WO2017090773A1

WO2017090773A1 - Cell culturing system and sterilization method

Info

Publication number: WO2017090773A1
Application number: PCT/JP2016/085234
Authority: WO
Inventors: 久五味; 敏光冨士; 成則尾▲崎▼; 高橋　勉; 明威田村; 竹内　晴紀; 小林　直樹; 佳雅須田
Original assignee: 東京エレクトロン株式会社; シンフォニアテクノロジー株式会社
Priority date: 2015-11-27
Filing date: 2016-11-28
Publication date: 2017-06-01
Also published as: JP2019013151A

Abstract

In the present invention, a single-system main gas line (10) installed in a conveyance module (2) is divided into a first region (11) and a second region (13) by an opening/closing valve for division (10V). A plurality of branch supply lines (12) branch in parallel from the first region and independently supply gas to corresponding operation modules. A plurality of branch discharge lines (14) branch in parallel from the second region and independently discharge the gas from corresponding operation modules to the main gas line. Each of the branch supply lines is provided with an opening/closing valve for supply (12V) and each of the branch discharge lines is provided with an opening/closing valve for discharge (14V).

Description

Cell culture system and sterilization method

The present invention relates to a cell culture system including a plurality of operation modules that automatically perform operations necessary for cell culture, and a transfer module that automatically transfers a culture container such as a medium plate between the operation modules. The present invention relates to a technique for sterilizing the inside.

In recent years, with the development of regenerative medicine, the regeneration of some human tissues (skin, bone, cartilage, etc.) has reached a practical level. In addition, development of a cell culture system that automatically performs a series of work related to cell culture, which has been dependent on the advanced techniques of workers and researchers so far (see, for example, Patent Document 1). In addition to an incubator module that is maintained in an environment suitable for cell culture in order to perform cell culture, the cell culture system includes a work module for performing various operations such as culture preparation, post-treatment, inspection, and observation. included. The cell culture system also includes a transfer module that transfers the culture container between the work modules.

In each process related to cell culture, contamination by microorganisms such as bacteria and viruses should not exist, and sterilization technology that guarantees sterility is a very important factor. It is desired to develop a sterilization technique for performing sterilization of each module included in the cell culture system efficiently and without causing harm to the worker by the sterilizing agent. However, no satisfactory one has been developed.

JP 2012-147893 A

An object of the present invention is to provide a technique capable of efficiently sterilizing each module of a cell culture system formed by combining a plurality of modules and efficiently purging sterilization gas after sterilization treatment. It is said.

According to one embodiment of the present invention, a transport module having a transport machine for transporting a culture container therein, and connected to the transport module, the culture container or the contents of the culture container are related to culture. A plurality of work modules, the plurality of work modules including at least a culture module for culturing cells and a test module for inspecting a state of cells cultured in the culture module. A plurality of work modules, one main gas line provided in the transfer module, and a dividing on-off valve interposed in the main gas line, by closing the dividing on-off valve, A dividing on-off valve in which the main gas line is divided into a first region and a second region on the boundary of the dividing on-off valve, and a parallel opening from the first region of the main gas line. A plurality of branch supply lines each branching into a plurality of branch supply lines each supplying gas from the main gas line to the corresponding work module, and branching in parallel from the second region of the main gas line A plurality of branch discharge lines, each of which discharges gas from the corresponding work module to the main gas line, and a supply on / off valve provided in each branch supply line, There is provided a cell culture system provided with a discharge on-off valve provided in each branch discharge line.

According to another embodiment of the present invention, in the sterilization method performed in the cell culture system, selecting a sterilization target module to be sterilized from the plurality of operation modules, and for dividing the main gas line With the on-off valve closed, the supply on-off valve of the branch supply line and the discharge on-off valve of the branch discharge line corresponding to the module to be sterilized are opened, and then the first region side of the main gas line Thereby supplying the sterilizing gas to the first region of the main gas line, the branch supply line corresponding to the module to be sterilized, the inside of the module to be sterilized, the branch discharge line corresponding to the module to be sterilized, and Sterilizing the inside of the module to be sterilized by sequentially flowing the sterilizing gas through the second region of the main gas line And then supplying a purge gas to the first region side of the main gas line, whereby the first region of the main gas line, the branch supply line corresponding to the module to be sterilized, and the module to be sterilized The purge gas in the interior of the sterilization target module by sequentially flowing the purge gas through the branch discharge line corresponding to the sterilization target module and the second region of the main gas line; Opening the split on-off valve of the main gas line, opening the supply on-off valve of the branch supply line and the discharge on-off valve of the branch discharge line corresponding to the module to be sterilized, and thereafter Purge gas is supplied to the first region of the gas line, thereby causing the first region and the front of the main gas line to By sequentially flowing the purge gas to the second region, and to displace the sterilizing gas in the interior of the main gas line, sterilization method comprising a are provided.

According to the above embodiment, the main gas line can be used as the first region for gas supply and the second region for gas discharge by closing the dividing on-off valve. For this reason, the sterilization gas can be supplied to any sterilization target module from a common gas supply source by opening the supply on / off valve of the gas supply line and the discharge on / off valve of the gas return line corresponding to the sterilization target module. It is possible to sterilize any module to be sterilized efficiently. On the other hand, the entire main gas line can be reliably purged by flowing the purge gas through the main gas line with the dividing on-off valve opened.

It is a piping system diagram of a cell culture system. It is the schematic which shows the mechanistic structure of a cell culture system. It is a schematic longitudinal cross-sectional view which shows the structural example of a work module. It is a figure explaining the flow of the gas in piping of a cell culture system. It is a figure explaining the flow of the gas in piping of a cell culture system. It is a figure explaining the flow of the gas in piping of a cell culture system. It is the schematic which shows other arrangement | positioning of a relief valve.

Embodiments of a cell culture system will be described below with reference to the drawings.

As shown in FIG. 1, the cell culture system 1 includes a transfer module 2, a plate loader module 3 connected to the transfer module 2, and a plurality of work modules 4. The transfer module 2 is formed by joining two transfer module parts 2A and a transfer module part 2B.

A transfer robot 82 (see FIG. 2) is provided in the internal space (transfer space) of the transfer module 2, and a culture plate (not shown) and a CI (chemical indicator) are provided between the plate loader module 3 and the work module 4. ) And BI (biological indicators) (both not shown).

The plate loader module 3 is configured as a load port (container loading / unloading section) that receives and dispenses a cassette (not illustrated) that accommodates a plurality of (for example, 16) culture plates (not illustrated) as a kind of culture container. Has been.

Each of the work modules 4 has a function specialized for executing various work (culture, pre-treatment and post-treatment of culture, post-culture inspection, etc.) performed for cell culture. The work module 4 includes a plate loader module, an incubator module (culture module), an imaging module, a microscope module, a remover module, a preparation module, a liquid exchange module, a medium analysis module, and the like.

簡単 Briefly explain each work module. The incubator module is a module in which cell culture is performed, and includes a device for maintaining an environment (temperature, humidity, carbon dioxide concentration, etc.) suitable for cell culture. The imaging module and the microscope module include devices for performing macroscopic and microscopic imaging of cultured cells. The preparation module has a device for performing processing for culture preparation (ECM (Extracellular matrix) coating, PBS (Phosphate buffered saline) processing, etc.) on the culture plate. The module has a device that removes defective cells based on imaging results of an imaging module, a microscope module, etc. The liquid exchange module is used for seeding, periodic medium replacement, taking out cultured cells, etc. The medium analysis module has a device for analyzing the medium, and the work module can be equipped with a device related to cell culture other than the above-described devices. .

The cell culture system 1 further includes a sterilization gas supply / recovery unit 50 for supplying sterilization gas to the transport module 2 and the work module 4. The sterilization gas supply / recovery unit 50 may be detachable from the transport module 2 so that it can be connected (incorporated) to the cell culture system 1 only when sterilization is performed.

The transfer module 2 is provided with one continuous main gas line 10. The main gas line 10 is connected to the sterilization gas supply / recovery unit 50 from an inlet end 10a serving as a gas supply connection port, and is an outlet serving as a gas discharge connection port connected to the sterilization gas supply / recovery unit 50. It extends continuously until it reaches the end 10b. A normally open (open when not energized) on-off valve (dividing on-off valve) 10V is provided in the middle of the main gas line 10, and the main gas line 10 is upstream of the first region with the on-off valve 10V as a boundary. 11 and the second region 13 on the downstream side. The first area 11 has a role as a main supply line, and the second area 13 has a role as a main discharge line.

The transfer module 2 is provided with a plurality of branch supply lines 12 branched from the first region 11 of the main gas line 10. The branch supply line 12 is for distributing the gas flowing through the first region 11 to each work module 4 connected to the transfer module 2.

The transfer module 2 has a plurality of branch discharge lines 14 branched from the second region 13 of the main gas line 10. The branch discharge line 14 is for allowing the gas discharged from each work module 4 connected to the transfer module 2 to flow into the second region 13.

The transport module 2 has a main pressure measurement line 15 for measuring the pressure in each work module 4 and a plurality of branch pressure measurement lines 16 branched from the main pressure measurement line 15. The pressure in each work module 4 is transmitted to the pressure gauge 59 provided in the sterilization gas supply / recovery unit 50 via the corresponding branch pressure measurement line 16 and the main pressure measurement line 15.

Each work module 4 is also provided with a pressure gauge 19 for measuring the pressure in each work module 4.

Each work module 4 is provided with a relief line 4L provided with a relief valve 4RV in order to release the pressure when the pressure in the internal space of the work module 4 becomes too high. The transfer module 2 includes a main exhaust line 17 for sending the gas discharged from each work module 4 through the relief line 4L to the abatement device 70, and a plurality of branch exhaust lines 18 branched from the main exhaust line 17. Have. Exhaust gas rendered harmless by the abatement apparatus 70 can be discharged into an exhaust system set outside the cell culture system 1 such as a building such as a research facility.

In addition, as shown in FIG. 7, the relief line 4L may be connected to the second region 13 of the main gas line 10, that is, the main discharge line. In this case, the main exhaust line 17 may not be provided.

The branch supply line 12, branch discharge line 14, and branch pressure measurement line 16 are provided with normally closed (non-energized) on-off

valves

12V, 14V, and 16V, respectively.

Since the transfer module 2 is also a sterilization target module, the transfer module 2 includes various lines (branch supply line 12, branch discharge line 14, branch pressure measurement line 16, branch exhaust line 18 and the like) leading to the transfer space of the transfer module 2. Equivalent). In addition, the transfer module 2 is provided with a relief line with a relief valve (equivalent to 4L and 4RV) communicating with the transfer space of the transfer module 2, and a pressure gauge 19 is also provided.

In FIG. 1, as with each work module 4, various lines (12, 14, 16, 18) and a pressure gauge 19 are provided corresponding to the plate loader module 3. The plate loader module 3 is provided in the case where it has a chamber that is isolated from an external environment such as a load lock module and can be adjusted in atmosphere. A configuration in which the plate loader module 3 is an open cassette mounting module, and the internal space of the cassette mounted on the plate loader module 3 can be directly and airtightly connected to the internal space of the transport module 2. The above-mentioned various lines and pressure gauges are not provided. In this case, the plate loader module 3 is a special module different from the work module 4, and the work module 4 can be attached to an attachment portion (mount, attachment port, etc.) of the plate loader module 3 in the transport module 2. Not.

The transfer module 2 (the transfer module part 2A and the transfer module part 2B) has a generally hollow rectangular parallelepiped or cubic shape as a whole. Each work module 4 also has a generally hollow rectangular parallelepiped or cubic shape. As shown in FIG. 2, the transport module 2 is provided with a plurality of connection ports 2 </ b> P (four are shown in the figure) for connecting the work module 4. The work module 4 is provided with a connection port 4P that can be airtightly connected to the connection port 2P of the transfer module 2.

Each connection port 2P of the transfer module 2 has the same structure and dimensions. The connection ports 4P of each work module 4 have the same structure and dimensions regardless of the type of the work module 4. Therefore, a system having a function corresponding to a user's request can be constructed by combining the transport module 2 with a desired type of work module 4.

The interconnection between the two

transfer module parts

2A and 2B can be performed, for example, by screwing the flanges together with the seals sandwiched between the flanges provided on the opposing end surfaces of the two transfer module parts. As for the connection between the transport module 2 and the work module 4, for example, the flanges are screwed together with a seal interposed between the flange provided in the connection port 10P of the transport module 2 and the flange provided in the work module 4. Can be performed. In addition, by connecting modules, the male connector provided in one module and the female connector provided in the other module may be connected, and corresponding lines may be connected.

Guide rails 81 are provided on the floor surfaces of the

transport module portions

2A and 2B. Guided by the guide rail 81, the base 83 of the medium plate transport robot 82 travels. The medium plate transport robot 82 can be constituted by, for example, a horizontal articulated robot, and the pick (medium plate holding member) at the tip thereof can be directed in any direction within a horizontal plane and can move in any direction. Can be moved up and down. When the transfer module 2 is configured by connecting the two

transfer module portions

2A and 2B, the two guide rails 81 are also connected so that the base 83 can travel from end to end of the transfer module 2. *

Each connection port 2P of the transfer module 2 is provided with a gate valve 85. By opening the gate valve 85, the culture medium plate can be carried in and out of the work module 4 through the

corresponding connection ports

2P and 4P. Depending on the configuration of the cell culture system 1, there may be a connection port 2P to which the work module 4 is not connected, but the gate valve 85 of such a connection port 2P may be always closed.

As described above, when the plate loader module 3 is an open special module, the plate loader module 3 can be connected to the transport module 2 by a connection structure different from the work module 4. In this case, it is not necessary to provide the gate valve 85 in the portion where the plate loader module 3 is attached.

Instead of forming the transfer module 2 by connecting a plurality of transfer module parts (2A, 2B) in series, the transfer module 2 may be configured by one module part. It is also possible to configure the transfer module 2 by combining three or more transfer module portions.

The configuration of each work module 4 will be briefly described with reference to FIG. The work module 4 includes a chamber 41, work equipment 42 provided in the chamber, and a partition wall 44 that defines a sterilization management area 43 that is a part of the internal space of the chamber 41. A part of the partition wall 44 may be movable. In the internal space of the chamber 41, the above-described relief line 4L in which the above-described relief valve 4RV is interposed is connected. The relief line 4L is connected to the branch exhaust line 18 of the transport module 2.

The work equipment 42 provided in the work module 4 differs depending on the type of the work module 4. The working device 42 includes a member that holds a medium plate, a device that adjusts the culture environment such as the temperature, humidity, and carbon dioxide gas concentration in the chamber, each device that performs medium adjustment, cell removal, and a microscope. Examples include observation equipment.

The work module 4 has a gas supply line 45. The gas supply line 45 is connected to the branch supply line 12 of the transport module 2. The gas supply line 45 opens in the sterilization management area 43 and discharges a gas such as sterilization gas into the sterilization management area 43. In the illustrated example, the sterilization management area 43 is not completely isolated from the surrounding space in the chamber 41 (the surrounding chamber space 41 ′), but partly communicates with the surrounding chamber space 41 ′. . That is, the partition wall 44 does not completely surround the sterilization management area 43. Accordingly, when the sterilization gas is supplied into the sterilization management area 43, the surrounding chamber inner space 41 'is also filled with the sterilizing gas, and the surrounding chamber inner space 41' is also sterilized.

A gas discharge line 46 is opened in the surrounding chamber inner space 41 ′. The gas discharge line 46 is connected to the branch discharge line 14 of the transport module 2. Therefore, the gas in the chamber 41 can be discharged to the outside of the chamber 41 through the gas discharge line 46. An FFU (fan filter unit) 48 is provided on the ceiling of the partition wall 44. The FFU 48 has a fan and a particle filter such as an ULPA filter (Ultra Low Low Penetration Air Air Filter), and filters and discharges the gas present in the surrounding chamber inner space 41 ′ into the sterilization management area 43. Thereby, the particle level of the gas in the chamber 41 can be kept below a certain level.

As described above, by setting the sterilization management area 43 partitioned by the partition wall 44 in the chamber 41, it is possible to improve the sterilization efficiency of the work equipment 42 directly related to the culture and its surroundings.

In FIG. 3, reference numeral 47 is a pressure detection line communicating with the branch pressure measurement line 16.

It is not necessary to set the sterilization management area 43 partitioned by the partition wall 44 in all the work modules 4, and the entire chamber 41 may be used as the sterilization management area. Further, it may be difficult to provide the FFU in the chamber 41 in the relatively small work module 4 (however, the size of the connection port 4P is the same). In this case, a circulation line in which a pump, a filter, a particle counter, and the like are connected to the chamber 41 (all are not shown), and the gas taken out from the chamber 41 is returned to the chamber 41 through the circulation line. The particles may be removed by a filter.

As shown in FIG. 1, the sterilization gas supply / recovery unit 50 is one of sterilization gas hydrogen peroxide vapor, dry air (dry air) and inert gas, or a mixture of two or more. The gas can be supplied to the inlet end 10 a of the main gas line 10. Further, the sterilization gas supply / recovery unit 50 can recover the exhaust gas discharged from the outlet end 10 b of the main gas line 10.

The sterilization gas supply / recovery unit 50 has a gas delivery line 51 connected to the inlet end 10 a of the main gas line 10. The gas delivery line 51 is provided with a flow rate control mechanism 52 and an emergency shutoff valve 53. Dry air or inert gas is supplied to the gas delivery line 51 from an external gas supply mechanism 54 connected to the sterilization gas supply / recovery unit 50. A hydrogen peroxide vapor generator 55 is connected to the gas delivery line 51. The flow rate of the gas (for example, dry air) supplied from the gas supply mechanism 54 is supplied by the flow rate control mechanism 52, and the hydrogen peroxide vapor supplied from the hydrogen peroxide vapor generator 55 is added to this flow rate-controlled gas flow. By being added, a sterilized gas having a predetermined hydrogen peroxide concentration is sent out to the main gas line 10. That is, the sterilization gas supply / recovery unit 50 receives from the gas delivery line 51 a mixed gas containing air (preferably dry air) (which may be an inert gas) and hydrogen peroxide as sterilization gas, and hydrogen peroxide gas. Any one of air (which may be an inert gas) not included is selectively supplied.

The sterilization gas supply / recovery unit 50 has a gas recovery line 56 connected to the outlet end 10 b of the main gas line 10. The gas recovery line 56 is provided with a detoxifying mechanism 57 for detoxifying components (for example, hydrogen peroxide) harmful to the human body contained in the exhaust gas. The gas recovery line 56 is provided with a hydrogen peroxide concentration meter 58 for measuring the hydrogen peroxide concentration in the gas flowing through the gas recovery line 56. The gas recovery line 56 is connected to the gas delivery line 51 via the flow rate control mechanism 52.

The exhaust gas recovered from the main gas line 10 is detoxified by the detoxification mechanism 57 and returned to the gas delivery line 51, and the hydrogen peroxide vapor supplied from the hydrogen peroxide vapor generator 55 is added to this gas. Then, a sterilized gas having a predetermined hydrogen peroxide concentration is sent to the main gas line 10. Note that a blower (not shown) is provided in the gas delivery line 51 in order to give the gas a driving force for sending the gas returned to the gas delivery line 51 to the main gas line 10 again. In addition, when gas with high humidity is returned to the gas delivery line 51, a dryer (not shown) is connected to the gas delivery line 51 from the connection point of the hydrogen peroxide vapor generator 55 in order to reduce the humidity of the gas. It is interposed at an upstream position.

The emergency shut-off valve 53 is closed to shut off the supply of sterilization gas when a dangerous situation such as an increase in the hydrogen peroxide concentration in the room where the cell culture system 1 is installed due to a leak.

The sterilization gas supply / recovery unit 50 further has a pressure gauge 59. The pressure gauge 59 is connected to a connection port of the main pressure measurement line 15 provided in the transfer module 2, and the pressure in each work module 4 of the transfer module 2 through the main pressure measurement line 15 and the branch pressure measurement line 16. Is detected. The pressure gauge 59 may be provided outside the sterilization gas supply / recovery unit 50.

Next, the operation of the cell culture system 1 will be described. A cassette (conveying container) containing a plurality of medium plates is placed on the plate loader module 3, and the conveying robot 82 takes out the medium plate (culture container) from the conveying container and conveys it to each work module 4 to each work module. 4, various operations such as preparation necessary for cell culture, observation of cultured cells, and removal of cultured cells are performed according to a predetermined schedule. The medium plate holding the cultured cells is returned to the cassette of the plate loader module 3. A description of the specific content of the work performed in each work module 4 is omitted.

When work is performed on cells or culture media in each work module 4, the sterilization level inside the chamber 41 of each work module 4 (at least inside the sterilization management area 43 when the sterilization management area 43 is set) is Must meet predetermined criteria. In addition, the sterilization level inside the transport module 2 that transports the culture plate to each work module 4 must also satisfy a predetermined standard. Further, if the medium plate is exposed to the ambient atmosphere in the plate loader module 3, the plate loader module 3 needs to be sterilized.

Therefore, (1) before the first operation after installation of the cell culture system 1, (2) after maintenance of the cell culture system 1, (3) after completion of lot processing, (4) abnormal value of particles is detected When necessary, the necessary module sterilization process is executed.

During one period during the sterilization process, one module selected from the plurality of

modules

2, 3 and 4 (hereinafter referred to as “sterilization target module”) is sterilized. That is, different modules are sterilized at different periods. Although it is possible to sterilize two modules in succession, it is preferred that only one module is sterilized at the same time.

The sterilization process is automatically performed according to the following procedure under the control of the system controller 101 that controls the operation of various devices constituting the cell culture system 1. Note that detection values of various measuring devices (pressure gauges 19 and 59, a thermometer, a hygrometer, a carbon dioxide gas concentration sensor, etc.) included in the cell culture system 1 are also input to the system controller 101. In the following description, it is assumed that the sterilization target module is one of the work modules 4 (hereinafter also referred to as “sterilization target module 4”).

[CI / BI carry-in process]
First, CI (chemical indicator) and BI (biological indicator) are carried into the plate loader module 3, and these CI and BI are conveyed from the plate loader module 3 to the sterilization management area 43 of the sterilization target module 4 by the conveyance robot 82. .

[Leak check process]
After all the gate valves 85 are closed, the leak check of the module 4 to be sterilized is performed. The open / close valve (supply open / close valve) 12V of the branch supply line 12 corresponding to the sterilization target module 4 is opened, and is supplied from the sterilization gas supply / recovery unit 50 via the first region 11 of the main gas line 10 and the branch supply line 12. Then, dry air not containing hydrogen peroxide vapor is supplied into the sterilization target module 4. At this time, the on-off valve of the branch discharge line 14 corresponding to the sterilization target module 4 is closed. The sterilization target module 4 is filled with air until the detected value of the pressure gauge 19 provided in the sterilization target module 4 reaches a predetermined pressure (pressure lower than the set pressure of the relief valve 30R), and then the open / close valve of the branch supply line 12 Close 12V. After the elapse of a predetermined time, if the pressure drop calculated based on the detection value of the pressure gauge 19 is equal to or less than a predetermined threshold value, it is determined that the sealing property of the sterilization target module 4 is not a problem. The pressure gauge 19 is also used for pressure management in the work module when performing various operations before and after culturing.

[Dehumidification process]
After the leak check, the on-off

valves

12V and 14V of the branch supply line 12 and the branch discharge line 14 corresponding to the sterilization target module 4 are opened. Next, dry air that does not contain hydrogen peroxide is sent from the sterilization gas supply / recovery unit 50 to the sterilization target module 4 via the first region 11 of the main gas line 10 and the corresponding branch supply line 12. The dry air sent to the sterilization target module 4 flows through the sterilization management area 43 (see FIG. 3), dehumidifies the inside of the chamber 41 including the sterilization management area 43, and then flows out of the sterilization target module 4 to branch off the discharge line. 14 and through the second region 13 of the main gas line 10 back to the sterilization gas supply / recovery unit 50. The gas that has returned to the gas recovery line 56 of the sterilization gas supply / recovery unit 50 flows again into the gas delivery line 51 and is dehumidified by a dryer (not shown) provided in the gas delivery line 51, and then the main gas line 10. To be supplied.

Thus, the dry air is supplied to the gas delivery line 51, the first region 11 of the main gas line 10, the branch supply line 12 corresponding to the sterilization target module 4, the sterilization target module 4, the branch discharge line 14 corresponding to the sterilization target module 4, A chamber including the sterilization management area 43 of the sterilization target module 4 by circulating through a circulation path (hereinafter also referred to as “first circulation path”) including the second region 13 of the main gas line 10 and the gas recovery line 56. 41 is dehumidified.

[Sterilization process]
After performing the dehumidifying process for a predetermined time, hydrogen peroxide vapor is added to the dry air flowing through the gas delivery line 51, and a sterilization gas comprising a mixed gas of dry air and hydrogen peroxide vapor is sent to the sterilization target module 4. It is done. The sterilization gas circulates through the first circulation path, and sterilizes the sterilization management area 43 and the surrounding chamber inner space 41 ′ when passing through the sterilization management area 43 of the module 4 to be sterilized. When passing through the gas delivery line 51, the generated hydrogen peroxide vapor is added to the sterilizing gas circulating in the first circulation path, and the sterilized gas having a higher hydrogen peroxide concentration is sent again to the sterilization target module 4. It is done.

Thus, sterilization of the sterilization management area 43 of the sterilization target module 4 is performed by flowing through the sterilization management area 43 when the sterilization gas circulates through the first circulation path.

The pressure in the sterilization target module 4 during the sterilization process is constantly monitored by the pressure gauge 59 of the sterilization gas supply / recovery unit 50, and the flow rate is set so that the pressure in the module 30 is maintained within a predetermined pressure range. The control mechanism 52 adjusts the sterilization gas supply flow rate from the sterilization gas supply / recovery unit 50.

The pressure in the sterilization target module 4 is propagated to the pressure gauge 59 via the branch pressure measurement line 16 and the main pressure measurement line 15 by opening the on-off valve 18V of the branch pressure measurement line 16 corresponding to the sterilization target module 4. . Therefore, the pressure in the sterilization target module 4 can be monitored by the pressure gauge 59 located away from the sterilization target module 4.

Also, during the sterilization process, the hydrogen peroxide concentration meter 58 constantly monitors the hydrogen peroxide concentration in the sterilization gas flowing through the gas recovery line 56. Based on the detection result of the hydrogen peroxide concentration meter 58, the flow rate of hydrogen peroxide vapor sent to the gas delivery line 51 from the hydrogen peroxide vapor generator 55 (that is, the hydrogen peroxide / air mixture ratio) is controlled.

By the way, during the sterilization of the module to be sterilized, cells may be cultured and inspected by other modules. In that case, since the module to be sterilized becomes a positive pressure, the sterilization target module is in the sterilization process because there is a possibility that the sterilization gas from the module to be sterilized is slightly leaked from the seal part of the gate valve to the transport module In this case, it is desirable not to open the gate valve of another module. In this case, the culture container cannot be transported or collected to each module, but the processing in each module can be continued.

[Aeration process]
After performing the sterilization process for a predetermined time, supply of hydrogen peroxide vapor to the gas delivery line 51 from the hydrogen peroxide vapor generator 55 of the sterilization gas supply / recovery unit 50 is stopped, and sterilization gas supply / recovery is performed. Only air (dry air) is sent out from the unit 50. That is, air is circulated in the first circulation path. As a result, the hydrogen peroxide vapor existing in the first circulation path is pushed out by the air, and the hydrogen peroxide vapor is decomposed into oxygen and water when circulating in the first circulation path together with the air. .

The gas flow during the dehumidification process, the sterilization process, and the aeration process is shown by bold lines in FIG.

[Main gas line purge process]
After performing the aeration process for a predetermined time (or when the hydrogen peroxide concentration detected by the hydrogen peroxide concentration meter 58 falls below a predetermined first threshold value), air is supplied from the sterilization gas supply / recovery unit 50 to the air. While maintaining the state where only (dry air) is being delivered, the open / close valve 10V of the main gas line 10 is opened, the open / close valve 12V of the branch supply line 12 corresponding to the sterilization target module 4 and the open / close valve of the branch discharge line 14 (discharge) Open / close valve) 14V is closed (that is, all the open / close valves 12V and all the open / close valves 14V are closed). As a result, the gas passes through the gas delivery line 51, the main gas line 10 (the entire area of the first region 11 and the entire area of the second region 13), and the gas recovery line 56 (hereinafter also referred to as “second circulation path”). Air will circulate.

As a result, the following two parts that have become dead spaces, that is, from the branch point of the branch supply line 12 connected to the sterilization target module 4 in the first region 11 of the main gas line 10 to the on-off valve 10V. The sterilization gas staying in the part and the part from the branch point of the branch discharge line 14 connected to the module 4 to be sterilized in the second region 13 to the on-off valve 10V is forced from the part by air. Extruded and circulated through the second circulation path with air, during which the hydrogen peroxide in the sterilization gas is broken down into oxygen and water.

When the above state is maintained and the hydrogen peroxide concentration detected by the hydrogen peroxide concentration meter 58 falls below a predetermined second threshold value (for example, 1 ppm which is a hydrogen peroxide concentration that is not harmful to the human body), a gas line purge is performed. The process ends.

The gas flow during the main gas line purge process is shown in bold lines in FIG.

¡The aeration process and the main gas line purge process may be integrated. That is, when performing the aeration process, the on-off valve 10V of the main gas line 10 may be opened. By doing so, air flows through both the first circulation path and the second circulation path, and the internal spaces of the first circulation path and the second circulation path can be purged with air at the same time (the gas at this time (Refer to the thick line in FIG. 6 for the flow.)

[Leak check process]
Next, the leak check process is performed again.

[CI / BI recovery process]
Thereafter, the CI and BI carried into the sterilization target module 4 in the CI / BI carrying-in process described above are carried out by the transport robot 82, and it is confirmed that the sterilization has been properly performed. Thus, a series of steps for sterilization of one sterilization target module 4 is completed. When it is confirmed that the necessary modules (for example, all the transport modules 2 and all the work modules 4) have been properly sterilized, work related to cell culture can be started. The sterilization of other modules can be performed in the same manner as described above. By the way, the effect on sterilization gas (H ₂ O _{2 in} this example) can be confirmed by BI, that is, whether the bacteria contained in BI are killed by the gas, but on the other hand, it is necessary to check sterility regularly. There is. In that case, in addition to the BI recovery, as a method for confirming the sterilized state after the sterilization treatment, the agar medium may be transported, the falling bacteria may be collected and cultured, and the presence of bacteria may be confirmed. If the agar medium is brought into the target module and left for a certain period of time, if there are bacteria, it falls to the surface of the agar medium. The presence or absence of bacteria can be confirmed by growing and cultivating this for a certain period of time and removing it from the apparatus for inspection. Proliferation culture may be performed in an incubator outside the apparatus.

According to the above embodiment, the open / close valve 10V is provided in the middle of one continuous main gas line 10, and the open / close valve 10V is closed during the sterilization process, whereby the main gas line 10 is connected to the gas supply line (first region). 11) and a gas exhaust line (second region 13). The on / off valve 10V is opened with all of the on / off

valves

12V and 14V of the branch supply line 12 and the branch discharge line 14 closed, and a purge gas (for example, dry air) is supplied from one end of the main gas line 10 to A purge gas can be allowed to flow from one end of 10 to the other end. For this reason, almost no sterilization gas remains in the main gas line 10. For this reason, it can prevent that the atmosphere in the laboratory where the cell culture apparatus was installed is contaminated by releasing the sterilized sterilization gas and harming a researcher, a worker, and the like.

Further, according to the above embodiment, the branch supply line 12 and the branch discharge line 14 each branching from the main gas line 10 and provided with the on-off

valves

12V and 14V are connected to the sterilization target module (2, 3, 4). Since air supply and exhaust are performed, a plurality of modules to be sterilized can be sequentially and efficiently sterilized by one sterilization gas supply / recovery unit 50 by switching the on-off valves (12V, 14V).

In addition, according to the above embodiment, the sterilization target module (2, 3, 3) is branched from the main pressure measurement line 15 through the branch pressure measurement line 16 provided with an on-off valve (pressure measurement on-off valve) 16V. 4) The internal pressure can be measured at a remote location. For this reason, the gas supply state from the sterilization gas supply / recovery unit 50 can be easily adjusted based on the detection value of the pressure gauge 59 installed at a position away from the sterilization target module (2, 3, 4). .

In the above embodiment, the sterilization gas supply / recovery unit 50 sends the sterilization gas returned after being supplied to the sterilization target module to the delivery line 51 again, but is not limited thereto. The sterilized gas that has returned may be discarded into an exhaust system set outside the cell culture system 1.

Claims

A transport module having a transport machine for transporting the culture container therein;
A plurality of operation modules connected to the transfer module and performing operations related to culture on the culture vessel or the contents of the culture vessel, the plurality of operation modules including a culture module for culturing cells And a plurality of work modules, including at least an inspection module for inspecting the state of cells cultured in the culture module,
One main gas line provided in the transfer module;
A split on / off valve interposed in the main gas line, wherein the main gas line is divided into a first region and a second region with the split on / off valve as a boundary by closing the split on / off valve. An on-off valve for splitting,
A plurality of branch supply lines branched in parallel from the first region of the main gas line, each of which supplies a gas to the corresponding work module from the main gas line,
A plurality of branch discharge lines branched in parallel from the second region of the main gas line, each of which discharges gas from the corresponding work module to the main gas line,
An on-off valve for supply provided in each branch supply line;
A discharge on-off valve provided in each branch discharge line;
A cell culture system.
A branch supply line that supplies gas from the first region of the main gas line to the transfer space inside the transfer module further branches, and further from the second region of the main gas line to the second region from the transfer space. The cell culture system according to claim 1, wherein a branch discharge line for discharging gas is branched.
The cell culture system according to claim 1, wherein the transfer module has a plurality of connection ports for connecting the work modules, and each connection port is provided with a gate valve.
A main pressure measurement line provided in the transfer module;
A plurality of branch pressure measurement lines branching in parallel from the main pressure measurement line, each of which is a plurality of branch pressure measurement lines for propagating the pressure in the work module corresponding to the main pressure measurement line;
A pressure measuring on-off valve provided in each branch pressure measuring line;
The cell culture system according to claim 1, further comprising:
The sterilization gas supply unit having a gas delivery line is further provided, and the gas delivery line is connected to an upstream end of the first region of the main gas line to supply a sterilization gas to the main gas line. Cell culture system.
The sterilization gas supply unit further includes a gas recovery line, the gas recovery line is connected to the downstream end of the second region of the main gas line, and recovers the sterilized gas discharged from the main gas line, The cell culture system according to claim 5, wherein the sterilized gas recovered in the gas recovery line is caused to flow again to the gas delivery line.
The cell culture system according to claim 1, wherein an end of the main gas line on the second region side is connected to an exhaust facility provided outside the cell culture system.
The cell culturing system according to claim 5, wherein the sterilizing gas supply unit is configured to be able to send air or inert gas independently from the gas delivery line.
The sterilization gas supply unit selectively selects, from the gas delivery line, a mixed gas as a sterilization gas containing air or an inert gas and hydrogen peroxide and air or an inert gas not containing hydrogen peroxide gas. The cell culture system of Claim 5 comprised so that supply is possible.
A main pressure measurement line provided in the transfer module;
A plurality of branch pressure measurement lines branching in parallel from the main pressure measurement line, each of which is a plurality of branch pressure measurement lines for propagating the pressure in the work module corresponding to the main pressure measurement line;
A pressure measuring on-off valve provided in each branch pressure measuring line;
Further comprising
The sterilization gas supply unit has a pressure gauge connected to the main pressure measurement line,
The apparatus further includes a control unit that controls the flow rate of the sterilization gas from the sterilization gas supply unit that maintains the pressure in the work module within a target pressure range based on the pressure in the work module measured by the pressure gauge. The cell culture system according to claim 5.
At least one of the work module and the transfer module includes a chamber, a partition wall defining a sterilization management area in a part of the chamber, and a gas outside the sterilization management area in an internal space of the chamber A fan filter unit for filtering and blowing into the sterilization management area, a gas supply port for supplying the gas supplied from the corresponding gas supply line to the sterilization management area in the chamber, and a corresponding gas return line to the gas return line. The cell culture system according to claim 1, further comprising a gas exhaust port that exhausts the gas in the chamber.
A transport module having a transport machine for transporting the culture container therein;
A plurality of operation modules connected to the transfer module and performing operations related to culture on the culture vessel or the contents of the culture vessel, wherein the plurality of operation modules are used for culturing cells. A plurality of work modules, including at least a module and an inspection module for inspecting a state of cells cultured in the culture module;
One main gas line provided in the transfer module;
A split on / off valve interposed in the main gas line, wherein the main gas line is divided into a first region and a second region with the split on / off valve as a boundary by closing the split on / off valve. An on-off valve for splitting,
A plurality of branch supply lines branched in parallel from the first region of the main gas line, each of which supplies a gas to the corresponding work module from the main gas line,
A plurality of branch discharge lines branched in parallel from the second region of the main gas line, each of which discharges gas from the corresponding work module to the main gas line,
An on-off valve for supply provided in each branch supply line;
A discharge on-off valve provided in each branch discharge line;
In a sterilization method performed in a cell culture system comprising:
Selecting a sterilization target module to be sterilized from the plurality of work modules;
Opening the supply on / off valve of the branch supply line and the discharge on / off valve of the branch discharge line corresponding to the module to be sterilized, with the split on / off valve of the main gas line closed;
Thereafter, sterilization gas is supplied to the first region side of the main gas line, whereby the first region of the main gas line, the branch supply line corresponding to the sterilization target module, the inside of the sterilization target module, Sterilizing the inside of the sterilization target module by sequentially flowing the sterilization gas to the second region of the branch discharge line and the main gas line corresponding to the sterilization target module;
Thereafter, purge gas is supplied to the first region side of the main gas line, whereby the first region of the main gas line, the branch supply line corresponding to the module to be sterilized, the inside of the module to be sterilized, the Expelling the sterilization gas inside the module to be sterilized by sequentially flowing the purge gas to the second region of the branch discharge line and the main gas line corresponding to the module to be sterilized;
Thereafter, opening the dividing on-off valve of the main gas line, opening the on-off valve for supplying the branch supply line corresponding to the module to be sterilized and the on-off valve for discharging the branch discharge line;
Thereafter, the purge gas is supplied to the first region of the main gas line, and thereby the purge gas is sequentially flowed to the first region and the second region of the main gas line, thereby being inside the main gas line. Expelling the sterilizing gas;
A sterilization method comprising:
The sterilization method according to claim 12, wherein the purge gas is air or an inert gas, and the sterilization gas contains hydrogen peroxide vapor.