 3. The method of claim 1, wherein the impeller is rotated such that the rotational speed is a tip speed of 0.5 m / s to 1.5 m / s.

4. The method according to claim 1 or 2, wherein the culture tank is applied with 50 L to 100,000 L of culture medium.

5. The method according to claim 1 or 2, wherein the lower part of the wing contacts the culture medium at a depth of 50% or more of the distance from the interface to the deepest part of the bottom surface of the culture tank.

6. The method according to claim 1 or 2, wherein the vanes have a predetermined spacing from the axis of rotation.

7. The method according to claim 1 or 2, wherein the connecting portion of the agitating impeller is a blade bottom portion that is arranged approximately vertically with a gap provided from the bottom portion of the culture tank.

8. The method according to claim 1 or 2, further comprising an oxygen control mechanism for controlling the amount of oxygen dissolved in the culture solution.

9. The method of claim 8 , wherein the oxygen control mechanism is a sparger.

10. 10. The method of claim 9, wherein the sparger is a sintered metal, an orifice, or an SPG membrane.

11. The method of claim 1 or 2, wherein the cell is an animal cell.

12. The method according to claim 1 or 2, wherein the cells are cultured while adding fresh culture medium.

13. The method according to claim 1 or 2, wherein the culture is carried out while adding fresh culture medium and discharging the culture medium from the culture tank.

14. The method according to claim 13, wherein the cell-containing concentrated culture medium after removal or reduction of any components from the discharged culture medium is added to the culture tank.

15. A cell culture device for use in the method according to claim 1 or 2, comprising: A culture tank for storing the culture medium; An agitator blade for agitating the culture solution, which is installed inside the culture tank and rotates around a rotation axis extending from the top of the culture tank toward the bottom of the culture tank; Equipped with The stirring blade has a blade portion and a connecting portion to the blade portion, The wing portion has an upper portion protruding into the gas phase above the interface of the culture solution, and a lower portion extending in the water depth direction from the upper portion through the interface. Cell culture equipment.

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