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WO2015111544A1 - Incubateur, système de culture cellulaire doté de celui-ci, et procédé d'alimentation d'eau d'humidification - Google Patents

Incubateur, système de culture cellulaire doté de celui-ci, et procédé d'alimentation d'eau d'humidification Download PDF

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Publication number
WO2015111544A1
WO2015111544A1 PCT/JP2015/051226 JP2015051226W WO2015111544A1 WO 2015111544 A1 WO2015111544 A1 WO 2015111544A1 JP 2015051226 W JP2015051226 W JP 2015051226W WO 2015111544 A1 WO2015111544 A1 WO 2015111544A1
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WO
WIPO (PCT)
Prior art keywords
container
space
humidifying
incubator
humidification
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2015/051226
Other languages
English (en)
Japanese (ja)
Inventor
章博 太田
利一 矢野
頼雄 高橋
高橋　秀幸
山本　宏
芳生 神谷
浩司 岡野
山本　博一
桑田　秀典
英樹 谷口
圭輔 関根
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
PHC Corp
Yokohama City University
Original Assignee
Yokohama City University
Panasonic Healthcare Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yokohama City University, Panasonic Healthcare Co Ltd filed Critical Yokohama City University
Priority to JP2015558840A priority Critical patent/JP6259838B2/ja
Publication of WO2015111544A1 publication Critical patent/WO2015111544A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas

Definitions

  • the present invention relates to an incubator having a culture space for culturing cells, a cell culture system provided with the same, and a method for supplying humidified water.
  • Patent Document 1 discloses a water supply tank mounted outside an incubator having a cell culture space, a water supply passage for supplying sterilized water stored in the water supply tank into the culture space in the incubator, and a water supply passage And a humidifying means for humidifying the culture space by gasifying, misting, or evaporating the sterilized water supplied via the culturing apparatus.
  • the water for humidifying the culture space needs to be supplied in a sterile state in order to ensure a sterile state in the incubator.
  • Patent Document 2 discloses an incubator in which a filter for removing bacteria and the like contained in water is provided on a supply path for supplying water for humidifying the culture space in the incubator. In this configuration, since it is sterilized by the filter immediately before entering the incubator, aseptic water is supplied into the culture space of the incubator.
  • the conventional incubator has the following problems. That is, in the incubator disclosed in the above publication, when the temporary side of the filter for sterilization is contaminated, it is difficult to maintain a sterilized state for a long period of use. Similarly, when the connection portion constituting the supply path is contaminated, it is difficult to maintain a sterilized state for a long period of use.
  • the subject of this invention is providing the incubator which can fully ensure the aseptic state of the water used for humidification in culture
  • An incubator is an incubator for receiving cells to be cultured from a clean space formed in a cell manipulation chamber and forming a culture space in which cells are cultured inside, a transport unit, A humidification space and a circulation part are provided.
  • the transport unit receives various articles transported from the cell operation chamber side, transports them to a predetermined position, and operates a container containing aseptically treated humidified water to maintain the humidity in the culture space. Receive from the chamber side. In the humidification space, a container containing the humidified water received by the transport unit is placed.
  • aseptically-humidified humidified water is supplied from a cell operation chamber having a clean space for performing predetermined pretreatment or the like to cells cultured in the incubator.
  • the placed container is received and placed in the humidification space, and the culture space is humidified by the circulation unit.
  • the clean space includes a sterilized or decontaminated space, a clean room with a high degree of cleanliness, and the like.
  • the humidified water subjected to the sterilization treatment is not limited to the sterilized water in which the bacteria contained in the water is 0, but also includes water containing a number of bacteria within an allowable range.
  • the cell manipulation chamber includes an isolator having a clean space inside, a biohazard cabinet, and the like.
  • the transport unit includes, for example, a robot arm and other transport mechanisms for receiving a container from the cell manipulation chamber side and transporting the container to a predetermined position.
  • the circulation unit includes a fan that circulates air (eg, steam, mist, gas, etc.) containing humidified water in the culture space.
  • An incubator according to a second invention is the incubator according to the first invention, further comprising a humidifying shelf and a moving mechanism.
  • the humidification shelf is provided in the humidification space, and the container is placed thereon.
  • the moving mechanism moves the humidifying shelf between a delivery position for delivering the container conveyed to the humidification space by the conveyance unit and a humidification position provided in the humidification space and through which the airflow formed by the circulation unit passes. Move.
  • the humidifying shelf is moved from the delivery position where the container carried into the incubator from the cell operation chamber side by the transport unit is placed on the humidifying shelf to a predetermined humidifying position provided in the humidifying space.
  • a moving mechanism is provided.
  • the delivery position of a container and a humidification position can be provided in a separate position by providing the moving mechanism which moves the humidification shelf in which a container is mounted separately from the conveyance part which conveys a container. Therefore, at the delivery position, the container can be delivered smoothly, and at the humidification position, the container can be installed near the air flow generated by the circulation section to efficiently humidify the culture space. it can.
  • An incubator according to a third invention is the incubator according to the first or second invention, wherein the open part of the container placed in the humidification space is irradiated with ultraviolet rays for sterilization to sterilize the humidified water.
  • a UV (Ultraviolet) irradiation unit to be processed is further provided.
  • a UV irradiation unit for irradiating ultraviolet rays for sterilization is provided on the open part of the container containing humidified water carried into the incubator.
  • ultraviolet irradiation by the UV irradiation unit can be set as appropriate, such as constant irradiation, irradiation at predetermined intervals, and the like.
  • the irradiation of ultraviolet rays by the UV irradiation unit is performed in a state where the container is in the humidification position. Thereby, the humidified water after carrying in in an incubator can be maintained more reliably in a sterile state.
  • An incubator according to a fourth aspect of the present invention is the incubator according to the third aspect of the present invention, further comprising a shielding member that shields the humidification space and the culture space irradiated with ultraviolet rays by the UV irradiation unit.
  • a shielding member is provided in order to prevent ultraviolet rays irradiated from the UV irradiation unit in the humidified space from leaking into the culture space.
  • the shielding member includes a lid-like member that covers a portion of the UV irradiation unit placed in the humidification space where the UV irradiation unit is irradiated. Thereby, it is possible to prevent the cells and the like placed in the culture space from being killed by the ultraviolet rays irradiated for sterilization.
  • An incubator according to a fifth invention is the incubator according to the fourth invention, further comprising a humidifying shelf and a moving mechanism. The humidification shelf is provided in the humidification space, and the container is placed thereon.
  • the moving mechanism moves the humidification shelf between a delivery position for delivering the container conveyed to the humidification space and a humidification position provided in the humidification space.
  • the shielding member prevents leakage of ultraviolet rays irradiated from the UV irradiation unit to the outside of the humidifying space.
  • An incubator according to a sixth aspect of the invention is the incubator according to the fourth or fifth aspect of the invention, wherein the shielding member comprises a lid member that covers the space on the open side of the container, and a side wall that covers the side of the container. Have.
  • the shielding member for preventing leakage of ultraviolet rays irradiated from the UV irradiation unit into the culture space covers the lid member covering the open space of the container and the side of the container at the humidified position. And a side wall portion.
  • An incubator according to a seventh invention is the incubator according to the sixth invention, further comprising a humidifying shelf and a moving mechanism.
  • the humidification shelf is provided in the humidification space, and the container is placed thereon.
  • the moving mechanism moves the humidification shelf between a delivery position for delivering the container conveyed to the humidification space and a humidification position provided in the humidification space.
  • the moving mechanism moves the humidifying shelf between the delivery position and the humidifying position
  • the moving mechanism integrally moves the humidifying shelf and the lid member.
  • the lid member constituting the shielding member moves integrally with the humidifying shelf.
  • the lid member that covers the open side of the container moves together with the humidifying shelf, while the side wall portion that covers the side of the container is fixedly arranged at the humidifying position, Since the side of the humidifying shelf is opened, the container can be easily put in and out. Therefore, in the humidification position where the ultraviolet ray is irradiated from the UV irradiation unit, the open side and the side of the container can be covered by the lid member and the side wall part of the shielding member, so that the ultraviolet ray leaks out of the humidification space. And the container can be taken in and out smoothly at the delivery position.
  • An incubator according to an eighth aspect is the incubator according to any one of the third to sixth aspects, further comprising a humidifying shelf and a moving mechanism.
  • the humidification shelf is provided in the humidification space, and the container is placed thereon.
  • the moving mechanism moves the humidification shelf between a delivery position for delivering the container conveyed to the humidification space and a humidification position provided in the humidification space. Except for the state where the humidifying shelf is at the humidifying position, the irradiation of ultraviolet rays from the UV irradiation unit is stopped.
  • An incubator according to a ninth aspect is the incubator according to any one of the first to eighth aspects, wherein the container detection unit discriminates between a culture container conveyed to the culture space and a container containing humidified water. Is further provided.
  • the container detection part for discriminating whether the container carried into the incubator from the cell operation chamber side is a culture container containing cells to be cultured or a container containing humidified water is provided.
  • the culture container containing the cells is mistakenly transported into the humidification space, and is killed by receiving ultraviolet rays from the UV irradiation unit, or the container containing the humidification water is mistakenly placed in the culture chamber. Problems such as being conveyed can be prevented.
  • An incubator according to a tenth invention is the incubator according to the ninth invention, wherein the container detection unit includes a plurality of sensors for detecting a difference in shape of the bottom surface of the culture container and the container containing the humidified water. .
  • the plurality of sensors include contact sensors such as mechanical switches, non-contact sensors such as proximity sensors, and the like.
  • An incubator according to an eleventh aspect of the invention is the incubator according to any one of the first to tenth aspects of the invention, in which the circulation unit is configured to generate a flow of air and a flow of air formed by the fan. And a circulation path that leads to the open side of the container placed in the humidification space.
  • a circulation unit for circulating air containing humidified water in the humidified space a fan that generates an air flow in the incubator, and an air flow generated by the fan to the open side of the container in the humidified space. A circulation path that leads.
  • a cell culture system includes the incubator according to any one of the first to eleventh aspects and a cell operation chamber.
  • pretreatment is performed on the cells in the incubator, and an operation of adding humidified humidified water to the container is performed.
  • a cell culture system including the above-described incubator and a cell operation chamber that pretreats cells and humidified water contained in a container transported to the incubator is configured.
  • the cell manipulation chamber includes an isolator having a clean space inside, a biohazard cabinet, and the like.
  • a cell culture system according to a thirteenth invention is the cell culture system according to the twelfth invention, and is provided between the incubator and the cell operation chamber, and a container is provided between the incubator and the cell operation chamber.
  • a connection box for carrying is further provided.
  • a connection box is provided between the incubator and the cell manipulation chamber for transporting the container in a sterile state.
  • the connection box has a transport mechanism for delivering an article such as a container pretreated in the cell manipulation chamber to the incubator side, and the inside is aseptic as the cell manipulation chamber.
  • a cell culture system according to a fourteenth aspect of the invention is the cell culture system according to the thirteenth aspect of the invention, wherein the connection box receives a container containing humidified water whose expiration date has passed from the incubator transport section, Has an opening for taking out the outside.
  • the connection box receives a container containing humidified water whose expiration date has passed from the incubator transport section, Has an opening for taking out the outside.
  • the container transported from the incubator side is taken out from the opening provided in the connection box.
  • a container can be taken out from the connection box adjacent to the incubator without transporting the container to the loading / unloading port on the cell manipulation chamber side.
  • a cell culture system is the cell culture system according to the thirteenth or fourteenth invention, wherein the connection box discriminates between a culture container transported to the culture space and a container containing humidified water.
  • a container detection unit is further provided.
  • a container detection unit for determining whether the container carried into the incubator from the cell operation chamber side is a culture container containing cells to be cultured or a container containing humidified water is provided in the connection box. Provided. As a result, by determining the type of container in the connection box, the culture container containing the cells is mistakenly transported into the humidified space without an incubator, and is killed by receiving ultraviolet rays from the UV irradiation unit. It is possible to prevent problems such as that the container containing the humidified water is erroneously transported to the culture chamber.
  • a cell culture system is the cell culture system according to the fifteenth aspect of the invention, wherein the container detection unit is a plurality of sensors for detecting differences in the shape of the bottom surface of the culture container and the container containing the humidified water.
  • the plurality of sensors include contact sensors such as mechanical switches, non-contact sensors such as proximity sensors, and the like.
  • contact sensors such as mechanical switches
  • non-contact sensors such as proximity sensors, and the like.
  • a humidifying water supply method is to humidify a culture space with respect to an incubator in which a culture space for culturing cells is formed from a cell operation chamber in which a clean space is formed.
  • a method of supplying humidified water comprising the following steps. In the cell manipulation chamber, a step of charging the humidified water into the container from a humidified water container in which aseptically treated humidified water is enclosed. Transferring the container from the cell operation chamber side to a predetermined humidification space formed in the incubator; A step of generating an air flow passing through the vicinity of an open portion of the container conveyed to the humidification space and circulating the air containing the humidification water in the culture space.
  • aseptically treated humidified water was introduced from a cell operation chamber having a clean space for performing predetermined pretreatment and the like on cells cultured in the incubator.
  • the container is received and placed in the humidification space, and the culture space is humidified.
  • a robot arm or other transfer mechanism for receiving the container from the cell operation chamber side and transferring it to a predetermined position is used.
  • a fan that circulates air (for example, steam, mist, gas, etc.) containing humidified water in the culture space is used.
  • the cell manipulation chamber includes an isolator having a clean space inside, a biohazard cabinet, and the like.
  • a method for supplying humidified water according to an eighteenth aspect of the invention is a method for supplying humidified water according to the seventeenth aspect of the invention, in which ultraviolet light for sterilization is applied to the open portion of the container conveyed to the humidified space, The method further includes the step of sterilizing the humidified water.
  • the method further includes the step of irradiating ultraviolet rays for sterilization on the open portion of the container containing the humidified water carried into the incubator.
  • irradiation with ultraviolet rays can be set as appropriate, such as continuous irradiation and irradiation at predetermined intervals. Moreover, it is more preferable that the irradiation with ultraviolet rays is performed in a state where the container is in a humidified position. Thereby, the humidified water after carrying in in an incubator can be maintained more reliably in a sterile state.
  • a humidifying water supply method according to a nineteenth aspect of the invention is a humidifying water supply method according to the eighteenth aspect of the invention, which is carried out between the step of transporting the container to the humidification space and the step of performing the sterilization treatment. The method further includes a step of inserting a shielding member for shielding the humidification space and the culture space.
  • a step of inserting a shielding member is provided between the container transporting step and the sterilizing step.
  • the shielding member includes a lid-like member that covers a portion of the UV irradiation unit placed in the humidification space where the UV irradiation unit is irradiated.
  • a humidifying water supply method is a humidifying water supply method according to any one of the seventeenth to nineteenth aspects of the invention, wherein the humidifying water is contained in a container conveyed to the humidifying space.
  • the method further includes the step of taking out the container from the connection box provided between the incubator and the cell manipulation chamber.
  • the expiration date of the humidified water transported into the incubator expires, the container transported from the incubator side is taken out from the opening provided in the connection box.
  • the incubator according to the present invention can sufficiently ensure the aseptic condition of water used for humidification in the culture space.
  • the front view which shows the whole structure of the cell culture system containing the incubator which concerns on one Embodiment of this invention.
  • the front view which shows the structure inside the incubator contained in the cell culture system of FIG.
  • the side view which shows the structure inside the incubator contained in the cell culture system of FIG.
  • the top view which shows the structure inside the incubator contained in the cell culture system of FIG.
  • the perspective view which shows a structure around the delivery position and humidification position of the container in which the humidification water provided in the incubator contained in the cell culture system of FIG. 1 was put.
  • the perspective view which shows the structure in the connection box contained in the cell culture system of FIG. (A) And (b) is a top view which shows the structure of the large / small tray in which a container is mounted.
  • FIGS. 9A to 9C are a plan view, a side view, and a front view showing a configuration of a claw member that is attached to the tip of a transport unit provided in an incubator that lifts the tray shown in FIG.
  • the flowchart which shows the flow of the supply method of humidification water in the cell culture system of FIG.
  • the flowchart which shows the flow of the replacement
  • the front view which shows the whole structure of the cell culture system which concerns on other embodiment of this invention.
  • a cell culture system 1 provided with an incubator 3 according to an embodiment of the present invention will be described below with reference to FIGS.
  • the clean spaces that appear in the following description include sterilized or decontaminated spaces, clean rooms with high cleanliness, and the like.
  • the sterilized humidified water is not limited to aseptic water in which the bacteria contained in the water is zero, but also includes water containing a number of bacteria within an acceptable range.
  • the cell culture system 1 according to the present embodiment is a system for culturing cells that have been pretreated in the clean operation space S1, and as shown in FIG. 1, an isolator (cell operation chamber) 2, an incubator 3, and The connection box 4 is provided.
  • the isolator 2 is an example of a cell operation chamber, and forms a clean operation space S1 that is isolated from the outside air, as shown in FIG.
  • the clean operation space S1 is maintained at a certain level of cleanliness or higher by circulating clean air that has flowed in through the HEPA filters installed above and below the isolator 2 from above to below.
  • the cleanliness is generally expressed by an index called a cleanliness grade. It is said that it is necessary to ensure grade A in the isolator 2 used for processing cultured cells.
  • Grade A This is a local area where work that needs to prevent the risk of contamination of products at a high level is performed.
  • Grade B A multi-purpose area where operations that require prevention of contamination risks at a relatively high level are performed.
  • Grades C and D Areas where work with a relatively high risk of product contamination is performed.
  • a pass box 20 is provided between the isolator 2 and the external space.
  • Various items are carried into the pass box 20 from the outside through the open / close door 21 while maintaining the cleanliness of the cleaning operation space S1 in the isolator 2.
  • the pass box 20 includes an internal door (not shown), an aeration fan, and an aeration filter.
  • the internal door and the opening / closing door 21 are structured so that they cannot be opened simultaneously.
  • the open / close door 21 is opened and the various articles are carried in. Then, when the open / close door 21 is closed, clean air flows in through the aeration filter by the operation of the aeration fan. The inside of the pass box 20 and the surfaces of various articles are cleaned. Thereafter, the internal door is opened, various articles are carried into the isolator 2 through the globes 22a and 22b, and the internal door is closed.
  • the open / close door 21 has a handle 21a, and the user opens and closes the open / close door 21 with the handle 21a. Specifically, in the cleaning operation space S1 in the isolator 2, cells cultured in the incubator 3 are opened. Various treatments such as seeding on the culture medium, medium exchange, passage, etc., and a process of putting humidified water that has been sterilized to humidify the culture space S2 formed in the incubator 3 into a predetermined container are performed.
  • the periphery of the isolator 2 has a grade C or D cleanliness.
  • the periphery of the isolator 2 is controlled so as to be approximately 20 to 25 ° C. and low humidity in order to prevent human work and bacterial growth.
  • the clean space S1 in the isolator 2 is also sealed with respect to the surrounding space, but has substantially the same temperature and humidity.
  • the process performed in the isolator 2 is performed using a tool or container that has been previously sterilized. As described above, these tools, containers, and the like are carried into the isolator 2 via the pass box 20 while maintaining the cleanliness of the clean space S1. (Incubator 3) As shown in FIG.
  • the incubator 3 has a culture space S2 for culturing cells received through the opening 3a (see FIG. 2) in a state of being placed in a container from the isolator 2 through the connection box 4.
  • a humidification space S3 (see FIG. 2) for controlling the humidity so that the culture space S2 is in a certain humidity range is formed inside.
  • the temperature of the culture space S2 in the incubator 3 is a temperature (about 37 ° C.) higher than the clean operation space S1 in the isolator 2 in order to promote cell culture. And it is managed to be high humidity.
  • connection box 4 is provided for delivering articles such as containers and various tools into which cells are introduced between the isolator 2 and the incubator 3. As shown in FIG. It is installed between. Specifically, the connection box 4 is configured so that the cells pretreated in the clean operation space S1 of the isolator 2, the container containing the sterilized humidified water, and the like are maintained in the incubator 3 while maintaining the sterility.
  • a transport mechanism 42 (see FIG. 7) is provided for transferring to the culture space S2 or the humidification space S3. The used humidification container 10 in the humidification space S3 of the incubator 3 is transported into the isolator 2 by the transport mechanism 42.
  • the humidified water that has been reduced by being used for humidification is replenished.
  • the used humidifying container 10 and the remaining humidified water may be once taken out via the pass box 20 and discarded in order to further reduce the possibility of propagation of bacteria or the like in the humidifying container 10.
  • a new humidification container 10 and humidified water having been subjected to aseptic processing are carried in.
  • the humidifying container 10 and the culture container 11 are cylindrical containers with one end opened, such as a petri dish, but the containers may have other shapes. Depending on the material of the container, it may be difficult to pass ultraviolet rays for performing a sterilization process described later. For this reason, it is desirable that the container has a shape in which one end is greatly opened so that the entire humidified water can be irradiated with ultraviolet rays.
  • the transport mechanism 42 is a mechanism for delivering various articles transported between the isolator 2 and the incubator 3 in the connection box 4 and transports the trays 51 to 53 on which various containers and the like are placed on the upper surface.
  • the transport mechanism 42 has a mounting table 42b that can rotate around a rotating shaft 42a. Thereby, the mounting table 42b can be brought closer to the isolator 2 side or the incubator 3 side by rotating the mounting table 42b around the rotation shaft 42a in the vicinity of the opening 4b or the opening 4c.
  • connection box 4 has a tray detection unit 50 (FIGS. 6 and 8A to 8) that determines the type of the container (tray) at a stage before the container received from the isolator 2 is delivered to the incubator 3. (See (c)).
  • the tray detection unit 50 is attached to the transport mechanism 42 and includes three sensor units SW1 to SW3.
  • three types of containers are used as the containers conveyed from the isolator 2 to the incubator 3, and each container is loaded on a different tray 51-53 and conveyed.
  • a culture container tray 51, a culture container tray 52, and a humidified water tray 53 are used.
  • the culture container tray 51 is a tray on which containers for cells are placed. As shown in FIG. 7A, the culture container tray 51 includes a main body 51a, detected parts 51ba and 51bb, and a gripping part 51c. is doing.
  • the main body 51a is a portion on which a culture vessel is placed, and has an opening inside, and is formed by a substantially rectangular frame portion in plan view.
  • the detected portions 51ba and 51bb are projecting surfaces formed so as to project inward from positions facing each other inside the frame portion of the main body 51a, and are detected by sensor units SW1 to SW3 described later.
  • the grip 51c is inserted from below into a claw member 32b (see FIG. 2 and the like) attached to the distal end of a transport part 32a included in the transport mechanism 32 on the incubator 3 described later, and transported to a desired position in the incubator 3. Is done.
  • the culture container tray 52 is a container larger than the culture container tray 51 or a tray on which a plurality of containers are placed among the containers in which cells are placed. As shown in FIG. And to-be-detected parts 52ba and 52bb and a grip part 52c.
  • the main body 52a is a portion on which a culture vessel is placed, and has an opening inside, and is formed by a substantially rectangular frame portion in plan view. Moreover, the main-body part 52a has a long dimension in the longitudinal direction compared with the main-body part 51a of the culture container tray 51 shown to Fig.7 (a).
  • the detected portions 52ba and 52bb are projecting surfaces formed so as to project inward from positions facing each other inside the frame portion of the main body 52a, and are detected by sensor units SW1 to SW3 described later. Specifically, as shown in FIG. 8A, only the sensor units SW1 and SW2 among the sensor units SW1 to SW3 are detected.
  • the detected parts 52ba and 52bb have a larger interval than the detected parts 51ba and 51bb of the culture container tray 51 shown in FIG. Has been placed.
  • the detected portions 51ba and 51bb and the sensor portions SW1 to SW3 that detect the detected portions 52ba and 52bb are different.
  • FIG. 8B only the sensor units SW1 and SW3 among the sensor units SW1 to SW3 are detected. Therefore, it is possible to easily determine which one of the culture container trays 51 and 52 is placed.
  • the gripping part 52c has the same size and shape as the gripping part 51c of the culture container tray 51 shown in FIG. 7A, and the tip of the transporting part 32a included in the transporting mechanism 32 on the incubator 3 side described later.
  • a claw member 32b (see FIG. 2 and the like) attached to the section is inserted from below and conveyed to a desired position in the incubator 3.
  • the humidifying water tray 53 is a tray for transporting the humidifying container 10 containing the humidifying water for maintaining the humidity of the culture space S2 of the incubator 3 within a predetermined range into the incubator 3. As shown in FIG. 7C, it has a main body portion 53a and a grip portion 53c.
  • the main body 53a is a portion where the humidifying container 10 is placed on the upper surface, and is formed by a substantially rectangular plane in plan view.
  • the main body 53a has substantially the same dimensions in plan view as compared with the main body 52a of the culture container tray 52 shown in FIG.
  • the main body portion 53a does not have an opening portion on the inner side as compared with the main body portions 51a and 52a of the culture container trays 51 and 52 described above. Thereby, when the humidifying water tray 53 is placed on the transport mechanism 42, the entire back surface side of the main body 53a functions as a detected portion.
  • the humidified water tray 53 has the same size in the longitudinal direction as the culture container tray 52, and the entire back surface of the main body 53a is detected by all the sensor units SW1 to SW3. Specifically, as shown in FIG. 8C, the sensor units SW1 to SW3 are detected by all the SW1 to SW3. Thereby, it is possible to easily determine which of the culture container trays 51 and 52 or the humidifying water tray 53 is placed. Therefore, it is possible to avoid the occurrence of a problem that the culture container is mistakenly transported into the humidification space S3 and cells are killed by ultraviolet irradiation.
  • the gripping part 53c has the same size and shape as the gripping parts 51c, 52c of the culture container trays 51, 52, and is attached to the tip of the transport part 32a included in the transport mechanism 32 on the incubator 3 side described later.
  • a claw member 32b (see FIG. 2 and the like) is inserted from below and is conveyed to a desired position in the incubator 3.
  • the tray detection unit 50 for determining the type of the container (tray) is provided. Accordingly, as described above, it is possible to detect that any of the trays 51 to 53 is placed on the transport mechanism 42 by the sensor unit SW1 being in a detection state, and which of the trays 51 to 53 is. Whether it is placed or not can be determined by the detection states of the sensor units SW2 and SW3.
  • the type of the container is a culture or humidified water. If it can be determined, it can be similarly implemented.
  • a discrimination method for example, a method of detecting the shape of a container using optical means such as transmitted light, or a culture specimen management system using an RFID and its non-contact reader or a barcode and a barcode reader system There is a method to determine in cooperation with.
  • the incubator 3 of the present embodiment receives a container in which cells pretreated in the isolator 2 described above are received and cultures the cells, and the culture space S2 in a predetermined humidity range.
  • a humidifying space S3 for maintaining the inner space is formed inside.
  • the incubator 3 includes culture shelves 31a and 31b, a transport mechanism 32, a humidifying shelf 33, a UV irradiation unit 34, and a shielding member (a lid member 35a and a side wall 35b). And a moving mechanism 36 and a fan 37.
  • the culture shelves 31a and 31b are shelves on which the culture vessels 11 (see FIG. 4 and the like) are placed.
  • a plurality of culture shelves 31a and 31b are arranged along the left side and right side of the incubator 3 in front view. Has been.
  • the culture shelf 31a is attached to the side surface opposite to the side surface on which the opening 3a through which various containers and the like are transported from the connection box 4 side is formed, and extends along the vertical direction. A plurality are provided.
  • the culture shelf 31b is a shelf having a smaller mounting area than the culture shelf 31a, and as shown in FIG. 2, a plurality of culture shelves are provided along the vertical direction above the opening 3a on the side surface where the opening 3a is formed. It has been.
  • the transport mechanism 32 is a mechanism that receives the various containers transported from the connection box 4 side together with the trays 51 to 53 and transports them to a desired position.
  • the transport mechanism 32 includes a transport unit 32a as a robot arm, a claw member 32b, and an attachment unit.
  • the transport unit 32a is a robot arm that is movable along the vertical direction and is driven by six axes, and as shown in FIG. 2, various containers transported from the connection box 4 side through the opening 3a. Is transferred to the humidifying shelf 33 and transferred to the delivery position P1. Further, the transport unit 32a has a claw member 32b for gripping and transporting the trays 51 to 53 at the hand portion of the robot arm.
  • the claw member 32b has a main body 32ba, a protrusion 32bb, and a connection 32bc.
  • the main body portion 32ba is a substantially U-shaped planar member, and is connected to the transport portion 32a via the connection portion 32ba.
  • the protrusion 32bb is a member that is inserted from below into the holes formed in the grips 51c, 52c, 53c of the various trays described above (culture container trays 51, 52 and humidified water tray 53).
  • the main body portion 32ba protrudes upward from the upper surface of the substantially U-shaped tip portion.
  • the various trays 51 to 53 are conveyed in the incubator 3 in a state where the two protrusions 32bb and 32bb are inserted and supported from below in the holes of the gripping portions 51c to 53c of the various trays 51 to 53. .
  • connection portion 32bc is a substantially columnar member, and is attached to the hand portion of the robot arm of the transfer portion 32a.
  • the transfer portion 32a and the claw member 32b are connected to the connection portion 32bc.
  • the attachment portion 32c is a member for attaching the conveyance portion 32a as a robot arm to the side surface of the incubator 3 (the side surface disposed on the front surface shown in FIG. 2), and is attached to the guide rail 32d.
  • the insertion portion 32ca is a portion protruding from the surface of the attachment portion 32c, and as shown in FIG. 2, is inserted into the guide rail 32d and is guided in the vertical direction to the guide rail 32d.
  • the guide rail 32d is a guide groove formed along the vertical direction, and moves in a state where the insertion portion 32ca of the attachment portion 32c to which the conveyance portion 32a is attached is inserted.
  • the transport unit 32a as a robot arm can be moved along the vertical direction.
  • description is abbreviate
  • the humidifying shelf 33 is a shelf on which the humidifying container 10 in which humidified water subjected to aseptic processing is placed, and as shown in FIG. 2, along the side surface on which the opening 3a is formed, the opening 3a It arrange
  • the UV irradiation unit 34 irradiates the open portion of the humidifying container 10 placed together with the humidifying water tray 53 on the humidifying shelf 33 so that the humidified water stored in the humidifying container 10 is not polluted by various bacteria or the like. So as to perform sterilization treatment.
  • the UV irradiation part 34 is arrange
  • Irradiation of ultraviolet rays is not limited to the elapse of a predetermined time, and may be always performed while the humidifying shelf 33 is at the humidifying position P2.
  • the shielding members (the lid member 35a and the side wall portion 35b) shield the ultraviolet rays emitted from the UV irradiation unit 34 from leaking directly to the outside of the humidification space S3.
  • the shielding member has a lid member 35a and a side wall portion 35b. That is, the shielding member is configured so that only the reflected light reaches the opening that is the connection portion between the humidification space S3 and the circulation portion.
  • the intensity of ultraviolet rays decreases due to repeated reflections, and even if the ultraviolet rays leak from the connection openings, the cultures in the culture vessels placed on the culture shelves 31a and 31b are harmed. The strength can be lowered to a level not given.
  • the shielding member is made of stainless steel, but is not limited to this.
  • a material having a low ultraviolet reflectance such as anodized aluminum may be used.
  • the influence of the leaked ultraviolet rays on the culture can be further reduced.
  • the lid member 35 a is a plate-like member, and is attached to the humidifying shelf 33 so as to cover the upper side of the humidifying shelf 33. Therefore, when the humidifying shelf 33 moves, the lid member 35a moves integrally with the humidifying shelf 33 together with the UV irradiation unit 34.
  • the UV irradiation part 34 is arrange
  • the side wall 35 b is fixedly disposed at a humidification position P ⁇ b> 2 in the humidification space S ⁇ b> 3 separately from the humidification shelf 33. For this reason, if the humidification shelf 33 moves to the humidification position P2, the side wall part 35b can cover the side of the humidification container 10 in which humidification water was stored.
  • the UV irradiation from the UV irradiation unit 34 is performed only at the humidifying position P2, and at the humidifying position P2, the upper surface of the humidifying container 10 placed on the humidifying shelf 33 and The sides can be covered with a lid member 35a and a side wall portion 35b constituting a shielding member.
  • the ultraviolet rays irradiated in the humidification space S3 hit the cells put in the culture container 11 placed on the culture shelves 31a and 31b of the culture space S2 outside the humidification space S3, and kill the cells.
  • the occurrence of such problems can be prevented.
  • the moving mechanism 36 is a mechanism that moves the humidifying shelf 33 along the vertical direction between the delivery position P1 and the humidifying position P2, and includes a drive motor 36a, a link mechanism 36b, a rotating shaft 36c, a connecting portion 36d, and It has a guide part 36e.
  • the drive motor 36 a is disposed outside the culture space S ⁇ b> 2 of the incubator 3 and supplies a driving force for moving the humidifying shelf 33 in the vertical direction.
  • the link mechanism 36b is disposed outside the culture space S2 of the incubator 3 together with the drive motor 36a.
  • the link mechanism 36b transmits the rotational driving force supplied from the drive motor 36a to the rotating shaft 36c.
  • the rotation shaft 36c is arranged from the outside to the inside of the culture space S2 of the incubator 3, and rotates by receiving the rotational driving force transmitted through the link mechanism 36b.
  • the rotating shaft 36c may be comprised by the coupling member which connects two axial members arrange
  • the connecting portion 36d is connected to the end of the rotating shaft 36c on the culture space S2 side, and rotates by the rotation of the rotating shaft 36c, thereby moving the humidifying shelf 33 in the vertical direction.
  • the guide portion 36e is a guide groove formed along the vertical direction so that the humidifying shelf 33 moved by the rotation of the connection portion 36d moves along the vertical direction, and the connection portion 36d extends along the vertical direction. To guide.
  • the fan 37 is disposed above the culture space S2 in the incubator 3, and enters the humidification space S3 via a circulation path (circulation part) 37a (see the dotted arrow in FIG. 2). Forms an air flow that is guided by In other words, the air flow formed by the fan 37 passes over the culture space S2 and travels downward along the side surface where the opening 3a is formed. Then, as shown in FIG. 5, the air flow passes through the rear side of the culture shelf 31 b and then flows from the humidification space S ⁇ b> 3 to the culture space S ⁇ b> 2 and circulates in the incubator 3.
  • the air flow formed by the fan 37 passes through the gap between the lid member 35a and the open side of the humidifying container 10 in the humidifying space S3, becomes air containing humidified water, and enters the culture space S2. It will flow.
  • cultivation space S2 can always maintain the humidified state, it can prevent that the humidity in culture
  • the incubator 3 of the present embodiment has the above-described configuration, and humidified water that has been sterilized to maintain the humidity of the culture space S2 is supplied from the isolator 2 side by the following procedure. That is, as shown in FIG. 10, first, in step ST1, as shown in FIG. 1, the humidified water container containing the humidified sterilized water is carried into the isolator 2 through the pass box 20.
  • the humidified water container may be a bottle or may be a sealed pack.
  • step ST2 in the cleaning operation space S1 of the isolator 2, humidified water is poured from the humidified water container into a predetermined container (humidified container 10) through the gloves 22a and 22b.
  • step ST ⁇ b> 3 the humidifying container 10 containing the humidified water is placed on the humidifying water tray 53, and then delivered to the connection box 4.
  • delivery of the humidification container 10 in step ST3 may be performed using the conveyance mechanism provided in the connection box 4 between the isolator 2 and the incubator 3, or a user's via globe 22a, 22b. It may be done by hand.
  • the gloves 21a and 21b are placed with the humidifying water tray 53 and the humidifying container 10 placed on the transport mechanism. It may be used to pour humidified water.
  • step ST4 the type of the container (tray) carried in is determined by the tray detector 50 provided in the connection box 4.
  • the humidifying water tray 53 on which the humidifying container 10 is placed is placed on the transport mechanism 42 in the connection box 4, the fact that the humidifying container 10 has been transported by the sensor units SW 1 to SW 3 described above. Can be detected.
  • the type of container detected here is transmitted to a control unit (not shown) that controls the entire cell culture system 1.
  • step ST ⁇ b> 5 the humidifying container 10 is transported together with the humidifying water tray 53 to the incubator 3 side by the transport mechanism 42 in the connection box 4. Specifically, the humidifying water tray 53 transported to the vicinity of the opening 3a on the incubator 3 side by the transport mechanism 42 is lifted from below by the claw member 32b of the transport mechanism 32 on the incubator 3 side, and the delivery position in the incubator 3 is reached. Transport to P1.
  • step ST ⁇ b> 6 after the humidifying water tray 53 transported to the delivery position P ⁇ b> 1 in the incubator 3 by the transport mechanism 32 is placed on the humidifying shelf 33, the humidifying shelf 33 moves downward. To move to the humidification position P2.
  • step ST7 when the humidifying shelf 33 moves to the humidifying position P2, in step ST7, the side wall 35b constituting the shielding member moves relatively to a position covering the side of the humidifying container 10. That is, since the side wall part 35b is fixedly arranged in the vicinity of the humidification position P2, the side of the humidification container 10 can be automatically covered by moving the humidification shelf 33 to the humidification position P3. Therefore, the upper surface of the humidification container 10 that has been transported to the humidification position P2 is covered with the lid member 35a, and the side is covered with the side wall portion 35b. As a result, the humidification space S3 can be a space shielded from the culture space S2.
  • step ST8 the UV irradiation unit 34 irradiates the humidified water stored in the humidification container 10 at the humidification position P2 with sterilizing ultraviolet rays every predetermined time.
  • the inside of the culture space S2 can be prevented from being contaminated by bacteria or the like due to the humidification.
  • ultraviolet irradiation may be performed frequently. Thereby, the humidification water stored in the humidification container 10 is warmed, and the humidity of the culture environment S2 can be kept higher.
  • step ST11 it is determined whether or not a predetermined period has passed since the humidified water was carried in.
  • the predetermined period is generally set to the shorter one of the expiration date for use of the humidified water set in advance or the period in which the carried-in humidified water is expected to be a certain amount or less due to humidification. .
  • the expected period according to the situation is set.
  • the humidifying container 10 containing the humidified water is transported together with the humidifying water tray 53 together with the humidifying shelf 33 from the humidifying position P2 to the delivery position P1.
  • step ST13 the transport mechanism 32 provided in the incubator 3 receives the humidified container 10 containing the humidified water together with the humidified water tray 53 at the delivery position P1, and the transport mechanism 42 on the connection box 4 side. Hand over to The used humidifying container 10 is transported into the isolator 2 by the transport mechanism 42. In the isolator 2, the used humidification container 10 and the remaining humidification are used to replenish the humidified water reduced for humidification or to reduce the possibility of propagation of bacteria and the like in the humidification container 10. Water is once taken out via the pass box 20 and discarded, and a new container and humidified water are carried in to continue humidification. ⁇ Main features> As shown in FIG.
  • the incubator 3 of this embodiment receives cells that have been pretreated in the cleaning operation space S1 formed in the isolator 2, and forms a culture space S2 in which cells are cultured. To do. And the incubator 3 is provided with the conveyance mechanism 32, humidification space S3, and the fan 37, as shown in FIG.
  • the transport mechanism 32 receives various articles transported from the isolator 2 side, transports them to a predetermined position, and the humidifying container 10 containing humidified water subjected to aseptic processing for maintaining the humidity in the culture space S2. Is received from the isolator 2 side. In the humidification space S3, the humidification container 10 containing the humidification water received by the transport mechanism 32 is placed.
  • the fan 37 generates an air flow that passes through the vicinity of the open side of the humidification container 10 placed in the humidification space S3, and circulates the air containing the humidification water in the culture space S2.
  • the humidified water is opened in the cleaning operation space S1 in the isolator 2 and placed in the humidified container 10, so that the humidified container 10 containing the humidified water is removed from the isolator 2 while maintaining the aseptic state of the humidified water. Can receive.
  • the humidified water is transported into the incubator 3 in a state in which sterility is ensured as compared with the conventional configuration in which sterilized water is supplied into the incubator through a pipe from a sterilized water tank attached outside the incubator.
  • the inside of the culture space S2 can be humidified.
  • a container detection unit may be provided in an incubator that receives containers from a connection box.
  • a container detector may be provided at the tip of the robot arm shown in FIG.
  • a cell culture system in which an incubator and an isolator are directly connected may be used.
  • C in order to humidify the culture space S2 in the incubator 3, an air flow that flows in the vicinity of the open side of the humidification container 10 storing the humidified water is formed by the fan 37, and humidification is performed while evaporating the humidified water.
  • the present invention is not limited to this.
  • the humidified water in addition to a method of promoting evaporation by an air flow passing in the vicinity of the humidified water, the humidified water is sprayed into the culture space S2 in the form of a mist or a gas containing the humidified water. Or may be circulated in the culture space S2.
  • the used humidification container 10 may be taken out via the open / close door 41 of the connection box 4 and the humidified water may be discarded.
  • the used humidification container 10 in the humidification space S3 of the incubator 3 is transported to the connection box 4 and taken out with the open / close door 41 opened with the shutter mechanism (not shown) closed.
  • the risk that the humidifying container 10 in which the bacteria or the like have propagated is transported to the isolator 2 and the clean space S1 in the isolator 2 is contaminated is minimized.
  • connection box 4 After the used humidifying container 10 is taken out, aeration operation for introducing clean air into the connection box 4 through the filter by the intake unit 16 and exhausting through the exhaust unit 18 or introduction of decontamination gas
  • the inside of the connection box 4 can be cleaned by a decontamination operation using a section (not shown).
  • the new humidification container 10 is carried into the humidification space S3 in the incubator 2 using the transport mechanism 42, so that the humidification in the humidification space S3 can be continued.
  • the cell culture system 1 containing the isolator 2, the incubator 3, and the connection box 4 was mentioned as an example, and was demonstrated.
  • the present invention is not limited to this.
  • a cell culture system 101 provided with a biohazard cabinet (cell manipulation chamber) 102 instead of an isolator it is good.
  • the environment in which the cell culture system 101 is installed is grade C or D, it is necessary to use an isolator, but if the environment is grade B, the biohazard cabinet 102 can be used.
  • the front glass can be opened and closed, and a predetermined amount of the door is opened and a hand is inserted from there to perform the work.
  • a clean air flow through a filter such as a HEPA filter is generated from the top to the bottom, and the work space S4 is kept clean.
  • an air curtain is formed by a faster air flow and a suction operation is performed at the lower end, so that no substance leaks from the inside to the outside even when the door is opened. Therefore, also in the intracellular system 101 shown in FIG. 12, humidified water can be supplied from the door of the biohazard cabinet 102 and supplied as humidified water as in the above embodiment.
  • the incubator according to the present invention has an effect that the aseptic condition of water used for humidification in the culture space can be sufficiently ensured at a level equivalent to or higher than that of the culture. Widely applicable.

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Abstract

　L'invention concerne un incubateur (3) doté d'un mécanisme de transport (32), d'un espace d'humidification (S3), et d'une unité de circulation (ventilateur (37), voie de circulation (37a)). Le mécanisme de transport (32) reçoit divers éléments transportés depuis le côté isolateur (2) et transporte les éléments jusqu'à des emplacements prédéterminés, et reçoit un récipient d'humidification (10) pour maintenir l'humidité au sein d'un espace de culture (S2), le récipient d'humidification (10) contenant de l'eau d'humidification traitée de manière aseptique, depuis l'isolateur (2). Le récipient d'humidification (10) reçu par le mécanisme de transport (32) est placé dans l'espace d'humidification (S3). Le ventilateur (37) génère un courant d'air qui passe à proximité du côté ouvert du récipient d'humidification (10) placé dans l'espace d'humidification (S3), et l'air contenant de l'eau d'humidification est fait circuler au sein de l'espace de culture (S2).
PCT/JP2015/051226 2014-01-24 2015-01-19 Incubateur, système de culture cellulaire doté de celui-ci, et procédé d'alimentation d'eau d'humidification Ceased WO2015111544A1 (fr)

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CN110612346A (zh) * 2017-05-15 2019-12-24 株式会社爱瑞思 恒温箱
EP3626813A4 (fr) * 2017-05-15 2021-02-24 Airex Co., Ltd. Incubateur
WO2025142843A1 (fr) * 2023-12-25 2025-07-03 三菱重工業株式会社 Système de transport, système de culture cellulaire, procédé de transport, mécanisme de support et plaque

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