JP5519994B2 - Cell culture device and cell culture method - Google Patents

Description

  The present invention relates to a culture apparatus and a culture method for culturing cells in a culture tank.

  In the production of biopharmaceuticals such as vaccines and antibodies, for example, animal cells are cultured, and active ingredients such as proteins are extracted and purified from the proliferated cells and products generated from these cells. The method used is known. In this culture, for example, cells stored in a batch or continuous culture tank are supplied with a medium containing cell nutrients (medium components such as sugar, vitamins and inorganic salts) and oxygen gas in the culture solution. Done.

  As a method for supplying the above-mentioned oxygen gas to the culture medium (culture solution), for example, a tubular sparger with a large number of gas discharge ports formed on the tip side is immersed in the culture medium, and the medium is passed through the sparger into the culture medium. A method of bubbling bubbles containing oxygen gas is known. However, in this method, the strength of the cell membrane that wraps the cells is small, and the cells may be damaged by physical impact caused by bubble breakage or the like. In particular, animal cells have the property of being vulnerable to such physical impacts.

  Therefore, as a method of dissolving oxygen gas without generating bubbles in the culture medium, for example, a permeable film that transmits oxygen gas, such as a silicon tube or a silicon film, is immersed in the culture medium, and oxygen is passed through the oxygen permeable film. A method for dissolving gas in a culture medium is known. However, in this method, in order to increase the amount of dissolved oxygen gas, it is necessary to increase the membrane surface area as much as possible. As a result, it is necessary to immerse many oxygen permeable membranes in the culture medium. On the other hand, since the volume of the culture medium (culture tank) is limited, the amount that can be immersed is limited, and it is extremely difficult to maintain the dissolved oxygen concentration at a sufficiently high value. Further, when the oxygen permeable membrane is immersed in the culture medium, it may be difficult to maintain the cleanability of the culture tank and the sterile environment, and contamination may occur through the oxygen permeable membrane. In addition, there is a drawback that handling of the oxygen permeable membrane becomes complicated.

  From the above, when the method of dissolving oxygen gas by a large amount of ventilation is adopted, cells are damaged by physical impact caused by bubble breakage, and oxygen gas is dissolved through the oxygen permeable membrane. Since it is difficult to supply a sufficient amount of oxygen gas to the medium in the method, even if it is an animal cell known to be able to be cultured at the experimental level, the yield is low due to the above reasons when it is industrially cultured. Therefore, it is not practically established as a business.

  Patent Document 1 describes a technique for supplying oxygen gas to a culture medium using an oxygen permeable membrane outside a culture tank in a method for culturing viruses with cells, but the above-mentioned problem cannot be solved. . Patent Document 2 describes a technique in which the culture tank 1 and the submerged aeration tank 2 are partitioned through the screen 3a, and oxygen gas is dissolved in the medium in the submerged aeration tank 2. The effect of oxygen gas on the medium has not been studied.

JP-A-11-187868 JP-A-7-203945

  The present invention has been made under such circumstances, and an object thereof is to provide a culture apparatus and a culture method capable of reducing damage caused by bubbles when oxygen is supplied to cells in a culture tank. is there.

The cell culture device of the present invention comprises:
In a culture apparatus for culturing cells using a culture solution in a culture tank,
A culture vessel for culturing cells;
A first preparation tank storing a first culture medium made of a solution containing at least one of an inorganic salt and sugar, which is a component that is difficult to oxidize in the culture solution ;
A second preparation tank storing a second medium containing at least one of an amino acid and a vitamin, which is an easily oxidizable component of the culture solution ;
Means for supplying oxygen gas into the first preparation tank;
And a means for supplying the first culture medium in which the oxygen gas in the first preparation tank is dissolved and the second culture medium in the second preparation tank to the culture tank. .
It may be provided with a means for supplying inert gas to at least one of the second medium in the first medium and the second preparation vessel of the first preparation vessel.

The cell culture method of the present invention comprises:
In a culture method of culturing cells using a culture solution in a culture tank,
Supplying oxygen gas into the first preparation tank in which the first culture medium made of a solution containing at least one of an inorganic salt and sugar, which is a component that is difficult to oxidize in the culture solution,
Next, a first medium in which oxygen is dissolved in the first preparation tank and a second medium containing at least one of an amino acid and a vitamin which is an easily oxidizable component of the culture solution are added to the culture tank. Each is supplied, and the cells are cultured in this culture tank.
Wherein at least one of the first of the first medium and the second medium in the second preparation vessel in preparation vessel, the inert gas may be a medium which is supplied.

  The present invention stores a medium containing a component that is difficult to oxidize and a medium containing a component that easily oxidizes in separate preparation tanks when culturing cells in a culture tank, and a medium containing a component that is difficult to oxidize Oxygen gas is dissolved in the preparation tank in which the gas is stored, and oxygen is supplied to the culture tank through the medium to culture the cells. Therefore, the generation of bubbles in the culture tank does not occur or is suppressed, so that the required oxygen amount can be supplied to the cells while reducing damage to the cells. Moreover, about the culture medium containing the component which is easy to oxidize, since it supplies in a culture tank from the preparation tank different from the preparation tank which supplies oxygen, oxidation can be suppressed about the said culture medium. In addition, since it is not necessary to provide an oxygen permeable membrane or the like in the culture tank, the culture tank can be easily washed and the aseptic environment can be easily maintained. Thus, the occurrence probability of contamination can be suppressed.

It is the schematic which shows an example of the cell culture apparatus of this invention. It is a block diagram which shows an example of the component of the culture medium used for culture | cultivation of a cell in said culture apparatus. It is a schematic diagram which shows the mode of the culture | cultivation of the cell performed using said culture apparatus. It is a schematic diagram which shows an example of the addition amount of the culture medium in said culture apparatus. It is a schematic diagram which shows an example of the addition amount of oxygen gas in said culture | cultivation apparatus. It is a schematic diagram which shows the effect | action in said culture apparatus. It is a schematic diagram which shows an example of the addition amount of the culture medium in said culture apparatus. It is a schematic diagram which shows an example of the addition amount of oxygen gas in said culture | cultivation apparatus. It is the schematic which shows the other example of said culture apparatus.

  An example of an embodiment of a cell culture device for culturing cells of the present invention, for example, animal cells, will be described with reference to FIG. The culture apparatus includes a culture tank 1 for culturing cells, and a first preparation tank 10 and a second preparation tank 20 for supplying a culture medium to the culture tank 1, respectively, which will be described later. Thus, the cells are cultured in the culture tank 1 by the medium supplied from the first preparation tank 10, the oxygen gas supplied together with the medium, and the medium supplied from the second preparation tank 20. Has been. An example of each medium component of the medium prepared (mixed) in the culture tank 1 is shown in FIG. 2. In the first preparation tank 10, a medium in which a component that is difficult to oxidize among these components is dissolved. A medium in which a component that is more easily oxidized than the component in the first preparation tank 10 is dissolved is stored in the second preparation tank 20. Specifically, the components that are difficult to oxidize are, for example, inorganic salts and saccharides, and the components that are easily oxidized are, for example, amino acids and vitamins. These medium components are adjusted to have a predetermined concentration and addition amount in the culture tank 1.

  Next, a specific configuration of the culture apparatus will be described. In the first preparation tank 10, for example, a gas discharge portion 11 a such as a sparger in which a large number of gas discharge ports are formed is immersed in the culture medium, and the gas supply path 11 extends from the gas discharge portion 11 a. Thus, oxygen gas or air and an inert gas such as nitrogen (N2) gas can be supplied (bubbled) into the culture medium from the oxygen supply source 12 and the inert gas supply source 13, respectively. Reference numerals 14 and 15 in FIG. 1 denote valves and a flow rate adjusting unit, respectively, and oxygen gas and nitrogen gas supplied to the first preparation tank 10 and flow rate adjustment are performed by a control unit 50 described later. Further, 16 in FIG. 1 is a dissolved oxygen measuring means such as a DO (dissolved oxygen) meter for measuring the dissolved oxygen concentration in the culture medium, and 17 is for stirring the culture medium in the first preparation tank 10. A stirrer.

  The first preparation tank 10 is provided with heating / cooling means 18 such as a jacket for heating / cooling the medium to a temperature suitable for culture. Although not shown in the first preparation tank 10, a supply port for replenishing the medium from the outside and an upper region of the medium in the first preparation tank 10 are maintained in a pressurized state, for example. A discharge port for discharging oxygen gas or nitrogen gas to the outside is formed, and a branch pipe 63 described later is connected. Between the first preparation tank 10 and the culture tank 1, a supply path 33 having a valve 31 and a flow rate adjusting unit 32 is provided, and oxygen gas is supplied to the culture tank 1 through the supply path 33. It is comprised so that the melt | dissolved culture medium may be supplied. In this example, means for supplying the culture medium from the first preparation tank 10 to the culture tank 1 is constituted by the valve 31, the flow rate adjusting unit 32, and the first supply path 33.

  One end side of an inert gas supply path 21 for supplying an inert gas, for example, nitrogen gas, is connected to the second preparation tank 20 in order to suppress oxidation of the medium stored therein, and the inert gas The other end side of the supply path 21 is connected to the above-described inert gas supply source 13 via the flow rate adjusting unit 22 and the valve 23. In addition, a supply path 26 provided with a valve 24 and a flow rate adjusting unit 25 is disposed between the second preparation tank 20 and the culture tank 1, and the culture tank 1 is connected via the supply path 26. Medium is supplied. In this example, the valve 24, the flow rate adjusting unit 25, and the second supply path 26 constitute means for supplying the culture medium from the preparation tank 20 to the culture tank 1. Note that the second preparation tank 20 may be provided with heating / cooling means in the same manner as the first preparation tank 10 described above.

  A large number of microcarriers 40 made of, for example, a polymer (resin) having a particle size of about several hundred μm are accommodated inside the culture tank 1. The microcarriers 40 allow cells to adhere to the surface of the microcarriers 40. It forms a carrier (scaffold) for proceeding culture. The culture tank 1 has dissolved oxygen measuring means 45 such as a DO meter for measuring the concentration of dissolved oxygen contained in the medium stored in the medium, as in the first preparation tank 10 described above. A stirrer 46 for stirring the culture medium and a heating / cooling means 47 for heating / cooling the culture medium in the culture tank 1 to a temperature suitable for culture are provided. Note that the microcarrier 40 is housed in the culture tank 1 and the cells are attached to the microcarrier 40 and cultured. However, in the case of cells that float and proliferate in the culture solution, the microcarrier 40 is disposed. You don't have to.

In addition, a culture medium extraction pipe (sedimentation pipe) 41 whose one end opens downward is provided inside the culture tank 1, and the other end side of the culture liquid extraction pipe 41 is connected to the culture tank 1. It extends to the outside and is connected to a collection unit (not shown) via a liquid feeding device 42 such as a tube pump, a valve 43 and a flow rate adjustment unit 44. In the culture solution taken out from the culture tank 1 through the culture solution extraction tube 41, effective components for producing biopharmaceuticals and the like, for example, cell products, are extracted or unnecessary components are discarded. Will be. A branch pipe 63 provided with a valve 61 and a flow rate adjustment unit 62 is connected to the culture solution extraction tube 41 between the liquid feeding device 42 and the flow rate adjustment unit 44, and the culture solution extraction tube 41 is connected to the culture solution extraction tube 41. A part of the culture solution collected through the medium is returned to the first preparation tank 10 described above.
In the case of cells proliferating by floating in a culture solution, the culture solution and cells can be separated by centrifugation or filtration separation.

  In addition, the culture apparatus outputs control signals to, for example, the valves 14, 23, 24, 31, 43, 61 and the flow rate adjusters 15, 22, 25, 32, 44, 62 to generate oxygen gas, nitrogen gas, A control unit 50 is provided for performing supply and disconnection of the medium and the culture solution and adjusting the flow rate. As will be described later, for example, according to a preset supply sequence or the cell metabolic rate (dissolved oxygen in the culture tank 1), the control unit 50 is configured so that cells are appropriately cultured in the culture tank 1. A program is set up to adjust the supply / amount of the culture medium, oxygen gas and culture solution from each of the preparation tanks 10 and 20 according to the concentration reduction rate. In this program, for example, the above-mentioned supply amount is set by conducting an experiment of cell culture in advance.

  Next, the cell culture method of the present invention will be described with reference to FIGS. First, an aqueous solution containing cells (microcarriers 40) is stored in the culture tank 1, and the culture medium is stored in each of the preparation tanks 10 and 20, and the heating tank 18 and 47 in FIG. 1 is maintained at a temperature suitable for cell culture, and nitrogen gas is supplied into the second preparation tank 20 for bubbling to remove dissolved oxygen from the medium. Next, as shown in FIG. 3A, oxygen gas is supplied to the first preparation tank 10 while being stirred in the first preparation tank 10, and the oxygen gas is dissolved. And the dissolved oxygen concentration in a culture medium becomes high gradually, and as shown to the same figure (b), the saturated aqueous solution with which oxygen concentration was saturated is obtained in the 1st preparation tank 10, for example. Next, as shown in FIG. 3C, a medium in which the oxygen concentration is saturated is supplied from the first preparation tank 10 to the culture tank 1, and the oxidation is suppressed from the second preparation tank 20 by nitrogen gas. Is supplied to the culture tank 1.

  When the culture medium in which the oxygen gas in the first preparation tank 10 is dissolved and the culture medium in the second preparation tank 20 are supplied into the culture tank 1, the medium and oxygen gas are quickly consumed by the cells. To go. And as a cell consumes a culture medium and oxygen gas, while producing a product, a waste product, etc., the number of individuals of a cell increases. At this time, when the amount of the culture solution containing these medium, products, wastes, and the like in the culture tank 1 exceeds a predetermined amount, the culture solution is passed through the culture solution extraction tube 41 by the liquid feeding device 42. Will be discharged to the outside. The liquid feeding (discharge) speed of the culture solution in the liquid feeding device 42 is such that the cells (microcarriers 40) in the culture tank 1 are not discharged to the outside together with the culture liquid, and the terminal sedimentation speed of the microcarriers 40 to which the cells are attached. Will be set to a slower speed. That is, when the culture medium in the culture tank 1 is aspirated from the culture liquid extraction tube 41, the microcarrier 40 is also aspirated together with the culture liquid and tries to rise in the culture tank 1. Since it is set slower than the sedimentation speed of 40, the microcarrier 40 stays in the lower part in the culture tank 1, and only the culture solution is discharged from the culture tank 1. Further, in order to effectively use unreacted oxygen gas remaining in the culture solution, a part of the culture solution extracted from the culture vessel 1 is returned to the first preparation vessel 10 via the branch pipe 63. In some cases.

  And as the number of cells increases with the passage of time, the consumption of the culture medium and oxygen gas also increases, so that the culture medium and oxygen gas in the culture tank 1 are not deficient in advance as described above. These supply amounts are adjusted according to the supply sequence of the culture medium and oxygen gas in each of the preparation tanks 10 and 20 set by a culture experiment or the like. As an example of such a supply sequence, as shown in FIG. 4, for example, the supply and stop of the medium in the first preparation tank 10 are performed a plurality of times at intervals, and the first preparation tank 10 The supply amount of the medium inside increases step by step. About the culture medium in the 2nd preparation tank 20, it sets to the supply amount of a fraction with respect to the supply amount of the culture medium in the 1st preparation tank 10, for example, as shown in FIG. For example, the supply amount according to the supply amount of the medium in the first preparation tank 10 is set. And when the storage amount of the culture medium in the 1st preparation tank 10 and the 2nd preparation tank 20 runs short, as shown in FIG. 6, for example, it is a culture medium from the external storage tank which is not illustrated to the preparation tanks 10 and 20. Are replenished respectively. In addition, about the oxygen concentration in the culture medium in the culture tank 1, it has shown with the dotted line in FIG. In addition, the culture tank 1 may be supplied with a medium and oxygen gas from a supply path (not shown) with respect to the cells.

  Thus, after culturing for a predetermined period, for example, several days to several weeks, the active ingredient contained in the inside is purified from the culture solution taken out from the culture tank 1 or the cells grown in the culture tank 1, It will be used as a raw material for biopharmaceuticals such as antibodies. In FIGS. 3B and 3C, the medium in which the oxygen gas is dissolved is hatched.

  According to the above-described embodiment, when culturing cells in the culture tank 1, a medium that is not easily oxidized and a medium that is easily oxidized are stored in the first preparation tank 10 and the second preparation tank 20, respectively. Oxygen gas is dissolved in the medium in the first preparation tank 10, and the medium is supplied from the first preparation tank 10 and the second preparation tank 20 into the culture tank 1. Is being cultured. For this reason, bubbles are not generated in the culture tank 1 or the generation of bubbles is suppressed as compared with direct bubbling in the culture tank 1, so that the required oxygen amount for the cells while reducing damage to the cells. Can be supplied. Moreover, since the culture medium which is easily oxidized is supplied into the culture tank 1 from the second preparation tank 20 different from the first preparation tank 10 to which oxygen is supplied, the environment in which oxidation is suppressed (second In the preparation tank 20), when the medium is supplied into the culture tank 1, it is quickly consumed by the cells. Therefore, compared with the case where the whole amount of the medium is supplied into the culture tank 1 at one time and oxygen gas is supplied into the culture tank 1 without dividing the medium, the oxidation of the medium is suppressed. Therefore, since the composition of the medium in the culture tank 1 can be kept at a value very close to a preset value, the cells can be rapidly cultured. From this, it is possible to supply a sufficient amount of oxygen gas and medium while suppressing damage to the cells, so even if the cells are conventionally considered to be low in yield and cannot be cultivated as a business, the yield is high. Can be cultured at a high rate.

Furthermore, since it is not necessary to immerse an oxygen permeable membrane or the like in the culture tank 1 when supplying oxygen gas to the cells, it is easy to handle and wash the culture tank 1 or maintain an aseptic environment in the culture tank 1. In addition, the generation of contamination through the oxygen permeable membrane can be suppressed.
Moreover, since oxygen gas is saturated in the culture medium in the first preparation tank 10, oxygen gas can be quickly supplied into the culture tank 1, and the oxygen gas concentration in the culture tank 1 can be adjusted. It becomes easy.

  In the above example, as described in FIGS. 4 and 5, the medium supplied from the first preparation tank 10 and the oxygen gas supplied together with the medium are supplied from the second preparation tank 20. Although the medium was intermittently supplied, for example, as shown in FIG. 7, the supply amount of the medium in the first preparation tank 10 may be increased continuously, or the second preparation tank may be used. For the medium in 20, the supply amount may be adjusted regardless of the supply amount of the medium in the first preparation tank 10. The supply amount of oxygen gas does not need to correspond to the supply amount of the culture medium in the first preparation tank 10 as shown in FIG. In this case, for example, when it is desired to reduce the supply amount of oxygen gas with respect to the supply amount of the medium in the first preparation tank 10, for example, nitrogen gas is supplied to the medium in the first preparation tank 10, The oxygen gas dissolved in the culture medium may be expelled, or the culture medium may be allowed to stand for a predetermined time so that the oxygen gas naturally escapes from the culture medium. In this way, by adjusting the supply amounts of the culture medium and oxygen gas independently of each other, it is possible to individually replenish the components that are deficient in the culture tank 1, so that, for example, changes in the environment in the culture tank 1 ( It is possible to respond flexibly to the rate of increase in cell population and changes in dissolved oxygen amount.

  Moreover, as a supply method of a culture medium and oxygen gas, you may adjust the supply amount of oxygen gas and the culture medium in the 1st preparation tank 10, for example so that the oxygen concentration in the culture medium in the culture tank 1 may become fixed. . Furthermore, the concentration of cells and products in the culture tank 1 or the concentration of carbon dioxide discharged from the culture tank 1 is measured, and together with the medium supplied from the first preparation tank 10 and this medium based on the measurement result The supply amount of the oxygen gas supplied and the medium supplied from the second preparation tank 20 may be adjusted. Furthermore, although the supply amount of the culture medium and the oxygen gas is adjusted by the control unit 50, for example, an operator may adjust these supply amounts manually.

  In the above example, one each of the first preparation tank 10 and the second preparation tank 20 is provided, but a plurality of each may be arranged. Specifically, for example, in FIG. 9, two second preparation tanks 20 are provided, and each of these second preparation tanks 20 and 20 includes the above-described oxidizable medium components. Different types of components are stored. In this culture apparatus, a medium in which oxygen gas is dissolved is supplied from the first preparation tank 10 into the culture tank 1, and mediums that are easily oxidized from the second preparation tanks 20 and 20 are respectively stored in the culture tank 1. The supplied cells will be cultured. By providing two second preparation tanks 20 in this way, only the components that are deficient in the culture tank 1 can be supplied into the culture tank 1 during cell culture. It is possible to respond more flexibly by cultivating the cell, and it is advantageous in terms of cost.

  In addition, although the first preparation tank 10 and the second preparation tank 20 each store a medium that is not easily oxidized and a medium that is easily oxidized, a part of the components of the medium that is not easily oxidized together with the medium that is easily oxidized is stored in the first preparation tank 10 and the second preparation tank 20. It may be stored in two preparation tanks 20. That is, as long as the first preparation tank 10 does not contain a component that easily oxidizes, the second preparation tank 20 may contain a component that hardly oxidizes. In this case, when the supply amount of the medium in the second preparation tank 20 is smaller than the supply amount of the medium in the first preparation tank 10, for example, among the medium components in the first preparation tank 10 Rare and expensive components may be supplied together with the medium in the second preparation tank 20. In addition, in each of the culture media in the first and second preparation tanks 10 and 20, for example, when components having greatly different solubility in water are included, for example, different from the preparation tank 10 (20). A large preparation tank 10 (20) is installed, a component having low solubility in water is dissolved in the large preparation tank 10 (20), and the component is separated from the other culture medium in the culture tank 1. You may make it supply.

  In the above example, the dissolved oxygen concentration in the first preparation tank 10 and the culture tank 1 was measured using the dissolved oxygen measuring means 16 and 45, but without the dissolved oxygen measuring means 16 and 45, for example, In the 1st preparation tank 10, you may set long supply time of oxygen gas until oxygen gas fully melt | dissolves in a culture medium. Further, when oxygen gas is dissolved in the culture medium in the first preparation tank 10, oxygen gas is supplied to the upper gas phase region of the culture medium instead of or in combination with bubbling of oxygen gas into the culture medium. The oxygen gas may be dissolved in the culture medium by keeping the oxygen gas in a pressurized state with respect to the liquid surface.

  Furthermore, the amount of medium in the culture tank 1 may be increased without extracting the culture medium from the culture tank 1. In addition, nitrogen gas is supplied in order to suppress the oxidation of the medium in the second preparation tank 20, but when the oxidation does not progress so much in the air atmosphere, the inside of the second preparation tank 20 may be an air atmosphere. . In addition, the microcarrier 40 is housed in the culture tank 1 and the cells are attached to the microcarrier 40 for culturing. However, in the case of cells that float and proliferate in the culture solution, the microcarrier 40 is disposed. You don't have to. In this case, when the medium is extracted from the culture tank 1, for example, membrane separation or centrifugation is performed in order to suppress the outflow of cells.

In order to circulate a part of the culture solution from the culture tank 1 to the first preparation tank 10 via the branch pipe 63, for example, a separation device provided with a membrane or the like is provided in the branch pipe 63. Carbon dioxide may be separated from the culture solution, or the culture solution may not be circulated. Furthermore, the culture medium in the first preparation tank 10 supplied the culture medium in which the oxygen gas concentration was saturated into the culture tank 1, but the culture medium in which the oxygen concentration was low (not saturated) was stored in the culture tank 1. May be supplied. If the amount of oxygen gas supplied from the first preparation tank 10 together with the medium is insufficient in the culture tank 1, oxygen gas is directly supplied to the medium (culture medium) in the culture tank 1. You may bubble. Even in this case, damage to the cells can be reduced as compared with the case where the entire amount of oxygen gas supplied into the culture tank 1 is bubbled in the culture tank 1.
In addition to culturing cells, the present invention may be applied to culturing microorganisms, for example.

DESCRIPTION OF SYMBOLS 1 Culture tank 10 1st preparation tank 12 Oxygen gas supply source 20 2nd preparation tank 21 Inert gas supply path 26 Supply path 24, 31 Valve 25, 32 Flow rate adjustment means 33 Supply path 40 Microcarrier 41 Culture solution extraction pipe 42 Liquid feeding device

Claims (4)

In a culture apparatus for culturing cells using a culture solution in a culture tank,
A culture vessel for culturing cells;
A first preparation tank storing a first culture medium made of a solution containing at least one of an inorganic salt and sugar, which is a component that is difficult to oxidize in the culture solution ;
A second preparation tank storing a second medium containing at least one of an amino acid and a vitamin, which is an easily oxidizable component of the culture solution ;
Means for supplying oxygen gas into the first preparation tank;
And a means for supplying the first culture medium in which the oxygen gas in the first preparation tank is dissolved and the second culture medium in the second preparation tank to the culture tank. Cell culture device. Claim 1, characterized in that it comprises a means for supplying inert gas to at least one of the first medium and the second second medium preparation tank in the first preparation vessel The cell culture device described in 1. In a culture method of culturing cells using a culture solution in a culture tank,
Supplying oxygen gas into the first preparation tank in which the first culture medium made of a solution containing at least one of an inorganic salt and sugar, which is a component that is difficult to oxidize in the culture solution,
Next, a first medium in which oxygen is dissolved in the first preparation tank and a second medium containing at least one of an amino acid and a vitamin which is an easily oxidizable component of the culture solution are added to the culture tank. A cell culturing method comprising supplying each of the cells and culturing the cells in the culture tank. Wherein at least one of the first of the first medium and the second medium in the second preparation vessel in preparation vessel, according to claim 3 in which the inert gas is characterized in that it is a medium that has been supplied Cell culture method.

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