WO2017154123A1

WO2017154123A1 - Culture container, culture device, and culture method

Info

Publication number: WO2017154123A1
Application number: PCT/JP2016/057263
Authority: WO
Inventors: 鈴木　大介; 広斌周
Original assignee: 株式会社日立製作所
Priority date: 2016-03-09
Filing date: 2016-03-09
Publication date: 2017-09-14
Also published as: JPWO2017154123A1; JP6554602B2

Abstract

The purpose of the present invention is to provide: a culture container that, even if the culture container is moved from a space having a high degree of cleanliness to a space having a lower degree of cleanliness, enables culture operations to be performed after said movement without affecting the culture state; a culture device; and a culture method. Provided is a culture container including: first containers which include culture areas, and in which openings are formed; a second container which accommodates the first containers; and a lid which is controlled and operated from the outside of the second container to close the openings of the first containers. Also provided are: a culture device including the culture container; and a culture method.

Description

Cultivation container, culture apparatus, culture method

The present invention relates to a culture vessel, a culture apparatus, and a culture method.

As a background art in this technical field, there is JP-T-2008-505629 (Patent Document 1). In this publication, “the basic structure for culturing cells includes well-like culture positions (10) separated from each other. Lid (3b) that limits the air space common to the culture position (10) together with the basic structure. The culture base structure is hermetically sealed from above with a lid having a gas inlet and a gas outlet, in order to maintain the humidity of the air space, the water is the culture base structure outside the culture position. Is added to the bottom of the ”(see summary).

Special table 2008-505629

In the configuration described in Patent Document 1, after performing an operation of seeding a cell suspension in a culture region in a culture vessel or an operation of replacing a medium in the culture region, it is sealed with a lid member having a gas injection / discharge port. The culture vessel is connected to the gas source.

However, when the space for performing the culture operation and the space having the gas source are different spaces, it is necessary to move the culture vessel, for example, from a culture operation space with a high cleanliness to a space with a lower cleanness than this. By moving the culture container, the outer surface of the culture container may be contaminated.
For example, when it is necessary to move the culture vessel from a region with a high degree of cleanliness to a region lower than this, an operation for culturing by connecting to a supply source such as a gas source or a culture medium after the transfer As a result, the culture area in the culture container may be contaminated.

Therefore, the present invention enables a culture operation after movement without affecting the culture state even when the culture container is moved from a clean space to a clean space. An object of the present invention is to provide a culture container, a culture apparatus, and a culture method.

In order to solve the above problems, for example, the configuration described in the claims is adopted. The present application includes a plurality of means for solving the above-described problems. For example, a first container having a culture region and having an opening formed therein and a second container for accommodating the first container are provided. And a lid for closing the opening of the first container by operating and operating from the outside of the second container.

According to the present invention, even when the culture container is moved from a clean space to a clean space, the culture operation after the movement can be performed without affecting the culture state. A culture vessel, a culture device, and a culture method can be provided.

It is sectional drawing of a culture container. It is sectional drawing of the culture container of the state which closed the cover part. It is a bird's-eye view of a culture container. It is sectional drawing of the culture container which shows arrangement | positioning of a cover part, a 1st container, and a liquid supply pipe | tube. It is sectional drawing of the culture container with a filter. It is sectional drawing of the culture container which shows the structure of a 1st container and a cover part. It is a figure which shows the structure of a culture | cultivation control system apparatus. It is a figure which shows the sterilization process with a culture | cultivation control system apparatus. It is a figure which shows the washing | cleaning process with a culture | cultivation control system apparatus. It is a figure which shows each tank and flow path in a culture | cultivation control system apparatus. It is a figure which shows the opening / closing mechanism of a cover part. It is a flowchart when culture | cultivating using the culture | cultivation container using the culture | cultivation control system apparatus in a normal environment. It is a figure which shows the edge part shape of the opening part of the 1st container of a culture container. It is a figure which shows the shape of the 1st protrusion part edge part of the cover part of a culture container. It is sectional drawing of the culture container which used the bellows etc. for the member of the cover part. It is a figure which shows the modification of the opening / closing mechanism of a cover part.

Hereinafter, with reference to FIGS. 1 to 16, the culture container, the culture apparatus, and the culture method according to this embodiment will be described in detail. In the following examples, the culture vessel is described as being particularly used for cell culture, but is not limited thereto.

Fig. 1 shows the cross-sectional structure of the container divided vertically. The culture container 10 shown in the present embodiment is a double-structured container, and a plurality of first containers 1 for accommodating and culturing cells are arranged inside the outer second container 2. It has a structure. The upper surface of the first container 1 is open, and the medium and the like can be supplied and discharged from this opening.

The medium is a liquid that functions to supply the cells with sugar, proteins, amino acids, oxygen, and other substances necessary for cell survival and proliferation, and to receive metabolites excreted from the cells. The medium in the container needs to be changed regularly.

A plurality of through holes 22 are formed on the upper surface of the second container 2 in accordance with the number of first containers and the position of the first container in the second container. The lid portion 3 made of an elastic body is disposed. The lid 3 is for opening and closing the opening of the first container 1, and can open and close the opening of the first container 1 by operating the lid 3 from the outside of the culture vessel 10. is there.

The lid 3 is a first projection formed of a plurality of protrusions according to the number and arrangement position of the through holes 22 or according to the number of first containers and the arrangement position of the first container in the second container. The first protrusion is inserted into the through-hole 22 and is disposed so as to protrude into the second container 2. The lid 3 has a second protrusion that corresponds to the position of the first protrusion and protrudes in a direction opposite to the protrusion direction of the first protrusion.

As shown in FIG. 2, by pressing the second protrusion, the first protrusion can be moved toward the inside of the second container by the elastic force. Due to the movement of the protrusion, the end of the first protrusion contacts and closes the opening of the first container, so that the first container can be held hermetically.

When the pressure on the second protrusion is released, the first protrusion moves toward the outside of the second container 2 and the opening of the first container 1 is opened. In addition, each 2nd projection part can be moved independently, and according to the projection part which gave the pressing force, each opening part of the 1st container 1 can be closed.

As shown in FIG. 3, the size of the lid 3 is large enough to cover a plurality of through holes provided in the second container 2, and the lid 3 and the second container 2 are It is joined so as to maintain airtightness by an adhesive portion 6 provided over the entire circumference of the lid portion 3, the second container 2 is sealed, and the inside of the second container 2 is an outer liquid, It is a structure that can be shielded from gas and bacteria.

In addition, as a material of the elastic body of the cover part 3, what is necessary is just a material which does not generate | occur | produce even if it uses for cell culture, such as silicone and polyvinyl chloride.

As shown in FIG. 1, a liquid supply pipe 4 that can supply liquid from the outside to the culture region of the first container 1 is connected to the second container 2. The outlet is arranged at the upper part of the opening of the first container 1 so that the liquid can be introduced into the first container 1 from the opening of the first container 1. The liquid discharge ports are provided at a plurality of locations according to the number of the first containers and the arrangement position of the first container in the second container.

An example of the configuration of the liquid discharge port of the liquid supply pipe 4 is shown in FIG. FIG. 4 is a longitudinal section of the container in a direction different from that of FIG. When the medium is put into the first container 1, the medium is fed through the liquid supply pipe 4, and the first container is passed through the liquid input pipe 36 extending from the liquid supply pipe 4 to the opening of the first container 1. The liquid is supplied to the inside of the container along the edge of 1.

The liquid discharge port of the liquid supply pipe 4 is not in direct contact with the opening of the first container 1 so that the lid 3 does not interfere when closing the opening of the first container 1. . When the liquid is supplied from the liquid supply pipe 4 with the lid 3 closing the opening of the first container 1, the liquid is not supplied to the culture region of the first container 1 and supplied to the inside of the second container. Is done.

The second container 2 is connected to a gas supply / exhaust pipe 7 for taking gas into and out of the second container 2 from the outside. The second container 2 is connected to a liquid discharge pipe 8 for discharging liquid from the inside of the second container 2 from the outside.

The liquid supply pipe 4, the gas supply / discharge pipe 7, and the liquid discharge pipe 8 are provided with a path switch 5 that can open and close each path, and the path switch 5 is disposed outside the second container 2. Moreover, as shown in FIG. 5, the structure by which the filter 21 is connected to the gas supply / discharge pipe | tube 7 of a container may be sufficient. Even if dust or bacteria are mixed in the air supply gas, the filter 21 can remove the gas and send clean gas into the container.

FIG. 1 shows an example of a configuration in which a plurality of first containers 1 are arranged in a second container, but the number of first containers 1 may be one. As shown in FIG. 6, a plurality of openings may be formed in the first container 1, and in this case, a plurality of lids 3 are provided in accordance with each opening. Also good. In FIG. 6, when the first container 1 is filled with the culture medium, the medium is charged from the upper opening 34, and when the medium is discharged, the medium is discharged from the side lower opening 35.

FIG. 7 shows a configuration in the case where the container according to the embodiment of the present invention is installed in the culture control system device 15 and culture is performed. The culture control system device 15 is provided with a medium tank 11, a cleaning liquid tank 12, and a sterilant tank 13, and each tank is connected to a liquid supply pipe 26.

In addition, a pipe valve 17 is installed between each tank and the liquid supply pipe 26 so that the opening and closing of the pipe can be controlled. The medium tank 11 is filled with a medium suitable for cells to be cultured. The cleaning liquid tank 12 is filled with a cleaning liquid that does not affect the cells, such as pure water or phosphate buffered saline (PBS). The sterilant tank 13 is filled with a chemical sterilant (glutaraldehyde preparation, orthophthalaldehyde preparation, hypochlorous acid preparation, peracetic acid preparation, etc.).

The liquid supply pipe 4 of the culture vessel 10 is connected to the liquid supply pipe 26 of the culture control system device 15. A gas supply / discharge pipe 27 and a liquid discharge pipe 28 are installed in the apparatus, and are connected to the gas supply / discharge pipe 7 and the liquid discharge pipe 8 of the container, respectively.

At the place where the culture container 10 is installed, a container lid opening / closing machine 14 is installed to open and close the first container 1 by applying a pressing force to the second protrusion of the lid 3. The culture control system device 15 is controlled by the controller 16 and executes the culture process.

FIG. 8 shows the opening of the first container 1 by pressing the second protrusion of the lid 3 in a clean environment (Cell Processing Center, cleanliness ISO class 5, EU-GMP grade A). Liquid supply pipe 26 that moves to a normal environment outside the clean environment (with a cleanliness level of ISO class 7 or so) and supplies gas and liquid necessary for culture with the first container closed and sealed The sterilization process after connecting to the gas supply / discharge pipe 27 and the liquid discharge pipe 28 is shown.

When the liquid supply pipe 26, the gas supply / discharge pipe 27, and the liquid discharge pipe 28 are connected to the liquid supply pipe 4, the gas supply / discharge pipe 7 and the liquid discharge pipe 8 of the culture container 10 in a normal environment, the second container 2 The sterility inside the container, the liquid supply pipe 4, the gas supply / discharge pipe 7, the liquid discharge pipe 8, and the liquid supply pipe 26, the gas supply / discharge pipe 27, and the liquid discharge pipe 28 cannot be maintained.

In order to sterilize the liquid supply pipe 4, the gas supply / discharge pipe 7, the liquid discharge pipe 8 and the liquid supply pipe 26, the gas supply / discharge pipe 27, and the liquid discharge pipe 28 of the culture vessel 10, a sterilant is filled from the sterilant tank 13. Shows the state. At this time, since the inside of the first container 1 is sealed by the first protrusion of the lid 3, the inside of the second container 2 is sterilized without any sterilizing agent being mixed into the inside of the first container 1. can do.

FIG. 9 shows a state in which, after the sterilization step shown in FIG. 8, the sterilant is discharged from the liquid discharge pipe 28, and the remaining sterilant is removed with the cleaning liquid. Since the sterilizing agent also kills cells to be cultured, if it remains even a little, the culture will be affected. Therefore, the sterilizing agent needs to be completely removed.

The cleaning liquid is filled from the cleaning liquid tank 12 into the liquid supply pipe 4, the gas supply / discharge pipe 7, the liquid discharge pipe 8, and the liquid supply pipe 26, the gas supply / discharge pipe 27, and the liquid discharge pipe 28 of the culture control system device 15. And wash away the sterilant.

Note that the tanks of the culture control system device 15 are not necessarily connected by a common liquid supply pipe. In some cases, as shown in FIG. 10, a dedicated sterilant supply pipe 30 for feeding a sterilant is used. The form which has this may be sufficient.

FIG. 11 shows details of the pressing mechanism of the lid 3 on the culture vessel 10 shown in FIG. FIG. 11 shows a cross-sectional view of the culture vessel 10 divided in the vertical direction.

The pressing portion 31 is movable, and has a structure in which the first container 1 is sealed by pressing the lid portion 3 with a pressing spring 9. When releasing the pressing to the lid 3 and maintaining the opening of the first container 1, the pressing portion 31 is pulled up, the stopper 23 is put on, and the pressing portion 31 is not moved. In addition, although not shown in figure, the operation | movement of the press part 31 and the clasp 23 is controllable by a control mechanism.

Next, an operation flow when culturing is performed using the culture control system device 15 in a normal environment equipped with the culture vessel 10 will be described with reference to FIG. In step 1, cells are seeded inside the first container 1 in a clean space, and an appropriate medium according to the seeded cells is supplied.
In step 2, in the liquid supply pipe 4, the gas supply / discharge pipe 7 and the liquid discharge pipe 8 connected to the second container 2 of the culture container 10, the path switch 5 of each pipe is closed, and the second container 2 The inside of is closed.

In step 3, the second protrusion of the lid 3 is pushed in, the opening of the first container 1 is closed, and the first container 1 is closed. In step 4, the culture vessel 10 is installed in the culture control system device 15 with the lid 3 pressed and the first vessel 1 closed, and the liquid supply pipe 4 connected to the vessel and the culture control system. Liquid supply pipe 26 on the apparatus side, gas supply / discharge pipe 7 connected to the container and gas supply / discharge pipe 27 on the culture control system apparatus side, liquid discharge pipe 8 connected to the container and liquid on the culture control system apparatus side Each discharge pipe 28 is connected.

In step 5, each of the gas supply and discharge pipes 7 and the liquid supply pipe 4 that holds the state in which the opening of the first container 1 is closed by the lid 3 and is connected to the second container 2 of the culture container 10 is provided. Open the path switch 5. In step 6, the opening of the first container 1 is held closed by the lid 3, and the tube valve 17 of the sterilant tank 13 connected to the liquid supply tube 26 on the culture control system device 15 side is opened. The inside of the second container 2, the inside of the liquid supply pipe 26, and the inside of the connection portion between the liquid supply pipe 4 and the liquid supply pipe 26 are filled with sterilizing agent, and the tube valve of the sterilizing agent tank 13 is closed and filled. Hold time for the space to be sufficiently sterilized.

In step 7, after sterilization, the path switch 5 of the liquid discharge pipe 8 that holds the opening of the first container 1 closed by the lid 3 and is connected to the second container 2 of the culture container 10. In step 8 of discharging the filled sterilant, the state in which the opening of the first container 1 is closed by the lid 3 is maintained, the second container 2 of the culture container 10 and the liquid supply pipe 26 are maintained. In order to clean the sterilant remaining inside, the pipe valve 17 of the cleaning liquid tank 12 is opened, and the inside of the second container 2, the inside of the liquid supply pipe 26, and the inside of the connection portion between the liquid supply pipe 4 and the liquid supply pipe 26 are opened. Wash. For cleaning, the path switch 5 of the liquid discharge pipe 8 is closed, the cleaning liquid is held in the space in contact with the sterilizing agent, the pipe valve 17 of the cleaning liquid tank 12 is closed, and then the path switch 5 of the liquid discharge pipe 8 is cleaned. The process of opening and discharging the cleaning liquid is repeated about 2 to 10 times, or after holding the cleaning liquid in the cleaning space, the path switch 5 of the liquid discharge pipe 8 is opened and the cleaning liquid is 2 to 10 times the volume of the cleaning space. Take a method of flowing through a volume of about.

In step 9, after washing the sterilant, the liquid supply pipe 4 connected to the second container 2 of the culture container 10 and the path switch 5 of the liquid discharge pipe 8 are closed, and the inside of the second container 2 and In order to remove the cleaning agent remaining in the liquid supply pipe 26 on the culture control system device 15 side by drying, dry air is introduced from the gas supply / discharge pipe 7 connected to the second container 2 of the culture container 10.

In step 10, after completion of drying, a gas suitable for the culture conditions is passed through the gas supply / discharge pipe 7 connected to the second container 2 of the culture container 10, and the inside of the second container 2 of the culture container 10 is cultured. After adjusting to the environmental conditions, the lid 3 that has closed the opening of the first container 1 is opened, and the culture inside the first container 1 starts the culture process in the culture control system device 15.

In step 11, the medium in the first container 1 is exchanged by opening the lid 3 and tilting the culture container 10 so that the culture container 10 is discharged from the first container 1 to the second container 2 and then the culture container. 10 is returned to the normal posture, and the medium is filled into the first container 1 from the liquid supply pipe 4 connected to the second container of the culture container 10 through the liquid supply pipe 4 from the medium tank 11. The medium discharged into the second container 2 is discharged out of the culture container through the liquid discharge pipe 8 connected to the second container 2 of the culture container 10. After the opening of the first container 1 is closed with the lid 3, the medium remaining in the second container 2 is washed and dried in the same manner as the washing process of the remaining sterilant. To do.

In step 12, when culturing is completed and the cells are collected, the opening of the first container 1 is kept closed with the lid 3, the inside of the first container 1 is closed, and the main body of the culture container 10 In the liquid supply pipe 4, the gas supply / discharge pipe 7 and the liquid discharge pipe 8 connected to the second container 2, the path switch 5 of each pipe is closed and the inside of the second container 2 is closed. To do.

In step 13, while maintaining the inside of the second container 2 closed, the culture container 10 is removed from the culture control system device 15 and the outside of the culture container 10 is sterilized before moving to a clean environment. After moving to the clean environment, the lid 3 and the path switch 5 of each pipe may be opened.
In step 14, the first container 1 is removed from the second container 2 of the culture container 10, and the cells cultured in the first container 1 are collected.

As described above, according to the culture container, the culture apparatus, and the culture method according to Example 1 of the present invention, the culture container was moved from a high clean space to a lower clean space. However, the culture operation after movement can be made possible without affecting the culture state. In addition, after moving the culture container to a low clean space, for example, the culture container has a configuration that can be kept clean by sterilizing the culture container. It can be installed without being limited to a high degree of space, and the degree of freedom of configuration and layout in the cell processing facility can be increased.

FIG. 13 shows the structure of the open end of the first container 1 of the culture container 10. As shown in FIG. 13, the first container 1 has a wall structure in which a part of the opening end portion protrudes. This wall structure is formed over the entire circumference of the end of the opening.

Compared with the flat open end of the first container 1 shown in FIG. 1, when the lid 3 is pressed with the same force, if the open end has the protrusion 29, the surface area of the upper portion of the protrusion 29 Is small, the pressing force per unit area is increased.

From the above, in addition to the effect described in Example 1, it has the effect of improving the hermeticity of the culture vessel 10 by the lid 3.

FIG. 14 shows the end shape of the first protrusion provided on the lid 3 of the culture vessel 10. A convex portion is provided at the end of the first projecting portion provided in the lid portion 3, and this convex portion functions as a guide for alignment with the opening of the first container 1. Since the area of the convex portion that comes into contact with the lid portion 3 is small, even if the lid portion 3 is pushed with the same force, the pressure that comes into contact increases, so that the sealing performance of the first container 1 is improved.

When the diameter of the convex portion provided at the center of the end of the first projecting portion of the lid 3 is Dr, the diameter on the opening side of the first container 1 is Dc, and the height of the convex portion is Hp, for example, Dr is slightly smaller than the inner diameter Dv of the opening of the first container 1, and the ratio is about 99% to 90%, Dc is about 89% to 70% of Dv, and Hp is about 10% to 50% of Dv. If it is.

FIG. 15 shows an embodiment in which the lid portion 3 and the second container 2 are connected via the bellows portion 19. The bellows portion 19 and the lid portion 3 and the bellows portion 19 and the second container 2 are hermetically bonded, and the airtightness of the second container 2 can be maintained.

15, a portion that contacts the opening of the first container 1 is formed of an elastic body 20, and the other portion is formed of a plastic material such as polystyrene.

From the above, in addition to the effects described in the first embodiment, the movable distance of the lid 3 can be increased more than in the first embodiment.

FIG. 16 shows a modification of the pressing mechanism shown in FIG. The example which attached the state holder 18 which hold | maintains the opening-and-closing state of the cover part 3 to the culture container 10 is shown. FIG. 16 shows a cross-sectional view of the culture vessel 10 divided in the vertical direction.

In FIG. 16, the pulling spring 32 is disposed on the pressing portion 31, and the pressing portion 31 is pulled up. When the lid 3 is closed, the pressing part 31 is pushed down by turning down the fixing screw 33 arranged on the upper part of the pressing part 31.

From the above, in addition to the effects described in the first embodiment, the container itself has the effect of maintaining the open / closed state of the lid 3.

1 First container
2 Second container
3 Lid
4 Liquid supply pipe
5 Path switch
6 Bonding part
7 Gas supply / discharge pipe
8 Liquid discharge pipe
9 Push spring
10 Culture vessel
11 Medium tank
12 Cleaning liquid tank
13 Sterilizer tank
14 Container lid opener
15 Culture control system equipment
16 Controller
17 Pipe valve
18 State holder
19 bellows
20 Elastic body
21 Filter
30 Connecting tube for sterilant
22 Lid through hole
23 Clasp
24 Culture area
25 Container opening
26 Liquid supply pipe
27 Gas supply / discharge pipe
28 Liquid discharge pipe
29 Projection
30 Sterilizer supply pipe
31 Pressing part
32 Extension spring
33 Fixing screw
34 Top opening
35 Side lower opening
36 Liquid input tube

Claims

A first container having a culture region and having an opening formed therein;
A second container containing the first container;
A culture vessel comprising: a lid that closes the opening of the first container by operating from the outside of the second container.
The culture container according to claim 1,
The culture container according to claim 1, wherein the lid portion includes a first protrusion protruding in the second container inner direction and a second protrusion protruding in the second container outer direction.
A culture container according to claim 2,
The culture vessel, wherein the lid is formed of an elastic body.
A culture container according to claim 3,
A culture container having a third protrusion formed over the entire circumference of the end at the end of the opening of the first container.
The culture container according to claim 3 or 4,
At the end of the first projecting portion of the lid portion, a first bottom having a diameter that is 1 to 10% smaller than the inner diameter of the opening of the first container, and the opening of the first container A culture container comprising a fourth protrusion comprising a second bottom having a diameter that is about 11 to 30% smaller than the inner diameter of the portion.
A first container having a culture region and having an opening formed therein, a second container containing the first container, and the first container operated by being operated from outside the second container. A lid capable of closing the opening of the container, and a culture container having
An operation mechanism for operating the lid from the outside of the culture vessel.
The culture apparatus according to claim 6,
The culture apparatus according to claim 1, wherein the operation mechanism is a mechanism that applies a pressing force to the lid.
The culture apparatus according to claim 6 or 7,
The lid portion of the culture container has a first protrusion protruding in the second container inner direction and a second protrusion protruding in the second container outer direction. Culture device.
The culture apparatus according to claim 8, wherein
The culture apparatus, wherein the lid portion of the culture container is formed of an elastic body.
The culture apparatus according to claim 9, wherein
An culturing apparatus comprising a third protrusion formed over the entire circumference of the end of the opening of the first container of the culture container.
The culture apparatus according to claim 9 or 10, wherein
At the end of the first protruding portion of the lid portion of the culture container, a first bottom having a diameter that is 1 to 10% smaller than the inner diameter of the opening of the first container, and the first A culture apparatus comprising a fourth protrusion comprising a second bottom having a diameter of about 11 to 30% smaller than the inner diameter of the opening of the container.
The culture apparatus according to claim 6,
A medium tank containing the medium;
A cleaning liquid tank for storing the cleaning liquid;
A sterilant tank for containing the sterilant;
A channel connected to each of the medium tank, the washing liquid tank, and the sterilant tank, and connected to the culture vessel;
A culture apparatus comprising:
The culture apparatus according to claim 6,
A medium tank containing the medium;
A cleaning liquid tank for storing the cleaning liquid;
A sterilant tank for containing the sterilant;
A first flow path connected to each of the medium tank and the washing liquid tank and connected to the culture vessel;
A second flow path connected to the sterilant tank and connected to the culture vessel;
A culture apparatus comprising:
A first container having a culture region and having an opening formed therein, a second container containing the first container, and the first container operated by being operated from outside the second container. A culture vessel having a lid for closing the opening of the vessel of
Close the opening of the first container in the first cleanliness space, and move the culture container to a second cleanliness space different from the first cleanliness with the opening closed. And a step of performing a predetermined culture operation in the second cleanliness space.
The culture method according to claim 14, comprising:
After moving the culture container to the second cleanliness space, the inside of the second container is sterilized in a state where the opening of the first container of the culture container is closed. Culture method.