JPH0728715B2 - Culture liquid supply method and culture device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for supplying a culture solution to an incubator for culturing cultured cells and the like, and a culture apparatus for carrying out this culture solution supply method. .

(Prior Art) In recent years, research has been actively conducted on a culture device for reproducing a biochemical reaction carried out in a living cell in an artificial container. With such a culture device, it is possible to create an artificial environment for carrying out in-vivo reactions,
Further, it efficiently and continuously produces a large amount of a substance useful for human society, such as immunoglobulin, which has been obtained by culturing and producing a seed culture in vivo in animals such as mice. It becomes possible.

Currently known culture methods include jar fermenter culture, roller bottle culture, membrane culture, and the like, and improvement studies on the culture apparatus for each method are underway.

FIG. 3 is a block diagram schematically showing a configuration example of a conventional culture device using a membrane.

This culture device is equipped with an incubator using a membrane (for example, hollow fiber) and a culture solution tank. From this culture solution tank, a predetermined culture solution is supplied to the incubator, and desired cells are supplied in the culture device. It is constructed so that it can be cultured.

In FIG. 3, reference numeral 11 denotes a hollow fiber, and this hollow fiber 11 is mainly composed of a flow passage 11a through which a culture solution supplied from a culture solution tank 13 passes and a culture chamber 11b in which cells are cultured. . Also, the flow passage 11a and the culture chamber 1
It is separated from 1b by a membrane capable of fractionating a predetermined molecular weight. Because it has such a partition 11c,
It is possible to prevent cells cultured in 1b from entering the flow passage 11a, supply the culture solution flowing in the flow passage 11a to the culture chamber 11b, and remove waste products of the culture chamber 11b from the flow passage. You can take it out to 11a.

Further, the culture solution tank 13 stores a culture solution in which pH (hydrogen ion concentration), DO (oxygen dissolved concentration) and the like are adjusted to have predetermined values. In addition, a culture apparatus having a means for automatically adjusting pH, DO and other parameters in a culture solution tank is also conventionally known.

Reference numeral 14 denotes a drive source for supplying the culture solution from the culture tank 13 to the incubator 11. The drive source 14 causes the culture solution to flow in from one side of the flow passage 11a of the incubator 11 and flow out from the other side.
As the drive source 14, conventionally, a peristaltic pump, a bellows pump, or a magnetic pump has been used.

By the way, the supply of the culture solution to the incubator 11 when a peristaltic pump is used as the drive source 14 is performed by sliding the outer surface of the silicon tube or the like provided in this pump with a sliding means to remove the culture solution inside the ironing tube. It was done by moving.

Further, the incubator 11 using a bellows pump as the drive source 14
The culture solution was supplied to the bellows by feeding the culture solution stored in the bellows to the outside of the bellows according to the expansion and contraction of the bellows.

When a magnetic pump is used as the drive source 14, the culture solution is supplied to the incubator 11 by rotating a magnetic rotor provided inside the pump by a magnetic force from outside the pump to culture by the rotational force of the rotor. This was done by moving the liquid.

(Problems to be Solved by the Invention) However, when a peristaltic pump is used as a drive source, the culture solution is moved by a sliding means,
The tube is damaged during long-term culture, which causes problems such as stopping the supply of the culture solution, draining the culture solution to a place other than the incubator, and contaminating the culture chamber at the time of damage. there were.

Therefore, in order to prevent the occurrence of such a problem, maintenance such as periodically moving the sliding means to a new position of the tube is required.

Further, since the tube is squeezed by the sliding means, the inner wall of the tube may be worn and the tube material may be mixed into the culture solution. Therefore, there is a problem in that it is necessary to take measures such as filtering the tube material mixed in the culture solution.

Further, due to the mechanism of the peristaltic pump, when the culture solution is supplied to the incubator, a pulsating flow is generated in the culture solution flow, so that there is a problem in that the supply of the culture solution to the incubator becomes irregular.

Further, when the bellows pump or the magnetic pump is used, the drawbacks of the peristaltic pump can be alleviated, but there is a problem that maintenance and operation of the pump are required.

Further, in the conventional culture device, particularly, one using a membrane such as an ultrafiltration membrane, the flow direction of the culture solution in the flow passage of the incubator was only one direction. Therefore, when the cells used for culturing are adherent cells, the cells adhering to the area corresponding to the upstream of the flow of the culture solution in the culture chamber ingest the pH-adjusted culture solution, and as a result, the culture in the area corresponding to the downstream. The liquid is likely to contain waste products generated by the metabolism of cells attached to the upstream side. When the cells used for culturing are floating cells, the flow of the culture solution facilitates transport of these cells to the downstream region. Therefore, there is a problem that a non-uniform culture environment is locally produced, which causes a deviation in the growth density of the culture.

An object of the first invention of this application is to solve the above-mentioned problems and to provide a method capable of stably supplying a culture solution at a constant flow rate for a long period when supplying the culture solution to an incubator.

An object of the second invention of this application is to provide a culture apparatus which can realize the culture solution supply method according to the first invention and has a configuration in which damage to parts is unlikely to occur.

(Means for Solving the Problems) In order to achieve these objects, according to the first invention of the present application, at least the first and second methods are used for supplying the culture solution to the incubator using gas pressure. 2) two culture solution supply / collection tanks are prepared, and each of the first and second culture solution supply / collection tanks is managed so as to have a gas phase and a culture solution phase. At least one incubator is connected to each culture liquid phase in the liquid supply / collection tank, and the pressure of one gas phase of the first and second culture liquid supply / collection tanks is changed to that of the gas phase of the other tank. The culture liquid is supplied to the incubator from one of the tanks at a pressure higher than the pressure, and the culture liquid is collected in the other tank, and the culture liquid phase in the one or the other tank in the supplying and collecting is increased. Level is a predetermined level When the temperature reaches the maximum, the high and low states of the gas phase pressure in the first and second culture solution supply and recovery tanks are reversed, and the culture solution is supplied from the other tank to the incubator and the culture solution is supplied. Is collected in the one tank, and the culture liquid phase is continuously supplied to the incubator by sequentially repeating the culture liquid phase level detection processing and the gas phase pressure high / low state inversion processing.

In carrying out the present invention, it is preferable to supply the culture solution by temporally switching the pumping direction of the culture solution. That is, the culture fluid flow in the incubator so that the culture fluid flows in from one side of the incubator and flows out from the other side during a certain time period, and flows in from the other side and flows out from one side during the next time period. It is preferable that the direction of is switched every time an arbitrary time elapses.

In addition, it is preferable that the switching time of the pumping direction is set to an arbitrary time depending on the kind of the seed culture (eg, animal skin, cells forming an organ, etc.).

Further, the culture device of the second invention of this application is for incubating a culture solution, a culture solution supply unit for supplying the culture solution to the culture apparatus, and for feeding the culture solution from the culture solution supply section to the culture apparatus under pressure. And a gas pressure supply unit for supplying gas pressure.

The above-mentioned culture solution supply unit will be specifically described with an example. The culture solution supply unit includes a culture solution adjustment tank equipped with means for adjusting pH, DO value, etc. of the culture solution, and first and second tanks for supply / collection / replenishment of the culture solution. Equipped with. Further, the culture solution supply unit includes separate flow paths for connecting the culture solution supply / collection first and second tanks and the culture solution adjustment tank, respectively, and these flow paths are selectively selected as required. And a flow path switching means for enabling the above. Further, the culture solution supply unit includes a flow path for connecting the culture solution adjusting tank and one side of the incubator, a flow path for connecting the culture solution adjusting tank and the other side of the incubator, and these. A flow path switching means for activating one of the flow paths is provided. Further, the culture solution supply unit includes a flow path for connecting between the first and second culture solution supply / collection tanks and the incubator, and one culture solution supply / collection tank and the culture solution adjustment tank. And a flow path switching means for switching the flow paths so that the culture solution from the incubator is collected in the other culture solution supply / collection tank when the flow path between them is effective. In addition, it is preferable that the above-mentioned flow path switching means is comprehensively managed by a control unit described later.

The gas pressure supply unit has a gas pressure generation unit including, for example, first and second atmospheric pressure chambers and a compressor for making the atmospheric pressures in the first and second atmospheric pressure chambers different from each other. However, the atmospheric pressure of both atmospheric pressure chambers is configured such that the atmospheric pressure of the first atmospheric pressure chamber is higher than that of the second atmospheric pressure chamber. Further, this gas pressure supply unit is provided with a plurality of gas supply passages for sending the gas from the respective atmospheric pressure chambers to the necessary portions of the culture apparatus including the above-mentioned culture solution supply unit, and switching for switching these gas passages. And means.

(Operation) According to the configuration of the first invention described above, the culture solution is pressure-fed by gas pressure and supplied to the incubator. Therefore, by making the gas pressure constant, the flow rate of the culture solution can be made a constant amount corresponding to the gas pressure, so that the flow of the culture solution can be prevented. Of course, one of the first and second culture solution supply / collection tanks was used as the culture solution supply source and the other was supplied as the culture solution recovery destination, and then the other tank was used as the culture solution supply source. Further, the supply of the culture solution is repeated so that one of the tanks serves as the collection destination of the culture solution, so that the continuous supply of the culture solution to the incubator can be stably performed for a long period of time. Furthermore, the supply amount of the culture solution can be controlled by changing the gas pressure.

Further, the amount of gas dissolved in the culture solution can be easily controlled by changing the type and / or the composition of the gas serving as the pressure source for supplying the culture solution.

Further, when the culture solution is supplied by temporally switching the pumping direction of the culture solution, the flow direction of the culture solution in the incubator is switched to the opposite direction accordingly. Therefore, the flow of the switched culture solution causes the inside of the culture chamber of the incubator to be agitated, so that the culture environment also changes and the culture conditions in the culture chamber can be made uniform.

Further, according to the configuration of the culture device of the above-mentioned second invention, the culture solution is supplied from the culture solution supply section to the incubator by the gas pressure of the gas pressure supply section.

Specifically, if a distribution path of the first atmospheric pressure chamber-first tank for supplying and recovering culture liquid-culture liquid adjusting tank-incubator-second tank for supplying and recovering culture liquid-second atmospheric pressure chamber is configured. The culture solution is pressure-fed from the first tank for supplying and collecting the culture solution to the second tank for supplying and collecting the culture solution via the culture solution adjusting tank and the incubator by the pressure difference between the first and second atmospheric pressure chambers.

Further, a flow path for connecting the culture solution adjusting tank and one side of the incubator, a flow path for connecting the culture solution adjusting tank and the other side of the incubator, and either one of these flow paths. A flow path switching means is provided which is provided with an effective flow path switching means and which switches the flow path so that the side of the incubator opposite to the side to which the culture solution is supplied is connected to the culture solution supply / collection tank. Therefore, the cell culture can be performed while the flow direction of the culture solution in the incubator is temporally switched to the opposite direction.

(Example) Hereinafter, the culture solution supply method of this invention and the culture apparatus suitable for implementing this supply method are demonstrated with reference to drawings.

Description of Embodiment of Culture Device FIG. 1 is a block diagram showing an embodiment of a culture device according to the present invention with respect to its main part. It should be noted that this figure is only schematically shown so that the present invention can be understood,
Each block having a function of performing sterilization, cleaning, maintenance, etc. of the culture device is omitted. Furthermore, the arrangement relationship of each component is not limited to the illustrated example.

<Incubator> In FIG. 1, reference numeral 11 denotes an incubator, and in the case of this embodiment, for example, a conventionally known hollow fiber is used. As already described with reference to FIG. 3, this hollow fiber 11 has a flow passage 11a for a culture solution and a culture chamber 11b, and a partition 11c capable of molecular weight fractionation between them. Equipped with. still,
In order to maintain the hollow fiber 11 at a desired environmental temperature, the culture device in the case of this embodiment comprises a first temperature control means 13 constituted by, for example, a water jacket.

Further, the hollow fiber 11 has an inlet 15 for receiving a seed culture and a product outlet 17, and the product outlet 17 is provided with a product storage tank 9 via a manual valve MV1.
Connect 1

<Culture liquid supply unit> Reference numeral 21 denotes a culture liquid supply unit. In the case of this embodiment, the culture solution supply unit 21 supplies the culture solution to the culture vessel and the culture solution adjusting tank 23 having a function of adjusting the pH and DO of the culture solution, and returns from the culture vessel. First and second tanks 25a, 25b for supplying and recovering the culture solution for recovering the culture solution (hereinafter, may be abbreviated as first tank 25a and second tank 25b, respectively), incubator 11, adjustment A culture solution flow channel provided in a predetermined relationship (details will be described later) between the tank 23, the first tank 25a, and the second tank 25b, and a switching means for switching these channels according to the purpose (details will be described later). Yes.) And. Further, in order to detect the upper and lower limits of the amount of culture solution stored, the culture solution adjustment tank 23 has a liquid level indicator LI2.
The first tank 25a has a liquid level gauge LI1 and the second tank 25b has a liquid level gauge LI3.

Further, in the case of this embodiment, the flow path switching means is used as the culture solution supply unit 21.
A valve (details of which will be described later) provided in the middle of a plurality of flow paths provided therein and a control unit 81 including functional means for opening and closing these valves are configured.

In the case of this embodiment, the above-mentioned valve is configured to be driven by the gas pressure from the gas pressure supply unit 61, but is not limited to this and may be configured to be electromagnetically driven. . Incidentally, these valves are indicated by AV1 to AV13 in FIG.

Next, the connection relationship of the culture fluid transfer pipes (flow paths) that are provided between the above-mentioned constituent components will be described.

A flow path 31 between the first tank 25a and the culture solution adjusting tank 23,
The valve AV1 and the channel 32 are connected to each other. The second tank 25b and the culture solution adjusting tank 23 are connected by the passage 33, the valve AV4 and the passage 34.

A flow path 35 having a flow meter 27 between the culture solution adjusting tank 23 and one side of the flow path 11a of the incubator 11, a valve AV7, a flow path 36, and a valve A.
Connection is made by the path of V9 and the flow path 37. Further, the culture solution adjusting tank 23 and the other side of the flow passage 11a of the incubator 11 are connected by a passage having a flow meter 27, a valve AV8, a passage 38, a valve AV10 and a passage 39. .

Further, the flow passage 31 and the flow passage 36 are connected via the valve AV3 and the flow passage 41, and the flow passage 33 and the flow passage 36 are connected via the valve AV5 and the flow passage 42.

Further, the flow passage 31 and the flow passage 38 are connected via the valve AV2 and the flow passage 43, and the flow passage 33 and the flow passage 38 are connected via the valve AV6 and the flow passage 44.

On the other hand, a separately prepared culture medium supply tank 51 is connected to the second tank 25b via the sterilizing filter F5, the valve AV11 and the flow path 45. Furthermore, the culture solution supply tank 51 is provided with a sterilizing filter F6,
It is connected to the first tank 25a via the valve AV12 and the flow path 46.

In addition, in the case of this embodiment, the flow passage 48 is provided in the culture medium adjusting tank 23.
Oxygen or carbon dioxide or nitrogen is supplied via
In addition, an alkaline aqueous solution (in this embodiment, 7.5% NaHCO 3
₃ ) is supplied from the storage tank indicated by 50 in FIG. 1, and the pH and DO are adjusted by these gases and chemicals. In FIG. 1, 29a indicates a pH meter sensor and 29b indicates a dissolved oxygen meter sensor. The gas pressure in the culture solution adjusting tank 23 may change due to the introduction of oxygen, carbon dioxide, or nitrogen gas, and as a result, the liquid level may change. Therefore, the culture solution adjusting tank 23 A liquid level gauge LI4 and a valve V8 for venting gas are provided in the to enable to deal with an abnormal state of the liquid level during gas introduction. The culture solution adjusting tank 23 also functions as a buffer device for suppressing flow rate fluctuations and the like when switching the flow paths.

Further, unnecessary culture liquid can be discarded from the culture liquid adjusting tank 23 via the flow path 47, the valve AV13 and the sterilizing filter F7.

In the case of this embodiment, the above-mentioned culture solution supply section and the incubator 11 already described are housed in a constant temperature bath 19 equipped with a second temperature control means.

<Gas pressure supply unit> 61 indicates a gas pressure supply unit. In the case of this embodiment, the gas pressure supply unit 61 includes a first and second atmospheric pressure chambers 63a and 63b, and a compressor 65 for making the atmospheric pressures in the atmospheric pressure chambers 63a and 63b different from each other. The generating unit 62 is included. Then, the gas from the outside, such as air, is filtered by the filter F1.
Via the electromagnetic three-way valve SV1 to the copresser 65, and the compressor 65 pressurizes this gas and sends it to the first atmospheric pressure chamber 63a for storage. Further, the other flow path of the three-way valve SV1 is connected to the second atmospheric pressure chamber 63b. Therefore, the atmospheric pressure of the first atmospheric pressure chamber can be made higher than that of the second atmospheric pressure chamber.

Further, pressure indicators with pressure switches PSI1 and PSI2 are provided in the first and second atmospheric pressure chambers 63a and 63b, respectively, and these atmospheric pressure chambers are connected via a temporary pressure adjusting valve RV2. Since these atmospheric pressure chambers supply the gas pressure for feeding the culture solution, they can be set to predetermined atmospheric pressures. When the pressure in the first atmospheric pressure chamber 63a reaches a predetermined value, the primary pressure regulating valve RV2
Adjust the pressure inside the room. Also, the first pressure chamber 63a
When the pressure of is lower than the specified value, SV1 is switched to the air intake side (F1 side) and the air is replenished to the specified pressure. Even when the atmospheric pressure in the second atmospheric pressure chamber 63b becomes equal to or lower than a predetermined value, the three-way valve SV1 is switched to the air intake side to introduce gas, and the pressure inside the chamber is adjusted by the primary pressure adjusting valve RV2.

Further, between the first atmospheric pressure chamber 63a and the first tank 25a of the culture solution supply unit 21 is a gas flow path 71 having a pressure adjusting valve RV1, a solenoid valve V1, a solenoid three-way valve SV2 and a sterilizing filter F2. Providing a configured route. A path formed by a gas flow path 72 having a valve V2, a three-way valve SV3 and a filter F3 is provided between the second atmospheric pressure chamber 63b and the second tank 25b. Furthermore, the three-way valve SV2
And the gas flow path 72 are connected by a gas flow path 73, and the three-way valve SV3 and the gas flow path 71 are connected by a gas flow path 74.

According to this structure, one culture solution supply / collection tank of the culture solution supply unit and the first atmospheric pressure chamber 63a are connected, and the other culture solution supply / collection tank and the second atmospheric pressure chamber 63b are connected. Since they are connected, a differential pressure is generated between the culture solution supply / collection tanks 25a and 25b, and the culture solution can be pressure-fed by this differential pressure.

In the case of such a configuration, by switching the three-way valves SV2, SV3 respectively, the first and second atmospheric pressure chambers 63
The culture solution supply / collection tanks 25a and 25b connected to a and 63b can be changed to each other. Therefore, gas flow path switching means for switching the gas flow path is realized by these valves.

In the above example, the gas for pumping the culture solution is air. However, depending on the type of culture such as cells, for example, the amount of dissolved oxygen in the culture medium used for culture may have a great influence on the growth of cells or the production of products. In other words, depending on the type of culture, the culture in which the dissolved oxygen content of the culture medium is the optimal environment when the dissolved oxygen content is higher than the equilibrium dissolved oxygen content in the atmosphere,
On the contrary, there are some cultures where the lower one is the optimal environment.

Therefore, when culturing such a culture, it is preferable that the amount of dissolved oxygen in the culture solution to be used is also appropriate for the culture.

In response to such a request, the culture solution supply method of the present invention is
Since gas is used as an upper supply source for pressure-feeding the culture solution, the equilibrium dissolved oxygen amount of the culture solution, for example, can be easily controlled by changing the type of gas used and / or the composition of the gas. That is, for a culture in which the dissolved oxygen content of the culture solution is higher than the equilibrium dissolved oxygen content in the atmosphere, the gas to be taken into the gas pressure generating unit via the filter F1 shown in FIG. For example, it can be dealt with by using air containing oxygen at a high concentration, which is obtained by mixing the atmosphere and oxygen. Further, for a culture in which the dissolved oxygen content of the culture broth prefers one that is lower than the equilibrium dissolved oxygen content in the atmosphere,
This can be dealt with by using, as the gas to be taken into the gas pressure generating portion, for example, air obtained by diluting the atmosphere with nitrogen gas and containing oxygen at a low concentration.

<Control Unit> The culture device according to the present invention includes a control unit for controlling each of the above-mentioned components. Since the structure of this control unit can be realized by a conventionally known control technique, it will be briefly described below.

In FIG. 1, reference numeral 81 denotes a control unit, and the control unit 81 is
For example, a microprocessor 83, a memory unit 85 for storing a program of a culture solution pressure feeding direction and a culture condition, and an input device for instructing a mode selection and a change of the pressure feeding direction and the culture condition.
87, the pressure data of the first and second atmospheric pressure chambers, the flow rate of the culture solution, the data such as pH and DO, and the culture solution volume data of each tank, and the reading of these data, pressure, pH,
An input / output (I / O) port 89 for outputting an instruction signal for correcting DO and operating each valve, and a display unit 90 for displaying various messages and the like are provided.

Description of Operation of Culture Device Hereinafter, an embodiment of the operation of the culture device of the present invention will be described. Incidentally, in the case of this embodiment, this operation is carried out by the following procedures. However, it is clear that the present invention is not limited to the following procedure. or,
It is apparent that the numerical conditions described in the following examples are merely examples, and various changes can be made depending on, for example, the type of seed culture.

Sterilization Supply of culture solution to the culture solution supply section Culture solution adjustment Injection of seed culture into incubator Supply of culture solution into incubator Sampling method of culture etc. during culture period In this example, incubator 11 For example, a molecular weight cutoff of 30 called “Visa Fiber II” manufactured by Grace, Inc. in the United States
000 holofiber cartridges are used.

<Sterilization> Prior to cell culturing, it is optional and suitable for the tanks 23, 25a, 25b and the flow passages in the inner part divided by the sterilizing filters F2, F3, F5, F6, F7, F9F10 and F11 related to the culture solution supply unit 21. Steam sterilization is performed at a temperature of about 120 ° C. for a certain period of time, for example, for 30 minutes.

<Supply of Culture Solution to Culture Solution Supply Unit> Next, the culture solution is supplied from the culture solution supply tank to the culture solution supply unit. In this case, this supply is from the culture solution supply tank 51 to one culture solution supply / collection tank, for example, the first tank 25.
First, the culture solution is replenished to a, and further the culture solution is supplied to the culture solution adjusting tank 23 via the first tank 25a and the second culture solution supplying / recovering second tank 25b via the culture solution adjusting tank 23. Do as an example.

Therefore, the valve V1 is opened, and the predetermined gas pressure adjusted by the pressure adjusting valve RV1 is supplied from the first atmospheric pressure chamber 63a to the flow path 75.
And the culture solution supply tank 51 via the sterilizing filter F8, while the second atmospheric pressure chamber 63b, the gas flow path 72, the second tank 25.
b, channel 33, AV6, channel 44, channel 38, AV8, channel 35, flow meter 27, culture solution adjusting tank 23, channel 32, so that the channels of AV1, and channel 31 become effective, Valve V2, three-way valve SV3, valve AV6, AV
By operating 8 and AV1 respectively, a pressure feeding system is formed between the culture solution supply tank 51, the first and second tanks 25a and 25b, and the culture solution adjusting tank 23. After forming the pumping system in this way,
The valve AV12 is opened and the culture solution is supplied from the culture solution supply tank 51 to the first tank 25a through the flow path 46, and the culture solution is pumped to the culture solution adjusting tank 23 and further to the second tank 25b through the first tank 25a. To do. The amount of the culture solution in the second tank 25b is controlled by the liquid level system LI3. If it is detected that the liquid level of the supplied culture solution has reached the lower limit level of LI3, valve A
Close V1, AV6 and AV8. Subsequently, in order to pump the culture solution from the culture solution supply tank 51 to the first tank 25a, the SV3 is switched to the flow path 74 side, and the second atmospheric pressure chamber 63b, V2, SV3, the flow path.
74, channel 71, filter F2, first tank 25a, channel 46 and A
Enable the V12 route. The liquid level of the culture solution in the first tank 25a is controlled by the liquid level meter LI1, and when it is detected that the liquid level has reached the upper limit level of LI1, AV12 and V6 are closed.

Next, the gas flow passage 71 is operated by operating the valve V1 and the three-way valve SV2 so that the gas pressure adjusted from the first atmospheric pressure chamber 63a to a predetermined pressure by the pressure adjusting valve RV1 (also referred to as the first gas pressure).
To the first tank 25a. Further, the valve V2 and the three-way valve SV3 are operated to supply the gas pressure in the second atmospheric pressure chamber 63b (also referred to as the second gas pressure) to the second tank 25b via the gas flow path 72. Further, AV1, AV8 and AV6 are opened, and the culture solution in the first tank 25a is pressure-fed to the second tank 25b through the culture solution adjusting tank 23. The amount of the culture solution in the culture solution adjusting tank 23 is controlled by the liquid level gauge LI2.

<Adjustment of culture solution> In the culture solution adjustment tank 23, various parameters such as pH and DO of the culture solution are measured by the respective sensors 29a and 29b. Further, these measurement results are taken into the control unit 81, and when the values of these parameters are values other than those suitable for the cultured cells, the culture solution is treated so as to have a predetermined value. For this treatment, for example, for DO, dissolved oxygen is measured with a dissolved oxygen meter, oxygen is supplied through the flow path 48 when supplementation of oxygen is required, and flow path 48 is used when the dissolved oxygen amount is desired to be lowered. Nitrogen is replenished to the culture solution adjusting tank 23 via. The pH value is measured with a pH meter, and if the pH is desired to be raised, the alkaline solution in the storage tank 50 is passed through the channel 49, and if the pH is desired to be lowered, the CO ₂ gas is passed through the channel 48. Supply each. For lowering the pH, an acidic aqueous solution may be used instead of CO ₂ gas. Further, since the culture solution adjusting tank 23 is housed in the second temperature control means (constant temperature bath) 19, the temperature of the culture solution in the culture solution adjusting tank 23 is controlled to a predetermined value. Further, the temperature of the culture solution in the incubator during culturing is managed with higher accuracy by the first temperature control means 13.

Here, the culture solution whose pH has been adjusted in the culture solution adjusting tank 23 is not directly supplied to the incubator 11 (AV9
And AV10 are kept closed) and circulated and stabilized in the culture solution supply unit 21.

To stabilize the culture solution, first, the flow path 35, valve AV8, valve A
The culture solution in the culture solution adjustment tank 23 is pressure-fed to the second tank 25b via V6 and the flow path 33. The upper limit level of the culture solution in the second tank 25b is controlled by the liquid level gauge LI3. After pumping the culture solution in the culture solution adjusting tank 23 to the second tank 25b, the valve AV6
Close and open AV4. At this time, the second gas pressure is supplied from the second atmospheric pressure chamber 63b to the first tank 25a, and the first gas pressure is supplied from the first atmospheric pressure chamber 63a to the second tank 25b, so that the three-way valve SV2, Operate SV3 etc. to switch the gas flow path. Furthermore, AV1 is closed and AV7 and AV3 are opened. Therefore, this time, the culture solution is pressure-fed from the second tank 25b to the culture solution adjustment tank 23, and the pH adjustment and the like described above are performed in the culture solution adjustment tank 23. Further, the adjusted culture solution is pressure-fed to the first tank 25a through the flow paths 35, AV7, flow paths 36, AV3, and the flow path 31.

Culture medium adjusting tank 23 and first and second tanks 25a and 25b
The circulation of the culture solution between and is cyclically performed as described above, and the culture solution is adjusted to a predetermined temperature, pH and DO.

<Injection of Seed Culture into Incubator> Next, for example, seed cells are introduced from the seed culture inlet 15 of the holofiber 11 into the culture chamber 11a in a treated environment where they are not contaminated. inject.

<Supply of Culture Solution to Incubator> Next, the culture solution is supplied to the incubator.

In the culture solution supply unit of the culture device, for example, the first gas pressure is supplied to the first tank 25a, the second gas pressure is supplied to the second tank 25b, and the supply is returned from the incubator 11. The case where the culture solution is collected in the second tank 25b (state shown in FIG. 1) will be described.

In such a case, in order to allow the culture solution to flow in both directions in the incubator, the culture solution in the first tank 25a is adjusted during a certain period of time during the cell culture. Through the tank 23, the flow path 35-valve AV7-flow path 36-valve AV9-flow path 37
-Incubator 11-Flow path 39-Valve AV10-Valve AV6-Flow path 33-Second tank 25b is pressure fed. Further, during the next time period, the culture solution in the first tank 25a is passed through the culture solution adjusting tank 23, and the flow path 35-valve AV8-flow path 38-valve AV10-flow path 39-incubator 11-flow path 37. -Valve AV9-Flow path 42-Valve AV5-Flow path 33-Second tank 25b
It is pumped by the route of. As a result, the flow of the culture solution during the culture period is bidirectional, that is, the case where it flows in from one side of the incubator and flows out from the other side, and the case where it flows in from the other side and flows out from one side. Can be
In addition, the number of times of switching and the switching cycle of the pumping direction are set to appropriate values according to the cultured cells.

Further, when the culture solution supplied to the incubator 11 is recovered in the second tank 25b and reaches the upper limit level of the second tank 25b,
This time, the first tank 25a is supplied with the second gas pressure, while the second tank 25b is supplied with the first gas pressure, and the incubator 1
By switching the above-mentioned culture solution supply flow passage so as to collect the culture solution returning from 1 in the first tank 25a, the same culture solution supply as in the above example can be performed.

Further, it is also possible to perform pressure feeding in only one direction without performing the operation of switching the flow direction of the culture solution in the incubator as described above.

<Sampling Method of Culture Product during Culture Period> In the case of this example, sampling is performed by the method described below in order to grow the culture product and evaluate the product.

In the mode in which the culture solution is supplied to the incubator 11, the AV9 (or AV10) that is downstream of the flow of the culture solution in the incubator 11 is closed, and the product take-out port 17 of the incubator 11 and the product storage tank 91 are closed. Open the MV1 of. By this operation, a differential pressure is generated between the culture solution adjusting tank 23 and the product storage tank 91, and thus the product in the culture chamber 11b is pressure-fed to the product storage tank 91 together with the culture solution. Cells and products sampled in this way, using a microscope for cells,
The products are observed using a predetermined reagent according to the products.

Further, by visually observing the culture in this way, by changing the pH and the amount of DO consumed, and visually observing the culture broth, the culture liquid exchange time can be determined, and the culture broth can be exchanged accordingly. . Further, a part of the culture solution is sampled through the path of the culture solution adjustment tank 23-flow path 47-AV13-sterilization filter F7, and the metabolites such as glucose and lactic acid in the sampled culture solution are quantified. Therefore, the changes in the consumed components and the waste products may be examined together, and the results may be used as information for determining the replacement time of the culture solution.

Modifications It is clear that the invention is not limited to the embodiments described above.

For example, in the above-described embodiment, the gas pressure generation unit 62 of the gas pressure supply unit 61 includes first and second atmospheric pressure chambers 63a and 63b, a compressor 65, a plurality of valves, and a pressure regulator. It has been described in the configuration example. However, this gas pressure generating unit 62
For example, a high-pressure gas cylinder 101 and a pressure regulator as shown in FIG. 2 may be provided, and a low-pressure chamber may not be provided, and instead, a direct connection to the atmospheric pressure may be made to provide a simpler configuration. I can. In the case of such a configuration, it is preferable to protect the sterilizing filters F2 and F3 by providing a coarse filter 103 at the inlet for introducing the atmosphere. Further, in a factory having facilities where various gases such as air and nitrogen are supplied as high-pressure gas, the gas pressure can be obtained from these facilities.

The gas taken into the gas supply unit is not limited to the atmosphere, and other suitable gas, for example, a gas such as N ₂ or Ar, or a mixed gas of these gases may be used.

Further, in the above-described embodiment, the example in which the culture solution supply unit includes the culture solution adjustment tank 23 has been described. However, the culture solution adjusting tank may be removed from the configuration, and instead, the pH of the culture solution may be adjusted in the culture solution supply / collection tank or in the flow path of the culture solution. The culture apparatus of the above-mentioned embodiment has been described as an example provided with means for adjusting the pH, DO, and liquid temperature of the culture solution. However, this culture device can also be provided with a means for adjusting other parameters, such as the dissolved concentration of CO ₂ , depending on the culture.

Further, operations such as supply of the culture solution and removal of the product can be performed in a suction mode with a negative gas pressure. In the following, the culture solution is supplied from the culture solution supply tank 51 to the first tank 25a.
An example of replenishing in suction mode will be briefly described.

In the case of this example, the flow path 75 between the sterile filter F8 and the valve V6 is removed, and the side of the F8 opposite to the culture solution supply tank 51 is opened to the atmosphere.

Compressor with SV1 switched to the second pressure chamber 63b side
Activate 65. V5 is open and V1 is closed.
The air in 63b is exhausted to the outside of the system via V5 to make the second atmospheric pressure chamber a negative pressure. After that, the second atmospheric pressure chamber 63b-V2-SV3-flow path 74
Each of the valves is switched so that the system of the flow passage 71-F2-the first tank 25a-the flow passage 46 becomes effective. Subsequently, when the AV 12 is opened, the culture solution in the culture solution supply tank 51 is pressure-fed to the first tank 25a in the suction mode through the flow path 46.

When the liquid level indicator LI1 of the first tank 25a detects that the culture solution in the first tank 25a has reached the upper limit after starting the pressure feeding, AV12 and V2 are closed.

Next, after the gas pressure generating section is brought into a state similar to the above-mentioned culture solution supply state, the first atmospheric pressure chamber 63a-RV1-V1-SV2-flow path 7
1-F2-First tank 25a-Flow passage 31-AV1-Flow passage 32- Culture medium adjusting tank 23-Flow meter 27-Flow passage 35-AV8-Flow passage 38-Flow passage 4
4-AV6-Flow channel 33-Second tank 25b-F3-Flow channel 72-SV3-V2
-When each valve is switched so that the system of the second atmospheric pressure chamber 63b becomes effective, the culture solution in the first tank 25a is supplied to the second tank 25b through the culture solution adjusting tank 23. The liquid level of the culture solution supplied to the second tank 25b is controlled by the liquid level gauge LI3, and when this liquid level reaches the lower limit, the entire system is closed and the gas pressure generating section is set to the suction mode state as described above. Return the culture solution in the culture solution supply tank 51 to the first tank 25 as described above.
supply to a. The amount of the culture solution in the first tank 25a is controlled by the liquid level gauge LI1, and when this liquid level reaches the upper limit, the AV12 is closed. By the above operation, the first tank 25a and the second tank 2
The liquid level of the culture solution replenished in 5b and the culture solution adjusting tank 23 is as shown in FIG.

In this way, the replenishment of the culture solution can also be performed in the suction mode.

Further, the culture solution supply unit and the gas pressure generation unit according to the present invention are not limited to the configuration of the embodiment, and various modifications can be made according to the way of supplying the culture solution.

For example, continuously introducing the culture solution from the outside of the culture device and continuously supplying the culture solution to the outside of the culture device, or continuously supplying the culture solution to the culture device while continuously supplying the culture solution to the culture device. Part of it can be circulated in the culture device or discharged to the outside of the culture device. Furthermore, while circulating the culture solution inside the culture device, part of it can be discharged to the outside of the culture device or supplied from the outside of the culture device. When it is desired to perform each of the above, it can be dealt with by configuring the culture device as shown in FIG. 4, for example.

FIG. 4 is a block diagram schematically showing an example of the structure of a culture apparatus having the function of supplying or circulating the culture solution in this manner. In addition, in FIG.
Constituent components similar to those shown in FIG. 1 are designated by the same reference numerals, but on the other hand, among the constituent components shown in FIG. Constituent components that are considered unnecessary, such as the control unit 81 and detailed constituent components, are omitted.

In FIG. 4, 162 indicates a gas pressure generating portion in the culture device of this modification. Gas pressure generation unit 162 in this case
In addition to the constituent components shown by 62 in FIG. 1 and already described, a negative pressure generating portion shown by 171 is added. In this case, the negative pressure generator 171 includes a vacuum pump 173, a third pressure chamber 65c, and
It has a three-way valve indicated by V4, solenoid valves indicated by V10 and V11, and a pressure indicator with a pressure switch indicated by PSI3. Then, by controlling the vacuum pump 173 according to the set value of PSI3, the gas pressure in the third atmospheric pressure chamber 63c can be set to a desired negative pressure. The third atmospheric pressure chamber 63c is connected to a portion between the V2 and the SV3 of the flow passage 72, which has already been described, by the gas flow passage 110 having an electromagnetic valve indicated by V12.

Here, the pressure inside the first atmospheric pressure chamber 63a is set to P ₁ , and the second atmospheric pressure chamber 6a
The pressure in the chamber of 3b is P ₂ , and the pressure in the chamber of the third atmospheric pressure chamber 63c is P _3.
When each of the above is expressed, the gas pressure generating unit 16 is arranged so that the pressures of the respective atmospheric pressure chambers satisfy the relation of P ₁ > P ₂ > 1 atmospheric pressure> P _3.
Operate the compressor, vacuum pump and valves of 2.

In this culture device including the gas pressure generating unit 162 having such a configuration, the gas pressure of P ₁ is supplied to the first tank 25a in response to the switching operation of the three-way valve SV2 and the three-way valve SV3, and the second tank 25b is supplied with P ₂ gas pressure, first tank 25a is supplied with P ₂ gas pressure and second tank 25b is supplied with P ₁ gas pressure, first tank 2
5a is supplied with the gas pressure of P ₁ and the second tank 25b is supplied with P _1.
3 gas pressure is supplied, or the first tank 25a
The gas pressure of P ₃ can be supplied and the gas pressure of P ₁ can be supplied to the second tank 25b.

Further, in FIG. 4, reference numeral 121 denotes a modified culture solution supply unit. The culture solution supply unit 121 is basically the same as the culture solution supply unit 21 shown in FIG. 1, but the arrangement of the culture solution flow paths and the arrangement of the valves are shown in FIG. It is different from the one.

In this case, the culture solution is supplied to the first tank 25a or the second tank 25b via the flow path indicated by 111 in FIG. 4, and the culture solution is further supplied from the first tank 25a or the second tank 25b. Culture medium adjusting tank 23-Flowmeter 27-Flow path 112-Flow direction switching bypass block (hereinafter also referred to as switching block) 123 shown in FIG. 4-Flow path 113 Can be done.

By the way, the above-mentioned switching block 123 is configured as described below.

A flow path 36 having AV7, a flow path 38 having AV8, a flow path 41 having AV3, and a flow path 44 having AV6 form a loop-shaped flow path, and the flow paths 36 and 38 are Channel 11 at the confluence
2, the flow channel 113 is connected to the confluence of the flow channel 41 and the flow channel 44, respectively. Furthermore, between the confluence of the flow paths 36 and 41 and the confluence of the flow paths 38 and 44, AV9, incubator 11 and A
It has a system composed of V10. Therefore, by switching the operation of each valve according to the purpose, this switching block 123
Also, as described in FIG. 1, it is possible to circulate the culture solution without switching the flow direction of the culture solution or supplying the culture solution to the incubator 11. To specifically explain such a route, the channel 112-AV7-AV9-incubator 11-AV10
-AV6-path for supplying the culture solution to the incubator 11 in the order of the flow path 113, or flow path 112-AV8-AV10-incubator 11-AV9-A
V3-A path for supplying the culture solution to the incubator 11 in the order of the flow path 113. Also, a bypass path that bypasses the incubator 11 consisting of the path 112-AV7-AV3-flow path 113, or the flow path 11
It is a similar bypass path composed of 2-AV8-AV6-channel 113.

Next, specific examples of use of the culture apparatus shown in FIG. 4 will be described separately for each mode of pumping the culture solution.

<Use Example 1> When the culture solution is continuously taken into the apparatus from the outside of the culture apparatus, the culture solution is continuously supplied to the incubator 11, and the culture solution is continuously discharged to the outside of the culture apparatus.

Gas at the pressure of P ₁ is stored in the first tank 25a and P _{3 is} stored in the second tank 25b.
Each valve is operated so that the gas of each pressure is supplied. Furthermore, AV11-channel 111-AV5-second tank 25b-
When the operation for activating the flow path is performed, the culture solution can be pressure-fed from the outside of the culture apparatus to the second tank 25b in the suction mode via the culture solution supply / supply line on the primary side of AV11. On the other hand, the first tank 25a-AV1-culture solution adjustment tank
23-Flowmeter 27-Route 112-Switching block 123-Flow path 113-A
When the path of V13 is made effective, the culture solution in the first tank 25a can be discharged to the outside of the culture device by positive pressure feeding through the path described above.

In addition, the gas having the pressure of P ₃ is stored in the first tank 25a and the second tank 25b.
Operate each valve so that the gas at the pressure of P ₁ is supplied to each. Furthermore, AV11-channel 111-AV2-first tank 25
When the operation for enabling the path of a is performed, the culture solution can be pressure-fed from the outside of the culture device to the first tank 25a in the suction mode. On the other hand, the second tank 25b-AV4-culture solution adjustment tank 23-flow meter 27-flow path 112-switch block 123-flow path
When the path of 113-AV13 is made effective, the culture solution in the second tank 25b can be discharged to the outside of the culture apparatus by positive pressure feeding through the above-mentioned path including the switching block 123.

Liquid level gauges LI1 to L
By switching and using under the control of I3, the supply and discharge of the culture solution can be performed by the method of Use Example 1.

<Use Example 2> When continuously circulating the culture solution taken into the culture device.

Since the method described in Use Example 1 can be used to take the culture solution into the culture device, the description thereof will be omitted.

Gas at the pressure of P ₁ is stored in the first tank 25a and P _{2 is} stored in the second tank 25b.
Each valve is operated so that the gas of each pressure is supplied. Furthermore, the first tank 25a-AV1-culture medium adjustment chamber 23-
Flowmeter 27-Flow path 112-Switch block 123-Flow path 113-AV14
-Flow path 111-AV5-When the operation of enabling the path of the second tank 25b is performed, the culture solution in the first tank 25a is fed to the second tank 25b by positive pressure feeding through the path including the switching block 123. Can be pumped.

In addition, the gas at the pressure of P ₂ is stored in the first tank 25a and the second tank 25b.
Operate each valve so that the gas at the pressure of P ₁ is supplied to each. In addition, the second tank 25b-AV4-culture medium preparation room
23-Flowmeter 27-Flow path 112-Switch block 123-Flow path 113-A
When the operation of enabling the path of V14-flow path 111-AV2-first tank 25a is performed, the culture solution in the second tank 25b is fed to the first tank 25a by positive pressure feeding through the path including the switching block 123. Can be pumped to.

Liquid level gauges LI1 to L
By switching and using it under the control of I3, the culture solution is circulated and pumped by the method of Use Example 2. still,
If the AV 13 is opened / closed as necessary while circulating the culture solution in this way, a part of the culture solution being circulated can be used without stopping the supply of the culture solution to the incubator 11. It can also be discharged to the outside.

Further, by appropriately switching between the operation of the above-mentioned Use Example 1 and the operation of Use Example 2, the culture solution is supplied to the culture apparatus from the outside of the culture apparatus without stopping the supply of the culture solution to the incubator 11. It can also be replenished.

As described above, according to the culture solution supply method of the present invention, continuous liquid supply is also possible.

In the culture device shown in FIG. 4, the gas pressure generation source is a compressor or a vacuum pump, but this gas pressure generation source is a gas pressure obtained from factory piping or the like, a gas pressure obtained from a pressurized cylinder, or the like. Can be any other suitable one, such as

Further, various modifications can be made to the culture solution supply section and the gas pressure supply section shown in the above-mentioned embodiments and modifications within the scope of the object of the present invention.

Features of the Invention According to the culture solution supply method or the culture apparatus of the present invention as described above, various kinds of cultured cells such as those attached to the culture chamber and those suspended in the culture chamber are used. Can be done. Regarding the supply of the culture solution to the incubator, it can be performed under two conditions: when the flow direction of the culture solution in the incubator is fixed in one direction and when the flow direction is switched to the opposite direction (bidirectional). I can.

For example, when culturing cells as cells that adhere to the culturing chamber and cells that float in the culturing chamber and culture is performed in one direction and two directions in the culture medium, the following Table 1 It seems that the features shown in each can be found.

In addition, according to the culture apparatus of the present invention, it is possible to sufficiently control these conditions even when the various parameters of the culture solution are set to the values shown in the following items, respectively.

Culture temperature: ± 0.1 ° C against set temperature DO value: ± 0.1 ppm according to cell growth amount ± 0.1 against set value (Effect of the invention) As is clear from the above description, According to the culture solution supply method and the culture device, the culture solution is pressure-fed using the gas pressure when the culture solution is supplied to the incubator. Of course, one of the first and second culture solution supply / collection tanks was used as the culture solution supply source and the other was supplied as the culture solution recovery destination, and then the other tank was used as the culture solution supply source. Further, the supply of the culture solution is repeated so that one of the tanks serves as the collection destination of the culture solution.

Therefore, the culture solution can be stably supplied to the incubator for a long period of time and smoothly without the veins of the culture solution.

Furthermore, according to the culture device of the present invention, since there is no mechanical sliding portion, maintenance is easy and the device life is long. Furthermore, there is no part where the culture medium is in mechanical contact. Therefore, even if the gas pressure supply unit fails,
Maintenance can be performed without impurities being mixed in the culture solution.

Further, when the culture solution is supplied to the incubator, the flow of the culture solution in the incubator is switched in the opposite direction with respect to time,
The culture environment can be made uniform throughout the culture room, and the culture yield can be improved.

Further, the culture yield can be further improved by changing the type of gas for pressure-feeding the culture solution or adjusting the gas composition according to the type of culture.

In this manner, by using the culture solution supply method and the culture device of the present invention, the culture for producing a substance useful for human beings such as immunoglobulin such as a monoclonal antibody, cells, etc. is performed more efficiently than before. You can

[Brief description of drawings]

FIG. 1 is a block diagram showing a main part of an embodiment of a culture apparatus of the present invention, FIG. 2 is a block diagram showing a modified example of a gas pressure supply unit according to the present invention, and FIG. 3 is a conventional culture apparatus. FIG. 4 is a block diagram schematically showing a configuration example, and FIG. 4 is a block diagram showing a main part of another example of the culture device of the present invention. 11 …… incubator, 11a …… flow passage 11b …… culture chamber, 11c …… partition (membrane capable of molecular weight fractionation) 13 …… first temperature control means (water jacket) 15 …… seed culture medium intake 17 …… Product outlet 19 …… Second temperature control means (constant temperature tank) 21 …… Culture solution supply unit, 23 …… Culture solution adjusting tank 25a …… First tank for supplying and collecting culture solution 25b …… Culturing Second tank for liquid supply and recovery 27 …… Flowmeter, 29a …… pH sensor 29b …… DO sensor 31-39,41-47,111,112,113 …… Culture fluid flow path 48 …… Gas flow path, 49 …… Chemical solution flow path 50 …… Chemical solution storage tank, 51 …… Culture solution supply tank 61 …… Gas pressure supply section, 62 …… Gas pressure generation section 63a …… First atmospheric pressure chamber, 63b …… Second atmospheric pressure chamber 63c …… Third atmospheric pressure Room, 65 ... Compressor 71 to 75,110 ... Gas flow path, 81 ... Control section 83 ... Microprocessor 85 ... Memory section, 87 ... Input device 89 ... I / O Ports, 90 ... Display section 91 ... Product storage tank 121 ... Alternative culture solution supply section 123 ... Culture solution flow direction switching and bypass block 162 ... Alternative gas pressure generation section 171 ... Negative Gas pressure generator 173 …… Vacuum pump AV1 to AV14 …… Gas drive valve LI1 to LI4 …… Liquid level gauge F1 to F12 …… Sterilization filter SV1 to SV4 …… Three-way valve PSI1 to PSI4 …… Pressure indicator with pressure switch RV1 to RV5 ...... Pressure adjusting valve V1 to V12 ...... Solenoid valve.

Claims (8)

[Claims] 1. When supplying a culture solution to a incubator using gas pressure, at least two first and second culture solution supply / collection tanks are prepared, and these first and second culture solution supplies are provided. Each of the combined recovery tanks is managed so as to have a gas phase and a culture solution phase, and at least one incubator is connected to each of the culture solution phases in the first and second culture solution supply and recovery tanks. The pressure of one gas phase of the first and second culture solution supply / collection tanks is made higher than the pressure of the gas phase of the other tank, and the culture solution is supplied from the one tank to the incubator and the culture solution is supplied. Is recovered in the other tank, and when the level of the culture solution phase in the one or the other tank reaches a predetermined level in the supply / recovery, the above-mentioned in the first and second culture solution supply / collection tanks Moth The high and low phase pressures are reversed, the culture solution is supplied from the other tank to the incubator, and the culture solution is recovered in the one tank, and the level detection process of these culture solution phases and the gas phase pressure A culture solution supply method, characterized in that the culture solution is continuously supplied to the incubator by sequentially repeating the high-low state inversion treatment. 2. The culture solution supply method according to claim 1, wherein the culture solution supply / collection tank on the side where the pressure of the gas phase is increased is always connected to one side of the incubator. A method for supplying a culture solution, which is characterized in that the supply of the culture solution in the incubator is made constant and the supply is performed. 3. The culture solution supply method according to claim 1, wherein the culture solution supply / collection tank on the side where the pressure of the gas phase is increased is switched to one side and the other side of the incubator at appropriate times. A method for supplying a culture solution, characterized in that the supply of the culture solution is performed by temporally switching the pressure-feeding direction of the culture solution in the incubator so as to be connected. 4. At least two first and second culture solution supply / recovery tanks in which a gas phase and a culture solution phase are formed, and these first and second culture solution supply / recovery tanks are connected. Incubator, a culture solution supply unit for supplying a culture solution to the incubator, and a gas phase pressure of one of the first and second culture solution supply / collection tanks to the other. A culture device comprising a gas pressure supply unit for increasing the pressure higher than that of the gas phase of the tank. 5. The culture apparatus according to claim 4, wherein the culture solution supply unit includes a plurality of the culture solution supply / collection tanks, and a plurality of tanks provided between these tanks and the incubator. And a switching means for switching these flow paths. 6. The culture apparatus according to claim 4, wherein the culture solution supply unit is provided between the at least two culture solution supply / collection tanks and the at least two tanks and the incubator. And a plurality of flow paths provided between the at least three tanks and between the at least three tanks and the incubator, and a switching means for switching the flow paths. Incubator. 7. The culture device according to claim 4, wherein the gas pressure supply unit includes a gas pressure generation unit and a plurality of gas pressure generation units provided between the gas pressure generation unit and the culture solution supply unit. An incubator comprising a gas flow path and a switching means for switching the gas flow paths. 8. The culture apparatus according to claim 4, wherein the gas pressure generating section includes a first and second atmospheric pressure chambers, and a compressor for making the atmospheric pressures in the atmospheric pressure chambers different from each other. A culturing apparatus comprising: