WO2022025042A1 - Cell-culturing system, cell producing method, and gas concentration control method - Google Patents

Abstract

Provided is a cell-culturing system in which gas concentration in a closed-system cell-culturing container is controlled via a gas-permeable film forming the container, whereby cells can be cultured at a high density and gas concentration can be finely adjusted according to the cells being cultured The cell-culturing system 1 controls the gas concentration in a culture solution filling a closed-system culturing container 20 having gas permeability, the cell-culturing system comprising: a container housing device 10 in which the culturing container 20 is accommodated in a closed state, the gas concentration around the culturing container 20 being varied; an oxygen concentration acquisition unit 30 for acquiring the oxygen concentration in the culturing container 20; an oxygen feeding device O installed so as to be capable of feeding oxygen to the container housing device 10; and a gas concentration control unit 40 connected to the oxygen concentration acquisition unit 30, the gas concentration control unit 40 controlling the feeding of oxygen from the oxygen feeding device O to the container housing device 10 on the basis of the acquired oxygen concentration.

Description

Cell culture system, cell production method, and gas concentration control method

The present invention relates to a cell culture technique, and particularly to a cell culture system for culturing cells at high density.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., it has been required to efficiently culture a large amount of cells and tissues in an artificial environment.
Under such circumstances, it has been proposed to automatically mass-culture cells in a closed system using a bag-shaped cell culture vessel.

When a large number of cells are cultured in a closed system using a cell culture vessel, the density of the cultured cells gradually increases to 500,000 cells / cm ² or more. Then, the gas permeation performance becomes insufficient in a general cell culture vessel having a thickness of 100 μm or more manufactured by using a gas permeable film such as polyethylene or ethylene vinyl acetate, and the oxygen concentration around the cells decreases. There was a problem that the growth efficiency was reduced.
On the other hand, for example, a cell culture vessel manufactured by using a gas permeable film having a thickness of 50 μm or less is suitable for culturing at a high density, but the strength of the vessel becomes insufficient and there is a risk of bag breakage, so that it is handled. There was a problem that it became difficult.

Here, as a material having excellent gas permeation performance, a silicone material can be mentioned. Although the silicone material has low strength, excellent gas permeation performance can be obtained even if the thickness is 300 μm or more, so that the strength at the time of handling can be ensured.
However, the silicone material has a problem that it is difficult to weld a port or the like. In addition, since harmful substances are eluted from the silicone material due to radiation sterilization or the like, there is a risk of adversely affecting the subsequent culture performance. Further, since the surface treatment required for culturing adhesive cells is difficult for the silicone material, it is possible to produce a culture vessel for floating cells, but it is difficult to produce a culture vessel for adhesive cells. There was also the problem.

Japanese Unexamined Patent Publication No. 2019-110767 Japanese Unexamined Patent Publication No. 2020-68741

Therefore, the present inventors have studied diligently and controlled the oxygen concentration around the closed cell culture vessel manufactured by using the gas permeable film so as to be higher than the oxygen concentration in the vessel. We succeeded in making it possible to control the oxygen concentration (dissolved oxygen concentration) in the culture solution in the container via the gas permeable film so as to be suitable for high-density culture.
In addition, by controlling the concentration of other gases around the cell culture vessel, it is also possible to control the concentration of other gases in the culture solution in the vessel via the gas permeable film, depending on the cells to be cultured. It has become possible to finely adjust the gas concentration in the culture solution.

By the way, in Patent Document 1, the oxygen concentration of the culture solution in the culture tank is measured, and the oxygen concentration of the culture solution is adjusted to a desired value by directly injecting gas into the culture tank and stirring the mixture. Control is disclosed.
Further, Patent Document 2 is provided with a circulation line for circulating the culture solution in the cell culture container, and the circulation line is provided with a gas exchange means for gas exchange between the gas of the culture solution and the adjusting gas. It is disclosed that the gas concentration in the cell culture vessel is controlled by this gas exchange means.

However, there is a risk of contamination when gas is directly injected into the culture tank, and when the culture solution is circulated in the cell culture vessel, it cannot be cultivated in a stationary state and always requires stirring and circulation, so the equipment is large-scale. There was a problem of becoming.
In addition, these methods have a problem that damage to the cells may be a concern because shear stress is applied to the cells due to stirring and circulation of the culture solution.
Furthermore, in the case of floating cells, there is a risk that the cells will move with the flow of the culture medium due to stirring or circulation, which will adversely affect the cells.

On the other hand, according to the present invention, it is possible to control the oxygen concentration in the cell culture vessel so as to be suitable for high-density culture without causing such a problem.
In addition, by controlling the concentration of other gases in the culture medium via the gas permeable film of the cell culture vessel, it is possible to finely adjust the gas concentration according to the cells to be cultured without damaging the cells. Is.

The present invention has been made in view of the above circumstances, and cells can be cultured at high density by controlling the gas concentration in the vessel via the gas permeable film of the closed cell culture vessel. It is an object of the present invention to provide a cell culture system capable of finely adjusting the gas concentration according to the cells to be cultured, a method for producing cells, and a method for controlling the gas concentration.

In order to achieve the above object, the cell culture system of the present invention is a cell culture system that controls the gas concentration in the culture solution filled in a closed culture container having gas permeability, and closes the culture container. A container accommodating device that changes the gas concentration around the culture container, an oxygen concentration acquisition unit that acquires the oxygen concentration in the culture container, and an oxygen concentration accommodating device that can supply oxygen to the container accommodating device. As a configuration including a gas concentration control unit connected to the oxygen concentration acquisition unit and controlling oxygen supply from the oxygen supply device to the container accommodating device based on the acquired oxygen concentration. be.

Further, the cell culture system of the present invention is provided with a nitrogen supply device arranged so as to be able to supply nitrogen to the container accommodating device, and the gas concentration control unit is provided with the nitrogen supply device based on the acquired oxygen concentration. It is preferable to have a configuration that controls the supply of nitrogen to the container accommodating device.

Further, the cell culture system of the present invention is provided with a carbon dioxide concentration acquisition unit for acquiring the carbon dioxide concentration in the culture container and a carbon dioxide supply device arranged so as to be able to supply carbon dioxide to the container storage device. It is preferable that the gas concentration control unit is connected to the carbon dioxide concentration acquisition unit and controls the supply of carbon dioxide from the carbon dioxide supply device to the container storage device based on the acquired carbon dioxide concentration. ..

Further, the cell culture system of the present invention is provided with the culture vessel, and the carbon dioxide concentration sensor for measuring the carbon dioxide concentration in the culture vessel is provided in the culture vessel, and the carbon dioxide concentration sensor is the carbon dioxide concentration. It is preferable that the carbon dioxide concentration acquisition unit is connected to the acquisition unit and acquires the carbon dioxide concentration in the culture vessel based on the input information from the carbon dioxide concentration sensor.

Further, the cell culture system of the present invention is provided with the culture vessel, an oxygen concentration sensor for measuring the oxygen concentration in the culture vessel is provided in the culture vessel, and the oxygen concentration sensor is connected to the oxygen concentration acquisition unit. Therefore, it is preferable that the oxygen concentration acquisition unit acquires the oxygen concentration in the culture vessel based on the input information from the oxygen concentration sensor.

Further, the cell culture system of the present invention is provided with the culture vessel, and a camera for inputting an image obtained by photographing the cells in the culture vessel to the oxygen concentration acquisition unit is connected to the oxygen concentration acquisition unit. It is preferable that the oxygen concentration acquisition unit calculates the number of cells based on the occupied area of the cells in the image, and calculates the oxygen concentration in the culture vessel based on the obtained number of cells.

Further, the cell culture system of the present invention is provided with the culture vessel, and the oxygen concentration acquisition unit calculates the number of cells in the culture vessel based on the cell culture time in the culture vessel, and the obtained cell number is obtained. It is preferable that the structure is such that the oxygen concentration in the culture vessel is calculated based on the above.

Further, in the cell culture system of the present invention, the gas concentration control unit maintains the difference between the oxygen concentration around the culture container in the container storage device and the oxygen concentration in the culture container at 5% or more. As such, it is preferable to have a configuration that controls the oxygen supply from the oxygen supply device to the container storage device.

Further, the method for producing cells of the present invention is a method for culturing cells using the above-mentioned cell culture system.

Further, the gas concentration control method of the present invention is a gas concentration control method in cell culture that controls the gas concentration in the culture solution filled in a closed culture vessel having gas permeability, and the culture vessel is used as the gas concentration control method. It is closedly housed in a container accommodating device that changes the gas concentration around the culture container, and the oxygen concentration acquisition unit acquires the oxygen concentration in the culture container and is connected to the oxygen concentration acquisition unit. However, based on the acquired oxygen concentration, the oxygen supply from the oxygen supply device that supplies oxygen to the container storage device to the container storage device is obtained, and the oxygen concentration around the culture container in the container storage device is the said. This is a method of controlling the target value to be higher than the oxygen concentration in the culture vessel.

Further, in the gas concentration control method of the present invention, the gas concentration control unit supplies nitrogen from the nitrogen supply device that supplies nitrogen to the container storage device to the container storage device based on the acquired oxygen concentration. It is preferable to use a method of controlling the oxygen concentration around the culture vessel in the container storage device so that the target value is higher than the oxygen concentration in the culture vessel.

Further, in the gas concentration control method of the present invention, the carbon dioxide concentration acquisition unit acquired the carbon dioxide concentration in the culture vessel, and the gas concentration control unit connected to the carbon dioxide concentration acquisition unit was acquired. Based on the carbon dioxide concentration, the carbon dioxide supply device that supplies carbon dioxide to the container storage device supplies carbon dioxide to the container storage device, and the carbon dioxide concentration around the culture container in the container storage device is the said. It is preferable to control the target value to be lower than the carbon dioxide concentration in the culture vessel.

According to the present invention, cells can be cultured at high density by controlling the gas concentration in the container through the gas permeable film of the closed cell culture container, and the gas concentration depends on the cells to be cultured. It becomes possible to provide a cell culture system, a method for producing cells, and a method for controlling gas concentration, which can be finely adjusted.

It is a schematic diagram which shows the structure of the cell culture system which concerns on 1st Embodiment of this invention. It is a schematic diagram which shows the structure of the cell culture system which concerns on the 2nd Embodiment of this invention. It is a schematic diagram which shows the structure of the cell culture system which concerns on 3rd Embodiment of this invention. It is a schematic diagram which shows the structure of the cell culture system which concerns on 4th Embodiment of this invention. It is a figure which shows the graph which shows the oxygen concentration control result in the cell culture container by the cell culture system of Test 1 and the gas concentration control method. It is a figure which shows the graph which shows the result of the cell culture using the cell culture system of Test 2 and the gas concentration control method.

Hereinafter, embodiments of the cell culture system, the cell production method, and the gas concentration control method of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and examples.

[First Embodiment]
The cell culture system of the present embodiment is a cell culture system that controls the gas concentration in the culture solution filled in a closed culture container having gas permeability, and the culture container is closedly stored and cultured. A container accommodating device that changes the gas concentration around the container, an oxygen concentration acquisition unit that acquires the oxygen concentration in the culture vessel, an oxygen supply device that is arranged so that oxygen can be supplied to the container accommodating device, and an oxygen concentration acquisition. It is characterized by being provided with a gas concentration control unit which is connected to the unit and controls the oxygen supply from the oxygen supply device to the container accommodating device based on the acquired oxygen concentration.
Further, the cell culture system of the present embodiment includes a nitrogen supply device arranged so as to be able to supply nitrogen to the container storage device, and the gas concentration control unit can supply the container storage device from the nitrogen supply device based on the acquired oxygen concentration. It is preferable to control the nitrogen supply to.

Specifically, as shown in FIG. 1, the cell culture system 1 of the present embodiment includes a container accommodating device 10, an oxygen concentration acquisition unit 30, a gas concentration control unit 40, and an oxygen cylinder O. Further, the cell culture system 1 of the present embodiment preferably includes a nitrogen cylinder N. The cell culture system 1 of the present embodiment is used by accommodating the culture vessel 20 in the container accommodating device 10.

As the container accommodating device 10, for example, a CO ₂ incubator, a culture double container (Patent No. 4665588), or the like can be used. In FIG. 1, a mounting table 11 made of punching metal or the like is installed in the container storage device 10, and the culture container 20 is arranged on the mounting table 11 and is stored in a closed manner. The mounting table 11 may be omitted.

Oxygen gas and nitrogen gas are supplied to the container accommodating device 10 from the oxygen cylinder O (oxygen supply device) and the nitrogen cylinder N (nitrogen supply device), respectively, so that the gas concentration inside the container storage device 10 is controlled to control the gas concentration inside the culture container. It is possible to adjust the gas concentration around 20.
An oxygen cylinder O for supplying oxygen gas and a nitrogen cylinder N for supplying nitrogen gas are connected to the container accommodating device 10 via valves, and the opening / closing operation of each valve is controlled by the control of the gas concentration control unit 40. The supply of gas from each gas cylinder to the container accommodating device 10 is controlled, and the oxygen concentration in the container accommodating device 10 can be adjusted.

The culture vessel 20 is a closed-type cell culture vessel having gas permeability, and for example, one formed by heat-sealing the peripheral edges of two rectangular gas-permeable films by heat sealing is used. Can be done. The space formed between the two gas permeable films is used as a culture space for culturing cells, and the region forming the culture space in the gas permeable film constitutes the culture section 21 in the culture vessel 20. do.

As the gas permeable film, for example, polyethylene such as LLDPE (Linear Low Density Polyethylene) or polyolefin resin such as polypropylene can be preferably used. Further, in order to make the inside of the culture vessel 20 visible, the gas permeable film is preferably a transparent material.

In FIG. 1, two ports 22 are provided facing both ends in the longitudinal direction of the culture vessel 20, but the number of ports is not limited to this, and may be one or three or more. As the material of the port, for example, polyethylene, polypropylene, vinyl chloride, polystyrene-based elastomer, thermoplastic resin such as FEP, or the like can be used.

The cells to be cultured using the culture vessel 20 are not particularly limited, and even floating cells such as lymphocytes and dendritic cells that are suspended in the culture medium and cultured can be placed in the culture section 21 in the vessel. Artificial pluripotent stem cells (iPS cells), nerve stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, hepatocytes, pancreatic islet cells, myocardial cells, corneal endothelial cells, and activation steps that are adhered and cultured. It may be an adhesive cell such as a lymphocyte.

The oxygen concentration acquisition unit 30 is a device for acquiring the oxygen concentration in the culture vessel 20, and can be configured by using a PLC (programmable logic controller), a microcomputer, a computer, or the like.
As the oxygen concentration acquisition unit 30, there are three types: one that acquires an oxygen concentration using an oxygen concentration sensor, one that calculates an oxygen concentration based on the number of cells, and one that estimates an oxygen concentration based on a culture time.

When an oxygen concentration acquisition unit 30 that acquires an oxygen concentration using an oxygen concentration sensor is used, the oxygen concentration sensor 31 is attached to the culture container 20 housed in the container storage device 10 as shown in FIG. Then, the oxygen concentration sensor 31 and the oxygen concentration acquisition unit 30 are connected, the dissolved oxygen concentration in the culture solution in the culture vessel 20 is measured by the oxygen concentration sensor 31, and the measured information is input to the oxygen concentration acquisition unit 30. do.

Examples of the oxygen concentration sensor 31 include a non-contact oxygen sensor and a dissolved oxygen meter. The non-contact oxygen sensor is coated with a fluorescent dye and measures the oxygen concentration by an optical method for measuring the fluorescent energy emitted from the fluorescent dye. Further, in the dissolved oxygen meter, an oxygen permeable membrane is used between the two poles filled with the electrolytic solution, and the oxygen concentration is measured based on the amount of oxygen permeated through the membrane and the current flowing between the two poles.

When the oxygen concentration acquisition unit 30 is used to calculate the oxygen concentration based on the number of cells, a CCD camera is connected to the oxygen concentration acquisition unit 30, and the cultured cells in the culture vessel 20 are connected to the oxygen concentration acquisition unit 30 via a phase-contrast microscope. The image obtained by taking a picture is input to the oxygen concentration acquisition unit 30, and the oxygen concentration acquisition unit 30 can calculate the number of cells based on the occupied area of the cells in the image.
Further, since the oxygen consumption per cell is generally determined by the cell type, the oxygen concentration acquisition unit 30 calculates the oxygen consumption in the container based on the obtained number of cells, and the oxygen concentration in the container. Can be calculated.

When an oxygen concentration acquisition unit 30 that estimates the oxygen concentration based on the culture time is used, the oxygen concentration acquisition unit 30 can calculate the number of cells based on the culture time (culture days).
That is, when culturing cells such as iPS cells having stable proliferation, the number of cultured cells is substantially constant with respect to the culturing time, so that the number of cells can be estimated from the culturing time. Then, the oxygen concentration acquisition unit 30 can calculate the oxygen consumption in the container based on the obtained number of cells, and calculate the oxygen concentration in the container.

The gas concentration control unit 40 is connected to the oxygen concentration acquisition unit 30, inputs information on the oxygen concentration acquired by the oxygen concentration acquisition unit 30 from the oxygen concentration acquisition unit 30, and based on this information, the container accommodating device 10 from the gas cylinder. It is a device that controls the gas supply to, and can be configured by using a PLC, a microcomputer, a computer, or the like.

When increasing the oxygen concentration in the container storage device 10, oxygen is supplied from the oxygen cylinder O to the container storage device 10, and the oxygen concentration around the culture container 20 in the container storage device 10 is a predetermined target value (culture container). Control is performed so that the target value is higher than the oxygen concentration in 20).

When the oxygen concentration in the container storage device 10 is first lowered, nitrogen is supplied from the nitrogen cylinder N to the container storage device 10, and the oxygen concentration around the culture container 20 in the container storage device 10 is a predetermined aim. Control is performed so as to be a value (a target value lower than the oxygen concentration in the culture vessel 20).
Then, when the oxygen concentration in the container storage device 10 is maintained at a low oxygen concentration, for example, the oxygen concentration in the container storage device 10 is increased by reducing the supply of nitrogen gas from the nitrogen cylinder N to the container storage device 10. Control is performed so that the target value becomes a predetermined target value (target value higher than the low oxygen concentration).

The gas supply from the gas cylinder to the container accommodating device 10 by the gas concentration control unit 40 can be controlled based on the number of times of opening and closing and the opening and closing time of the valve of the gas cylinder per unit time.
Specifically, in the case of a general CO ₂ incubator, it is made so as to leak slightly so that gas can be introduced, and the gas originally filled by a weak pressing force is introduced while being expelled from the chamber. For example, when maintaining the gas concentration in the container accommodating device 10, the gas concentration is monitored every few seconds, and when the gas concentration differs from the target value, the valve of the gas cylinder is opened for a moment to the container. Gas can be supplied to the accommodating device 10. If the gas concentration in the container accommodating device 10 has a difference of 1% or more from the target concentration, the opening time is 5 seconds, and if there is a difference of 1% to 0.2%, the opening time is 1 second. In the case of less than that, gas can be supplied to the container accommodating device 10 by setting the opening time to 0.5 seconds or the like.

Further, in the present embodiment, the gas concentration control unit 40 maintains the difference between the oxygen concentration around the culture container 20 in the container storage device 10 and the oxygen concentration in the culture container 20 at 5% or more. , It is preferable to control the oxygen supply from the oxygen cylinder O to the container accommodating device 11.

Here, when the cell density in the culture container 20 is about 1 million cells / cm ² , the oxygen concentration in the culture container 20 is usually 10% or more lower than the oxygen concentration in the container storage device 10. I know it.
Therefore, when culturing at a high density of 1 million cells / cm ² or more, the oxygen concentration in the container accommodating device 10 is controlled so as to be a target value of, for example, 25% to 40%, and the inside of the culture container 20. It is preferable to bring the oxygen concentration of the above to 21%.

Further, when culturing in the culture vessel 20 at a low oxygen concentration of about 5%, the oxygen concentration in the culture vessel 20 is the oxygen in the container accommodating device 10 even if the cell density is as low as about 100,000 cells / cm ² . It is experimentally known that the concentration is about 2% lower than the concentration.
Therefore, in such a case, it is preferable to control the oxygen concentration in the container accommodating device 10 to be, for example, a target value of 7% so that the oxygen concentration in the culture container 20 approaches 5%.

As described above, according to the present embodiment, a predetermined target value is set as the oxygen concentration in the container storage device 10 according to the cells to be cultured and the cell density, and the oxygen concentration in the container storage device 10 is set as the target value. By controlling so as to be, it is possible to finely adjust the oxygen concentration in the culture vessel 20. The specific target value is not limited to these, and can be set as appropriate. This also applies to the following embodiments.

The method for producing cells of this embodiment is characterized in that cells are produced using the cell culture system 1 of this embodiment described above.

The gas concentration control method of the present embodiment is a gas concentration control method in cell culture that controls the gas concentration in the culture solution filled in a closed culture container having gas permeability, and the culture container 20 is used for the culture. It is closedly housed in a container accommodating device 10 that changes the gas concentration around the container 20, and the oxygen concentration acquisition unit 30 acquires the oxygen concentration in the culture container 20 and is connected to the oxygen concentration acquisition unit 30. The control unit 40 supplies oxygen from the oxygen supply device O that supplies oxygen to the container storage device 10 to the container storage device 10 based on the acquired oxygen concentration, and oxygen around the culture container 20 in the container storage device 10. It is characterized in that the concentration is controlled so as to be a target value higher than the oxygen concentration in the culture vessel 20.

Further, in the gas concentration control method of the present embodiment, the gas concentration control unit 40 supplies nitrogen from the nitrogen supply device N that supplies nitrogen to the container storage device 10 to the container storage device 10 based on the acquired oxygen concentration. Is preferably controlled so that the oxygen concentration around the culture vessel 20 in the container storage device 10 becomes a target value higher than the oxygen concentration in the culture vessel 20.

According to this embodiment, the gas concentration in the container storage device is maintained in a normal state by controlling the supply of oxygen gas and nitrogen gas to the container storage device according to the cells to be cultured and the cell density. It can be controlled to a high oxygen concentration or a low oxygen concentration. Therefore, it is possible to finely and appropriately control the oxygen concentration in the container through the gas permeable film of the culture container.

[Second Embodiment]
The cell culture system of the present embodiment includes a carbon dioxide concentration acquisition unit for acquiring the carbon dioxide concentration in the culture container and a carbon dioxide supply device arranged so as to be able to supply carbon dioxide to the container storage device, and gas concentration control is provided. The unit is connected to the carbon dioxide concentration acquisition unit, and is different from the first embodiment in that the carbon dioxide supply from the carbon dioxide supply device to the container storage device is controlled based on the acquired carbon dioxide concentration. Other points are the same as those of the first embodiment except for the points described below.

Specifically, as shown in FIG. 2, the cell culture system 1a of the present embodiment includes a container accommodating device 10a, an oxygen concentration acquisition unit 30a, a gas concentration control unit 40a, a carbon dioxide concentration acquisition unit 50a, an oxygen cylinder O, and the like. It is equipped with a nitrogen cylinder N and a carbon dioxide cylinder C. The cell culture system 1a of the present embodiment is used by accommodating the culture vessel 20a in the container accommodating device 10a.

Oxygen gas, nitrogen gas, and carbon dioxide gas are supplied to the container accommodating device 10a from an oxygen cylinder O (oxygen supply device), a nitrogen cylinder N (nitrogen supply device), and a carbon dioxide cylinder C (carbon dioxide supply device), respectively. As a result, the gas concentration inside the cylinder is controlled, and the gas concentration around the culture vessel 20a can be adjusted.

An oxygen cylinder O for supplying oxygen gas, a nitrogen cylinder N for supplying nitrogen gas, and a carbon dioxide cylinder C for supplying carbon dioxide gas are connected to the container accommodating device 10a via valves, respectively, and a gas concentration control unit is used. The opening / closing operation of each valve is controlled by the control of 40a, the supply of gas from each gas cylinder to the container accommodating device 10a is controlled, and the oxygen concentration and the carbon dioxide concentration in the container accommodating device 10a can be adjusted. It has become.

The carbon dioxide concentration acquisition unit 50a is a device for acquiring the carbon dioxide concentration in the culture vessel 20a, and can be configured by using a PLC, a microcomputer, a computer, or the like.
As the carbon dioxide concentration acquisition unit 50a, a unit that acquires the carbon dioxide concentration using a carbon dioxide concentration sensor can be used. In this case, as shown in FIG. 2, the carbon dioxide concentration sensor 51a is attached to the culture container 20a housed in the container storage device 10a. Then, the carbon dioxide concentration sensor 51a and the carbon dioxide concentration acquisition unit 50a are connected, the dissolved carbon dioxide concentration in the culture solution in the culture vessel 20a is measured by the carbon dioxide concentration sensor 51a, and the measured information is used as the carbon dioxide concentration. Input to the acquisition unit 50a.
In addition, in the same manner as the oxygen concentration acquisition unit 30 described above in the first embodiment, the carbon dioxide concentration acquisition unit 50a may acquire the carbon dioxide concentration based on the number of cells and the culture time.

The gas concentration control unit 40a is connected to the oxygen concentration acquisition unit 30a and the carbon dioxide concentration acquisition unit 50a, and inputs the oxygen concentration information acquired by the oxygen concentration acquisition unit 30a from the oxygen concentration acquisition unit 30 and also inputs the carbon dioxide concentration. Information on the carbon dioxide concentration acquired by the acquisition unit 50a is input from the carbon dioxide concentration acquisition unit 50a, and the gas supply from the gas cylinder to the container accommodating device 10 is controlled based on this information.

That is, when the oxygen concentration in the container accommodating device 10a is increased, control is performed so that oxygen is supplied from the oxygen cylinder O to the container accommodating device 10a, and when the oxygen concentration in the container accommodating device 10a is decreased, control is performed. Control is performed so that oxygen is supplied from the nitrogen cylinder N to the container accommodating device 10a.
Further, since the concentration of carbon dioxide in the atmosphere is about 0.04% and the concentration of dissolved carbon dioxide in the culture solution suitable for cell culture is about 5%, carbon dioxide is transferred from the carbon dioxide cylinder C to the container storage device 10a. Is controlled to supply.

Here, as described above, when culturing at a high density of 1 million cells / cm ² or more, the oxygen concentration in the container accommodating device 10a is controlled so as to be a target value of, for example, 25% to 40%. , It is preferable to bring the oxygen concentration in the culture vessel 20a close to 21%.
Further, when culturing in the culture vessel 20a at a low oxygen concentration of about 5%, the oxygen concentration in the container accommodating device 10a is controlled so as to be a target value of, for example, 7%, and the oxygen concentration in the culture vessel 20a is controlled. Is preferably close to 5%.

Further, when culturing at a high density of 1 million cells / cm ² or more, the carbon dioxide concentration in the container accommodating device 10a is controlled so as to be a target value of, for example, 3%, and the carbon dioxide in the culture container 20a is controlled. It is preferable to bring the carbon concentration close to 5%, and when culturing at a low density of 100,000 cells / cm ² or more, the carbon dioxide concentration in the container accommodating device 10a should be a target value of, for example, 4.5%. It is preferable to control the carbon dioxide concentration in the culture vessel 20a to be close to 5%.

As an example of gas concentration control of the container accommodating device 10a, there are three cases: (1) maintaining the gas concentration in a normal state, (2) maintaining a low oxygen concentration, and (3) maintaining a high oxygen concentration. The method can be mentioned.

(1) When maintaining the gas concentration in a normal state, it is preferable to control the gas concentration in the culture vessel 20a to, for example, 21% oxygen and 5% carbon dioxide.
In this case, the gas concentration control unit 40a does not supply oxygen and nitrogen, but supplies carbon dioxide from the carbon dioxide cylinder C to the container storage unit 10a based on the carbon dioxide concentration information from the carbon dioxide concentration acquisition unit 50a. , The carbon dioxide concentration in the culture vessel 20a can be controlled to be 5%.
Specifically, for example, when the capacity of the container accommodating device 10a is 50 L, about 2 L of carbon dioxide gas is supplied to the container accommodating portion 10a.

(2) When maintaining a low oxygen concentration, it is preferable to control the gas concentration in the culture vessel 20a to, for example, 5% oxygen and 5% carbon dioxide. It has been reported that cardiomyocytes were efficiently induced by culturing iPS cells at a low oxygen concentration (Patent No. 6429280).
In this case, the gas concentration control unit 40a does not supply oxygen, but supplies nitrogen from the nitrogen bomb N to the container storage unit 10a based on the oxygen concentration information from the oxygen concentration acquisition unit 30a, and is in the culture container 20a. The oxygen concentration can be controlled to be 5%.

Further, carbon dioxide is supplied from the carbon dioxide cylinder C to the container container 10a based on the information of the carbon dioxide concentration from the carbon dioxide concentration acquisition unit 50a, and the carbon dioxide concentration in the culture container 20a is controlled to be 5%. can do.
Specifically, for example, when the capacity of the container accommodating device 10a is 50 L, about 40 L of nitrogen gas and about 3 L of carbon dioxide gas are supplied to the container accommodating portion 10a.

(3) When maintaining a high oxygen concentration, it is preferable to control the gas concentration in the culture vessel 20a to, for example, 40% oxygen and 5% carbon dioxide.
In this case, the gas concentration control unit 40a does not supply nitrogen, but supplies oxygen from the oxygen cylinder O to the container storage unit 10a based on the oxygen concentration information from the oxygen concentration acquisition unit 30a, and enters the culture container 20a. The oxygen concentration can be controlled to be 40%.

Further, carbon dioxide is supplied from the carbon dioxide cylinder C to the container container 10a based on the information of the carbon dioxide concentration from the carbon dioxide concentration acquisition unit 50a, and the carbon dioxide concentration in the culture container 20a is controlled to be 5%. can do.
Specifically, for example, when the capacity of the container accommodating device 10a is 50 L, about 15 L of oxygen gas and about 3 L of carbon dioxide gas are supplied to the container accommodating portion 10a.

Further, when the gas concentration is maintained in a normal state, the following procedure can be executed, for example, as a control example when the cell density in the culture vessel 20a is as low as about 100,000 cells / cm ² .
(A) The oxygen concentration acquisition unit 30a acquires information that the oxygen concentration has become (decreased), for example, 19%.
(A) The gas concentration control unit 40a inputs this information from the oxygen concentration acquisition unit 30a to supply oxygen gas from the oxygen cylinder O to the container storage device 10a, and the oxygen concentration in the container storage device 10a is, for example, 23. It is controlled to be the target value of% (21% + 2%).

(C) The carbon dioxide concentration acquisition unit 50a acquires information that the carbon dioxide concentration has become (increased), for example, 5.5%.
(D) The gas concentration control unit 40a inputs this information from the carbon dioxide concentration acquisition unit 50a to supply carbon dioxide gas from the carbon dioxide cylinder C to the container storage device 10a, and carbon dioxide in the container storage device 10a. The concentration is controlled to be, for example, a target value of 4.5% (5% -0.5%).

Further, when the gas concentration is maintained in a normal state, the following procedure can be executed, for example, as a control example when the cell density in the culture vessel 20a is as high as 2 million cells / cm ² .
(F) The oxygen concentration acquisition unit 30a acquires information that the oxygen concentration has become (decreased), for example, 2%.
(G) The gas concentration control unit 40a inputs this information from the oxygen concentration acquisition unit 30a to supply oxygen gas from the oxygen cylinder O to the container storage device 10a, and the oxygen concentration in the container storage device 10a is, for example, 40. It is controlled to be the target value of% (21% + 19%).

(H) The carbon dioxide concentration acquisition unit 50a acquires information that the carbon dioxide concentration has become (increased), for example, 7%.
(K) The gas concentration control unit 40a inputs this information from the carbon dioxide concentration acquisition unit 50a to supply carbon dioxide gas from the carbon dioxide cylinder C to the container storage device 10a, and carbon dioxide in the container storage device 10a. The concentration is controlled to be, for example, a target value of 3% (5% -2%).

Further, in the case of maintaining a low oxygen concentration, when the information that the oxygen concentration has become low is acquired by the oxygen concentration acquisition unit 30a, the gas concentration control unit 40a inputs the information of the oxygen concentration from the oxygen concentration acquisition unit 30a. Therefore, by reducing the amount of nitrogen gas supplied from the nitrogen bomb N to the container accommodating device 10a, it is possible to control the oxygen concentration in the container accommodating device 10a to increase.

Specifically, as a control example when the cell density in the culture vessel 20a is as low as 100,000 cells / cm ² , the following procedure can be executed, for example.
(S) The oxygen concentration acquisition unit 30a acquires information that the oxygen concentration has become (decreased), for example, 4%.
(S) The gas concentration control unit 40a inputs this information from the oxygen concentration acquisition unit 30a to reduce the supply of nitrogen gas from the nitrogen cylinder N to the container storage device 10a, so that the oxygen concentration in the container storage device 10a is increased. For example, the target value of 6% (5% + 1%) is controlled.

(S) The carbon dioxide concentration acquisition unit 50a acquires information that the carbon dioxide concentration has become (increased), for example, 5.5%.
(C) The gas concentration control unit 40a inputs this information from the carbon dioxide concentration acquisition unit 50a to supply carbon dioxide gas from the carbon dioxide cylinder C to the container storage device 10a, and carbon dioxide in the container storage device 10a. The concentration is controlled to be, for example, a target value of 4.5% (5% -0.5%).

Further, as a control example when the cell density in the culture vessel 20a is as high as 2 million cells / cm ² , the following procedure can be executed, for example.
(T) The oxygen concentration acquisition unit 30a acquires information that the oxygen concentration has become (lowered), for example, 0%.
(H) The gas concentration control unit 40a inputs this information from the oxygen concentration acquisition unit 30a to reduce the supply of nitrogen gas from the nitrogen cylinder N to the container storage device 10a, so that the oxygen concentration in the container storage device 10a is increased. For example, the target value of 8% (5% + 3%) is controlled.

(T) The carbon dioxide concentration acquisition unit 50a acquires information that the carbon dioxide concentration has become (increased), for example, 7%.
(T) The gas concentration control unit 40a inputs this information from the carbon dioxide concentration acquisition unit 50a to supply carbon dioxide gas from the carbon dioxide cylinder C to the container storage device 10a, and carbon dioxide in the container storage device 10a. The concentration is controlled to be, for example, a target value of 3% (5% -2%).

The method for producing cells of the present embodiment is characterized in that cells are produced using the cell culture system 1a of the present embodiment described above.

The gas concentration control method of the present embodiment is characterized by the following method in addition to the method of the first embodiment.
That is, in the gas concentration control method of the present embodiment, the carbon dioxide concentration acquisition unit 50a acquires the carbon dioxide concentration in the culture vessel 20a, and the gas concentration control unit 40a connected to the carbon dioxide concentration acquisition unit 50a acquires the carbon dioxide concentration. The carbon dioxide supply from the carbon dioxide supply device C that supplies carbon dioxide to the container storage device 10a to the container storage device 10a is based on the carbon dioxide concentration, and the carbon dioxide concentration around the culture container 20a in the container storage device 10a. It is characterized by controlling so that is the target value.

According to the present embodiment as described above, the gas concentration in the container storage device is increased by controlling the supply of oxygen gas, nitrogen gas and carbon dioxide gas to the container storage device according to the cells to be cultured and the cell density. Can be controlled. Therefore, it is possible to finely and appropriately control the oxygen concentration and the carbon dioxide concentration in the container through the gas permeable film of the culture container.

[Third Embodiment]
The cell culture system of the present embodiment is provided with an internal storage device for closedly storing the culture container in the container storage device, and is configured to control the gas concentration in the internal storage device to perform cell culture. It differs from the second embodiment. Other points are the same as those of the second embodiment except for the points described below.

Specifically, as shown in FIG. 3, the cell culture system 1b of the present embodiment includes a container accommodating device 10b, an oxygen concentration acquisition unit 30b, a gas concentration control unit 40b, a carbon dioxide concentration acquisition unit 50b, an oxygen cylinder O, and the like. It is equipped with a nitrogen cylinder N and a carbon dioxide cylinder C.
Further, an internal storage device 12b is provided in the container storage device 10b, and the culture container 20b is closedly housed in the internal storage device 12b and used for cell culture.

As the internal accommodating device 12b, for example, a small gas control chamber or the like can be used.
Oxygen gas, nitrogen gas, and carbon dioxide gas are supplied to the internal accommodating device 12b from an oxygen cylinder O (oxygen supply device), a nitrogen cylinder N (nitrogen supply device), and a carbon dioxide cylinder C (carbon dioxide supply device), respectively. As a result, the gas concentration inside the cylinder is controlled, and the gas concentration around the culture vessel 20b can be adjusted.

An oxygen cylinder O for supplying oxygen gas, a nitrogen cylinder N for supplying nitrogen gas, and a carbon dioxide cylinder C for supplying carbon dioxide gas are connected to the internal accommodating device 12b via valves, respectively, and a gas concentration control unit is provided. The opening / closing operation of each valve is controlled by the control of 40b, the supply of gas from each gas cylinder to the internal storage device 12b is controlled, and it is possible to adjust the oxygen concentration and the carbon dioxide concentration in the internal storage device 12b. It has become.

According to the cell culture system and the gas concentration control method of the present embodiment as described above, the gas concentration inside the container is controlled by using the internal storage device 12b having a capacity smaller than that of the container storage device 10b. It is possible to reduce gas consumption as compared with the cell culture system of the second embodiment.

[Fourth Embodiment]
The cell culture system of the present embodiment is provided with a plurality of internal storage devices for closedly storing the culture container in the container storage device, and cell culture is performed by controlling the gas concentration in each internal storage device. It differs from the second embodiment in that it has a configuration. Other points are the same as those of the second embodiment except for the points described below.

Specifically, as shown in FIG. 4, the cell culture system 1c of the present embodiment includes a container accommodating device 10c, an oxygen concentration acquisition unit 30c, a gas concentration control unit 40c, a carbon dioxide concentration acquisition unit 50c, an oxygen cylinder O, and the like. It is equipped with a nitrogen cylinder N and a carbon dioxide cylinder C.
Further, the container accommodating device 10c is provided with internal accommodating devices 12c-1, 12c-2, 12c-3. Then, the culture vessels 20c-1, 20c-2, and 20c-3 are closedly housed in each internal storage device and used for cell culture. The number of internal storage devices in the container storage device 10c is not limited, and may be two or four or more.

As the internal accommodating devices 12c-1 to 12c-3, for example, a small gas control chamber or the like can be used.
The internal accommodating devices 12c-1 to 12c-3 are provided with oxygen gas, nitrogen gas, and carbon dioxide from the oxygen cylinder O (oxygen supply device), the nitrogen cylinder N (nitrogen supply device), and the carbon dioxide cylinder C (carbon dioxide supply device), respectively. By supplying the carbon gas, the gas concentration inside the carbon gas is controlled, and it is possible to individually adjust the gas concentration around each of the culture containers 20c-1 to 20c-3.

Oxygen cylinder O for supplying oxygen gas, nitrogen cylinder N for supplying nitrogen gas, and carbon dioxide cylinder C for supplying carbon dioxide gas are connected to the internal accommodating devices 12c-1 to 12c-3 via valves, respectively. The opening / closing operation of each valve is controlled by the control of the gas concentration control unit 40c, the supply of gas from each gas cylinder to the internal accommodating devices 12c-1 to 12c-3 is controlled, and the internal accommodating devices 12c-1 to 12c-1 to It is possible to adjust the oxygen concentration and the carbon dioxide concentration in 12c-3, respectively.

According to the cell culture system and the gas concentration control method of the present embodiment as described above, a plurality of internal storage devices 12c are provided in the container storage device 10c, and the culture container 20c is housed in each internal storage device 12c to accommodate cells. By culturing, it is possible to individually control the gas for each culture container 20c.

Hereinafter, the tests conducted to confirm the effects of the cell culture system, the cell production method, and the gas concentration control method according to the embodiment of the present invention will be described.

[Test 1]
First, a test was conducted to confirm whether the oxygen concentration in the culture vessel could be appropriately controlled when high-density culture was performed by the cell culture system and the gas concentration control method of the present embodiment.
Specifically, as a culture container, a culture bag made of linear low-density polyethylene having a thickness of 110 μm, an outer size of 120 mm × 65 mm, and a bottom area of the culture space of 48 cm ² (manufactured by Toyo Kan Group Holdings Co., Ltd.). ) Was prepared. This culture bag is filled with 48 ml of AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) containing 2% FBS as a culture solution, and 2 million Jurkat (cell line derived from human leukemia T cells) as test cells / The seeds were sown at a high density of cm ² .

In addition, a non-contact oxygen sensor (PreSens, Oxy-4) was arranged in this culture bag, and the non-contact oxygen sensor was connected to the oxygen concentration acquisition unit. Further, the oxygen concentration acquisition unit and the gas concentration control unit are connected, and the oxygen supply from the oxygen cylinder O to the CO ₂ incubator (PHC Corporation, model MCO-5M-PJ) can be controlled by the gas concentration control unit.

Then, this culture bag is placed on a mounting table of a CO ₂ incubator and housed in a closed manner, and cell culture is performed at 37 ° C. with the oxygen concentration in the incubator remaining at 21%, which is the oxygen concentration in the atmosphere. At the same time, the measurement of the oxygen concentration input to the oxygen concentration acquisition unit from the non-contact oxygen sensor was started.

Approximately 190 minutes after the start of measurement when the oxygen concentration was maintained at about 10%, the gas concentration control unit controlled the oxygen concentration in the incubator to increase to the target value of 33% (oxygen concentration set value). Was changed from 21% to 33%), and oxygen gas was supplied from the oxygen cylinder to the container accommodating device. The change in oxygen concentration in the culture bag is shown in FIG.

As shown in the figure, as a result of culturing the cells at high density, the oxygen concentration in the culture vessel decreased to about 10%, which was a concentration at which efficient culture could not be performed. After that, by supplying oxygen gas into the incubator as described above, the equilibrium between the oxygen consumption of the cells and the gas permeation into the culture vessel can be maintained, and the oxygen concentration in the culture vessel is 21%. I was able to maintain it properly.

[Test 2]
Next, a test was conducted to confirm whether the cell culture efficiency was improved when high-density culture was performed by the cell culture system and the gas concentration control method of the present embodiment.
Specifically, as a culture container, a culture bag made of linear low-density polyethylene having a thickness of 110 μm, an outer size of 120 mm × 65 mm, and a bottom area of the culture space of 48 cm ² (manufactured by Toyo Kan Group Holdings Co., Ltd.). ) Was prepared.
Each culture bag was filled with 48 ml of AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) containing 2% FBS as a culture solution, and 3 million Jurkat (cell line derived from human leukemia T cells) as test cells. The seeds were sown at a high density of / cm ² .

Then, one of the culture bags is placed on the mounting table of the CO ₂ incubator and housed in a closed manner, and the oxygen supply from the oxygen cylinder O to the CO ₂ incubator can be controlled by the gas concentration control unit as in Test 1. , Cell culture was performed at 37 ° C.
At this time, the gas concentration control unit controls the oxygen concentration in the incubator to increase to the target value of 35% (changes the oxygen concentration setting value from 21% to 35%), and the oxygen cylinder O is used as a container accommodating device. Oxygen gas was supplied to. (Example 1).

In addition, the cells in the other culture bag were placed on a mounting table of a CO ₂ incubator and housed in a closed manner, and the cells in the incubator remained at 21%, which is the oxygen concentration in the atmosphere, at 37 ° C. Culturing was performed (Comparative Example 1).
Then, using these culture bags, the entire amount of the culture solution was exchanged twice a day, and the cells were cultured for 3 days. The change in cell density in the culture bag is shown in FIG.

As shown in the figure, after 3 days of culturing, the oxygen density in the culture vessel of Example 1 was about 8.2 million / ^cm2 , whereas the oxygen density in the culture vessel of Comparative Example 1 was 6.45 million. It was about / ^cm2 , and it was found that the cell proliferation efficiency of Example 1 was improved by nearly 30% as compared with Comparative Example 1.

It goes without saying that the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention. For example, the specific control content of the gas concentration is not limited to the illustrated one, and can be appropriately changed according to the purpose of the culture.

The present invention can be suitably used when cells are mass-cultured at high density using a cell culture bag.

The documents described in this specification and the contents of the Japanese application specification, which is the basis of the priority given to Paris in the present application, are all incorporated herein by reference.

1,1a, 1b, 1c Cell culture system 10,10a, 10b, 10c Container storage device 11,11a Mounting table 12b, 12c-1 to 12c-3 Internal storage device 20,20a, 20b, 20c-1 to 20c-3 Culture container 21, 21a Culture section 22, 22a Port 30, 30a, 30b, 30c Oxygen concentration acquisition section 31, 31a Oxygen concentration sensor 40, 40a, 40b, 40c Gas concentration control section 50a, 50b, 50c Carbon dioxide concentration acquisition section 51a Carbon dioxide concentration sensor O Oxygen cylinder (oxygen supply device)
N Nitrogen cylinder (nitrogen supply device)
C carbon dioxide cylinder (carbon dioxide supply device)

Claims (12)

1. A cell culture system that controls the gas concentration in a culture solution filled in a closed culture vessel with gas permeability.
   A container accommodating device that closely stores the culture container and changes the gas concentration around the culture container.
   An oxygen concentration acquisition unit that acquires the oxygen concentration in the culture vessel,
   An oxygen supply device arranged so as to be able to supply oxygen to the container storage device,
   A cell culture system including a gas concentration control unit connected to the oxygen concentration acquisition unit and controlling oxygen supply from the oxygen supply device to the container storage device based on the acquired oxygen concentration. ..
2. The container accommodating device is provided with a nitrogen supply device arranged so as to be able to supply nitrogen.
   The cell culture system according to claim 1, wherein the gas concentration control unit controls the supply of nitrogen from the nitrogen supply device to the container storage device based on the acquired oxygen concentration.
3. A carbon dioxide concentration acquisition unit that acquires the carbon dioxide concentration in the culture vessel,
   The container accommodating device is provided with a carbon dioxide supply device arranged so as to be able to supply carbon dioxide.
   A claim characterized in that the gas concentration control unit is connected to the carbon dioxide concentration acquisition unit and controls the supply of carbon dioxide from the carbon dioxide supply device to the container storage device based on the acquired carbon dioxide concentration. Item 2. The cell culture system according to Item 1 or 2.
4. Provided with the culture vessel
   A carbon dioxide concentration sensor for measuring the carbon dioxide concentration in the culture container is arranged in the culture container, and the carbon dioxide concentration sensor is connected to the carbon dioxide concentration acquisition unit.
   The cell culture system according to claim 3, wherein the carbon dioxide concentration acquisition unit acquires the carbon dioxide concentration in the culture vessel based on the input information from the carbon dioxide concentration sensor.
5. Provided with the culture vessel
   An oxygen concentration sensor for measuring the oxygen concentration in the culture container is provided in the culture container, and the oxygen concentration sensor is connected to the oxygen concentration acquisition unit.
   The cell culture system according to any one of claims 1 to 4, wherein the oxygen concentration acquisition unit acquires the oxygen concentration in the culture vessel based on the input information from the oxygen concentration sensor.
6. Provided with the culture vessel
   A camera for inputting an image obtained by photographing cells in the culture vessel to the oxygen concentration acquisition unit is connected to the oxygen concentration acquisition unit.
   Claims 1 to 4 are characterized in that the oxygen concentration acquisition unit calculates the number of cells based on the occupied area of cells in the image, and calculates the oxygen concentration in the culture vessel based on the obtained number of cells. The cell culture system according to any one of.
7. Provided with the culture vessel
   The oxygen concentration acquisition unit calculates the number of cells in the culture vessel based on the cell culture time in the culture vessel, and calculates the oxygen concentration in the culture vessel based on the obtained number of cells. The cell culture system according to any one of claims 1 to 4.
8. From the oxygen supply device, the gas concentration control unit can maintain the difference between the oxygen concentration around the culture vessel in the container accommodating device and the oxygen concentration in the culture vessel at 5% or more. The cell culture system according to any one of claims 1 to 7, wherein the oxygen supply to the container accommodating device is controlled.
9. A method for producing cells, which comprises culturing cells using the cell culture system according to any one of claims 1 to 8.
10. A gas concentration control method in cell culture that controls the gas concentration in a culture solution filled in a closed culture vessel having gas permeability.
    The culture container is closedly stored in a container storage device that changes the gas concentration around the culture container.
    The oxygen concentration acquisition unit acquires the oxygen concentration in the culture vessel and obtains the oxygen concentration.
    The gas concentration control unit connected to the oxygen concentration acquisition unit supplies oxygen from the oxygen supply device that supplies oxygen to the container storage device to the container storage device based on the acquired oxygen concentration. A gas concentration control method characterized in that the oxygen concentration around the culture vessel in the accommodating device is controlled to be a target value higher than the oxygen concentration in the culture vessel.
11. Based on the acquired oxygen concentration, the gas concentration control unit supplies nitrogen from the nitrogen supply device that supplies nitrogen to the container storage device to the container storage device around the culture container in the container storage device. The gas concentration control method according to claim 10, further comprising controlling the oxygen concentration of the above to a target value higher than the oxygen concentration in the culture vessel.
12. The carbon dioxide concentration acquisition unit acquires the carbon dioxide concentration in the culture vessel and obtains the carbon dioxide concentration.
    The gas concentration control unit connected to the carbon dioxide concentration acquisition unit supplies carbon dioxide to the container storage device based on the acquired carbon dioxide concentration, and the carbon dioxide from the carbon dioxide supply device to the container storage device. The gas according to claim 10 or 11, wherein the carbon supply is controlled so that the carbon dioxide concentration around the culture vessel in the container accommodating device is controlled to be a target value lower than the carbon dioxide concentration in the culture vessel. Concentration control method.
    

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