WO2016042743A1 - 細胞培養装置 - Google Patents
細胞培養装置 Download PDFInfo
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- WO2016042743A1 WO2016042743A1 PCT/JP2015/004591 JP2015004591W WO2016042743A1 WO 2016042743 A1 WO2016042743 A1 WO 2016042743A1 JP 2015004591 W JP2015004591 W JP 2015004591W WO 2016042743 A1 WO2016042743 A1 WO 2016042743A1
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- 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
- C12M23/02—Form or structure of the vessel
- C12M23/04—Flat or tray type, drawers
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- 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
- C12M23/48—Holding appliances; Racks; Supports
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- 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
- C12M23/02—Form or structure of the vessel
- C12M23/14—Bags
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- 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
- C12M23/34—Internal compartments or partitions
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- 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
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- 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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
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- 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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/16—Vibrating; Shaking; Tilting
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- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/20—Degassing; Venting; Bubble traps
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/12—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by pressure
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- 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
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- 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
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- 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
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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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
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
Definitions
- the present invention relates to a cell culture apparatus.
- a medium container for supplying the medium to the culture container and a culture container for use in cell culture are connected by a tube to form a cell culture unit, which is closed using this cell culture unit.
- Patent Document 1 discloses a culture apparatus that stores this type of culture unit in multiple stages and performs cell culture.
- the present invention has been made in view of the above circumstances, and a plurality of cell culture units including a culture vessel for supplying a culture medium together with cells to perform cell culture, and a culture vessel for supplying the culture medium to the culture vessel.
- the purpose of this study is to provide a cell culture device that can measure the progress of cell culture in all cell culture units with a single measurement unit.
- a cell culture device comprises at least a culture container for supplying a culture medium together with cells to perform cell culture, and a culture medium container for supplying the culture medium to the culture container. And a plurality of cell culture units connected to each other by a transfer tube in a multistage, and a cell culture apparatus for individually cultivating each cell culture unit in the height direction.
- a support frame in which a plurality of slide rails are arranged in a multi-stage is installed in the housing, and a culture vessel storage shelf for storing the culture vessel is slidably attached to each of the slide rails,
- a measurement unit including an imaging unit is movably installed between the back plate portion of the housing and the culture container storage shelf, and the measurement unit is a cell culture to be measured.
- the culture container storage shelf that stores the culture container included in the cell culture unit so that the measurement site of the culture container included in the cell culture unit falls within the imaging range of the imaging unit. The part moves toward the measurement unit.
- a plurality of cell culture units each including a culture container and a medium container are stored in multiple stages, and cell culture is performed separately for each cell culture unit.
- the progress of cell culture in the measurement target cell culture unit can be measured with one measurement unit without interfering with the cell culture unit to be measured.
- the culture medium container (medium bag) and the culture container (culture bag) are stored in the same thermostatic chamber, and the temperature suitable for each of these containers. Cell culture could not be performed in the environment.
- the present invention stores in multiple stages a plurality of cell culture units each including a culture container that supplies a culture medium together with cells to perform cell culture, and a culture medium container for supplying the culture medium to the culture container.
- a culture container that supplies a culture medium together with cells to perform cell culture
- a culture medium container for supplying the culture medium to the culture container.
- the cell culture device includes an incubation tank in which the culture container is stored and a cold storage tank in which the culture medium container is stored, and each of the incubation tank and the cold storage tank has a height direction.
- a plurality of slide rails are arranged in multiple stages along the incubation tank side support frame and the cold storage tank side support frame, and each of the slide rails arranged in multiple stages on the incubation tank side support frame is installed.
- the culture vessel storage shelf is slidably attached, and the culture vessel storage shelf is slidably attached to each of the slide rails arranged in multiple stages on the cold storage tank support frame.
- the culture vessel storage shelf and the culture vessel storage shelf are paired with the incubation bath and the cold storage bath, respectively.
- It may have a configuration which is provided to.
- a plurality of cell culture units each having a culture container and a medium container are stored in multiple stages, and cell culture is performed separately for each cell culture unit.
- Cell culture can be efficiently performed in a suitable temperature environment.
- a cell culture unit is configured by connecting a culture medium container and a culture container by a tube and performing cell culture in a closed system
- Air bubbles may enter the culture vessel. If air bubbles enter the culture container, there is a concern that the culture environment in the culture container is not uniform and the efficiency of cell culture is reduced.
- the contents of the culture vessel are taken out after the cell culture is finished, it is conceivable that bubbles may interfere with the liquid feeding.
- these are completely taken into consideration. Not.
- the present invention stores a cell culture unit that includes a culture container that supplies a culture medium together with cells to perform cell culture, and a culture medium container that supplies the culture medium to the culture container.
- a cell culture apparatus that can determine the timing of ending the operation when performing the operation of removing bubbles that have entered the culture container during the cell culture process when performing cell culture in the culture unit. It can also be configured.
- the culture container storage shelf or the culture medium container storage shelf is provided with a weight detection means, and when the bubbles that have entered the culture container in the process of cell culture are removed, While transferring air bubbles to the culture medium container through the transfer tube, the weight detection means provided in the culture container storage shelf or the culture medium container storage shelf detects a change in the weight of the culture container or the culture medium container, It is good also as a structure which complete
- the present invention stores a cell culture unit that includes a culture container that supplies a culture medium together with cells to perform cell culture, and a culture medium container that supplies the culture medium to the culture container.
- a culture container that supplies a culture medium together with cells to perform cell culture
- a culture medium container that supplies the culture medium to the culture container.
- the cell culture device includes a tilting means for tilting the culture container so that the culture container storage shelf is positioned at the lower or upper position of the liquid feeding port provided in the culture container. It is good also as a structure provided. With this configuration, when a cell culture unit including a culture container and a medium container is stored and cell culture is performed in the cell culture unit, bubbles that have entered the culture container during the cell culture process can be easily obtained. While removing, the content can be prevented from remaining when the content in the culture vessel is taken out.
- the present invention when a plurality of cell culture units each including a culture container and a medium container are stored in multiple stages and cell culture is performed individually for each cell culture unit, all cell culture units are adjacent to each other.
- the progress of cell culture in the measurement target cell culture unit can be measured with one measurement unit without interfering with the unit.
- 1 is a schematic external view of a cell culture device according to an embodiment of the present invention.
- 1 is a schematic external view of a cell culture device according to an embodiment of the present invention. It is explanatory drawing which shows an example of the cell culture unit stored in the cell culture apparatus which concerns on embodiment of this invention. It is explanatory drawing which carries out the cross sectional view of the inside of the incubation tank of the cell culture apparatus which concerns on embodiment of this invention. It is explanatory drawing which shows in plan view the state which pulled out the culture medium container accommodation shelf part of the cell culture apparatus which concerns on embodiment of this invention. It is explanatory drawing which shows in plan view the state which the culture container accommodation shelf part of the cell culture apparatus which concerns on embodiment of this invention slides and moves.
- FIG. 7 is a sectional view taken along line BB in FIG.
- FIG. 6 is a cross-sectional view taken along line AA in FIG. 5.
- the cell culture device 1 is a device for storing a plurality of cell culture units U in multiple stages and individually performing cell culture for each cell culture unit U.
- FIG. 1 and FIG. 2 show schematic external views of such a cell culture device 1.
- the cell culture device 1 includes an incubation tank 3 and a cold storage tank 4 that can be adjusted to a predetermined temperature by a heat insulating wall 20 inside the housing 2. It has a two-tank structure divided into two.
- the incubation tank 3 and the cold storage tank 4 are provided with front doors 3a and 4a that can be opened and closed independently.
- the cell culture unit U stored in the cell culture apparatus 1 includes a first culture container U20 and a second culture container U30 for use in cell culture, and these culture containers U20, A medium container U10 for supplying a medium to U30, and a transfer tube U40 and a culture container connection tube U42 for connecting these containers U10, U20, U30 can be provided.
- the first culture container U20 is a culture container in which cells are injected and cell culture is first performed.
- the first culture container U20 is preferably used as an activated culture container used for cell culture for activating cells. it can.
- a substance that activates cells such as anti-CD3 antibody is provided on the bottom surface of the first culture container U20. Is immobilized.
- the second culture vessel U30 is a vessel for performing cell culture by transferring the cell suspension in the first culture vessel after cell culture in the first culture vessel, for example, for proliferating cells. Therefore, it can be suitably used as an amplification culture vessel used for cell culture.
- the first culture vessel U20 as an activation culture vessel and using the second culture vessel U30 as an amplification culture vessel, it is possible to suitably propagate floating cells such as lymphocytes. Become.
- these containers are connected to the transfer tube U40 in the order of the culture medium container U10, the second culture container U30, and the first culture container U20.
- the first culture container U20, the second culture container U30, and the culture medium container U10 are connected to the transfer tube U40 in this order, and the order is the same.
- the medium container U10 includes a port U11 for liquid feeding (including feeding and sending), and is connected to the transfer tube U40 at this port U11.
- the first culture vessel U20 includes a liquid feeding port U21, and is connected to the transfer tube U40 at the port U21.
- the port U21 is preferably provided on the transfer tube U40 side at the peripheral edge of the first culture vessel U20 and on the most opposite side with respect to the second culture vessel U30. In this way, when the contents are sent out from the first culture container U20, that is, when the cell suspension is transferred from the first culture container U20 to the second culture container U30, the first culture container is used. By tilting the U20 so that the port U21 faces downward, it is possible to suppress the cell suspension from remaining in the first culture vessel U20.
- the first is set so that the port U21 faces upward.
- the bubbles that have entered the first culture vessel U20 can be collected in the vicinity of the port U21 and easily removed from the port U21.
- the second culture container U30 includes a port U31 and a port U32 for feeding liquid, and one end of two culture container connection tubes U42 is connected to these ports, and the other end is connected to the transfer tube U40. It is connected.
- a pump U41 is attached between the connection portions of the culture vessel connection tube U42 in the transfer tube U40. That is, the cell culture unit U is provided with only one pump, and the second culture container U30 is transferred by a culture container connection tube U42 that connects the second culture container U30 and the transfer tube U40. It is the structure connected to the transfer tube U40 on both sides of the pump U41 in U40.
- the port U31 is preferably provided on the transfer tube U40 side in the peripheral portion of the second culture vessel U30 and on the most opposite side with respect to the medium vessel U10. In this way, when the contents are sent out from the second culture container U30, for example, when the cell suspension is transferred from the second culture container U30 to the emptied medium container U10 and collected. In this case, it is possible to prevent the cell suspension from remaining in the second culture container U30 by inclining the second culture container U30 so that the port U31 faces downward.
- the second culture container U30 is tilted so that the port U31 faces upward, so that the air bubbles that have entered the second culture container U30 are collected in the vicinity of the port U31. Can be easily removed from the port U31.
- the number of ports in these containers is not particularly limited, and the number can be arbitrary.
- the first culture vessel U20 is provided with a port U22 for cell injection in addition to the port U21.
- the second culture vessel 30 is provided with a spare port U33 for injecting a growth factor and the like and a sampling port U34.
- a closing member U44 such as a clip. That is, in the example of FIG. 3, the flow path between each container and the pump U41 is clipped. Then, when the liquid is fed between the containers, only the closing member U44 for the two containers to be fed is unblocked, the flow path is opened, and the liquid is fed by the pump U41.
- cells are injected from the port U22 into the first culture container U20.
- the pump U41 is operated, and the medium is transferred from the medium container U10 to the first culture container U20 by the pump U41 (arrow (1) in the figure).
- cell culture is performed in the 1st culture container U20 for a predetermined period.
- the cell suspension is transferred from the first culture container U20 to the second culture container U30 by the pump U41 (arrow (2) in the figure), and then the culture medium container U10. To the second culture vessel U30 (arrow (3) in the figure).
- the cell suspension is transferred from the first culture container U20 to the second culture container U30 via the culture container connection tube U42 connected to the transfer tube U40 on the medium container U10 side of the pump U41.
- the culture medium is transferred from the culture medium container U10 to the second culture container U30 via a culture container connection tube U42 connected to the transfer tube U40 on the first culture container U20 side of the pump U41.
- the three systems of medium transfer from the medium container U10 to the second culture container U30 can be appropriately performed by using only one pump U41.
- a medium having a gas barrier property against oxygen and carbon dioxide is generally used so that the pH of the stored culture medium does not change greatly during the culture period.
- carbon dioxide is removed from the inside of the culture medium container U10. This is because it is desirable to minimize leakage of the medium and to prevent oxidation of the medium.
- the first culture container U20 and the second culture container U30 are formed in a bag shape (bag shape) using a soft packaging material, and a part or all of them are transparent so that the contents can be confirmed. is doing.
- these culture vessels must have gas permeability (oxygen and carbon dioxide permeability) necessary for cell culture, and are used in a culture environment of 37 ° C. and 5% carbon dioxide concentration. It is preferable to do.
- gas permeability oxygen and carbon dioxide permeability
- a polyethylene resin As a material for the culture vessel satisfying such conditions, a polyethylene resin is preferable.
- the polyethylene resin include polyethylene, a copolymer of ethylene and ⁇ -olefin, a copolymer of ethylene and vinyl acetate, an ionomer using an ethylene and acrylic acid or methacrylic acid copolymer and a metal ion.
- polyolefin, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, silicone resin and the like can also be used.
- the shape of the culture medium container U10, the first culture container U20, and the second culture container U30 and the shape of these storage portions are not particularly limited, and in the example of FIG. These containers can be manufactured by sealing four sides by heat sealing, and can also be manufactured as an integrally molded bag by blow molding. Each port is preferably arranged on the transfer tube U40 side, and it is also preferable that the accommodating portion is gradually narrowed toward the tube attachment portion.
- the material of the transfer tube U40 and the culture vessel connection tube U42 may be appropriately selected according to the use environment, but is preferably excellent in gas permeability.
- silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, etc. are used. be able to.
- styrene elastomer examples include SBS (styrene / butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene), and the like. Can be used.
- a silicone tube can be suitably used for the portion where the pump U41 is attached in the transfer tube U40, and a soft polyvinyl chloride resin tube can be suitably used for the other portions.
- the connection between the tubes in the transfer tube U40 can be performed by a luer connector or other connecting means.
- the transfer tube U40 may be provided with channel opening / closing means such as a pinch valve, a two-way stopcock, and a three-way stopcock.
- the type of the pump U41 is not particularly limited, but a tube roller pump such as a peristaltic pump (R) can be suitably used because it is easy to realize a closed system in the cell culture unit U.
- a tube roller pump such as a peristaltic pump (R) can be suitably used because it is easy to realize a closed system in the cell culture unit U.
- such a cell culture unit U is stored in the cell culture apparatus 1, but the first culture container U20 and the second culture container U30 are set to a temperature suitable for cell culture (for example, 37 ° C.).
- the culture vessel U10 is stored in the cool bath 4 whose temperature is adjusted so as to be maintained at a temperature suitable for storage of the culture medium (for example, 4 ° C.).
- an incubation tank in which a plurality of slide rails 31 are arranged in multiple stages along the height direction.
- a side support frame 30 is installed.
- a culture container storage shelf 32 for storing the first culture container U20 and the second culture container U30 is slidably attached to each of the slide rails 31 arranged in the incubation tank side support frame 30. It can be pulled out toward you. By doing in this way, when installing or removing the first culture container U20 and the second culture container U30 on the culture container storage shelf 32, the culture container storage shelf 32 is pulled out to the front, It is made easy to work.
- the culture container housing shelf 32 is provided with a pump U41 and a closing member U44 constituting the cell culture unit U on the front side, but the transfer tube U40 and the like are not shown. It is.
- a cool storage tank side support frame 40 in which a plurality of slide rails 41 are arranged in multiple stages along the height direction is installed.
- a medium container storage shelf 42 for storing the medium container U10 is slidably attached to each of the slide rails 41 arranged in the cold storage tank side support frame 40 so that it can be pulled out to the front.
- FIG. 5 shows a plan view of the state in which the medium container storage shelf 42 is pulled out to the front, and the culture medium container storage shelf 42 has a closing member U44 that constitutes the cell culture unit U on the front side thereof.
- illustration of the port U11 and the transfer tube U40 provided in the culture medium container U10 is omitted.
- the culture vessel storage shelf 32 and the medium supply container storage shelf 42 are paired with the incubation bath 3 and the cold storage bath 4 adjusted to a predetermined temperature, respectively.
- Unit U can be stored in multiple stages. By doing in this way, according to the cell culture apparatus 1 of this embodiment, a plurality of cell cultures are performed in a temperature environment suitable for each of the first culture container U20, the second culture container U30, and the culture medium container U10.
- the units U can be stored in multiple stages in an independent state, and cell culture can be performed individually for each cell culture unit U.
- FIG. 2 although the state which pulled out the culture container accommodation shelf part 32 and the culture medium container accommodation shelf part 42 which were paired is shown to this side, the cell culture unit U accommodated in these on drawing, The illustration of other attached devices is omitted.
- the culture vessel storage shelf 32 and other attached devices attached to the incubation tank side support frame 30 can be taken out from the incubation tank 3 together with the incubation tank side support frame 30.
- the incubation tank side support frame 30 is installed so as to be freely inserted into and removed from the incubation tank 3.
- the cold storage tank side support frame 40 is also installed in the cold storage tank 4 so as to be freely inserted into and removed from the cold storage tank side support frame 40. All 40 can be taken out from the cold storage tank 4.
- the equipment installed in the housing 2 of the cell culture device 1 can be taken out together with the support frames 30 and 40 or installed in each tank.
- the devices installed in the housing 2 are taken out together with the support frames 30 and 40, and the housing 2 side
- the workability when maintaining the cell culture device 1 can be improved.
- the entire cell culture unit U is taken out together with the support frames 30 and 40 without taking out the cell culture units U one by one. It becomes possible to cope with.
- the support frames 30 and 40 are taken out from the housing 2, they may be moved on the carriage, but the support frames 30 and 40 are moved by a caster or the like so that they can be easily moved. Means may be provided.
- the first culture container provided in the cell culture unit U
- a measurement unit 5 for measuring the progress of cell culture in the U20 and the second culture vessel U30 is installed between the back plate portion 21 of the casing 2 of the cell culture device 1 and the culture vessel storage shelf 32. (See FIG. 4).
- the measurement unit 5 includes an imaging unit 51 that images the measurement sites of the culture vessels U20 and U30, and measures the progress of cell culture by analyzing the imaging data and calculating the number of cells, cell density, and the like.
- an imaging unit 51 having a CCD camera and an objective lens, and an illumination unit 52 arranged to face the imaging unit 51 are provided, and the imaging sites of the culture vessels U20 and U30 are illuminated from above. While irradiating light, the imaged portions of the culture vessels U20 and U30 can be imaged from below.
- the measurement unit 5 In order to image the imaged parts of the culture vessels U20 and U30, the imaged parts are pressed so that the inside of the culture containers U20 and U30 can be clearly imaged while the bending is eliminated. You may make it provide a member.
- the measurement unit 5 is installed so as to be able to move up and down in the housing 2 so that the progress of cell culture can be measured for all the cell culture units U stored in the cell culture device 1. .
- it can be installed on the support 50 attached to the incubation tank side support frame 30 so that it can be moved up and down by a power source such as a motor.
- the cell culture unit U includes a first culture container U20 and a second culture container U30, and the progress of cell culture in each of the culture containers U20 and U30 can be measured.
- the measuring unit 5 can be moved in the horizontal direction as indicated by an arrow in FIG. In the example shown in FIG.
- the measurement unit 5 is held by guide members 50 a and 50 b so that the measurement unit 5 can be moved in the horizontal direction, and the guide members 50 a and 50 b are attached to the column 50 so as to be vertically movable. Is not limited to this.
- the culture container storage shelf part 32 which accommodates the 1st culture container U20 and the 2nd culture container U30 can be slid not only to the front but also to the back side. . Thereby, when the measurement unit 5 moves in the housing 2 and reaches the measurement position of the cell culture unit U to be measured, the first culture container U20 and the second culture container U30 of the cell culture unit U are moved. The culture vessel storage shelf 32 to be stored is moved toward the measurement unit 5 (see FIGS. 4 and 8).
- the measurement unit 5 Measurement of the progress of cell culture is started. By doing in this way, the progress of cell culture in the cell culture unit U to be measured is measured by one measurement unit 5 without interfering with the adjacent cell culture units U for all the cell culture units U. Can do.
- FIGS. 6 to 8 show the state in which the culture vessel storage shelf 32 slides and moves in a plan view.
- the state shown in FIG. 7 is obtained, which corresponds to the position of the culture vessel storage shelf 32 shown in the lowermost stage in FIG.
- the culture vessel storage shelf 32 is provided with a pump U41 and a closing member U44 constituting the cell culture unit U on the front side thereof, but the first culture vessel U20.
- Ports U21 and U22 provided in the port, ports U31, U32, U33 and U34 provided in the second culture vessel U30, transfer tube U40 and culture vessel connection tube U42 are not shown.
- the culture container U20 when the contents of the first culture container U20 or the second culture container U30 are transferred from the respective ports U21, U31, the culture container U20 is arranged so that these ports U21, U31 are at the bottom. , U30 can be tilted to prevent the contents from remaining. At the same time, when removing bubbles that have entered the culture vessels U20 and U30, the culture vessels U20 and U30 are tilted so that the ports U21 and U31 are at the top, so that the bubbles that have entered the culture vessels U20 and U30 are removed from the port U21. , U31 can be collected and easily removed.
- the culture container storage shelf 32 tilts the first culture container U20 and the second culture container U30 so that the ports U21 and U31 are positioned in the lower or upper position.
- An oscillating plate 33 is provided as a tilting means.
- the culture vessels U20 and U30 are accommodated in the culture vessel accommodating shelf 32 by installing the culture vessels U20 and U30 on the swing plate 33.
- two swinging plates 33 are attached to each of the opposing side edges of the four swinging arms 34 that support the swinging plate 33.
- the swing plate 33 swings and the culture vessels U20 and U30 are inclined with respect to the horizontal plane. Is not limited to this.
- the culture vessels U20, U30 can be tilted with a height difference so that the ports U21, U31 are lower or higher. It only has to be.
- the swinging plate 33 supported by the swinging arm 34 is provided as a tilting means.
- the swinging plate 33 is rotated around a predetermined axis.
- the culture vessels U20 and U30 may be installed on a plate configured to rotate and tilt, and this may be used as a tilting means.
- the rocking plate 33 may be provided with a stirring roller 36 for stirring the contents of the second culture vessel U30, as shown in FIGS.
- the specific configuration of the agitation roller 36 is not particularly limited, and the contents are moved back and forth while being pressed against the upper surface of the second culture vessel U30, and the second culture vessel U30 made of soft packaging material is bent and deformed. It only needs to be able to stir things.
- illustration of the stirring roller is omitted.
- the removed bubbles are, for example, the first culture container U20 or the second culture container.
- the blocking member U44 between U30 and the culture medium container U10 is unblocked, the flow path is opened, and the pump U41 is operated so that it is transferred through the transfer tube U40, thereby flowing into the culture medium container U10. it can.
- the medium container U10 accommodates.
- the culture medium container storage shelf 42 includes weight detection means 43 that detects a change in the weight of the culture medium container U10 (see FIGS. 5 and 10).
- the contents of the first culture container U20 or the second culture container U30 are transferred to the medium container U10 side through the transfer tube U40 together with bubbles, the contents increase in weight. If the increase in weight at this time is detected and the pump U41 is automatically or manually stopped to end the bubble transfer operation, the contents of the culture containers U20 and U30 are transferred to the culture medium container U10. Inflow can be suppressed. And by reversely operating the pump U41, the contents remaining in the transfer tube U40 can be returned to the culture containers U20, U30, and the contents are not wasted.
- the period from when the increase in weight is detected until the pump U41 stops and the period during which the pump U41 is reversely operated may be appropriately set according to the volume in the transfer tube U40, the liquid feeding speed by the pump U41, and the like. it can.
- the culture medium container storage shelf 42 is provided with the weight detection means 43
- the culture container storage shelf 32 is provided with the weight detection means so that the first culture container U20 or the second culture container U20 is provided. Even if the weight loss when the contents of the culture container U30 flows out is detected and the bubble transfer operation is automatically or manually stopped, the contents of the culture containers U20 and U30 flow into the culture medium container U10. Can be prevented.
- the culture medium container U10 is placed on the tray 44, and the culture medium container U10 is stored in the culture medium container storage shelf 42 via the tray 44. That is, instead of directly mounting the culture medium container U10 on the culture medium container storage shelf 42, the culture medium container U10 is placed on the tray 44, and the entire tray 44 is stored in the culture medium container storage shelf 42. In this way, for example, when the cell culture unit U is stored in the cell culture apparatus 1, the first culture container U20 and the second culture container U30 can also be carried on the tray 44 together. it can. Thus, after the tray 44 is placed on the medium container storage shelf 42 and the medium container U10 is stored, the first culture container U20 and the second culture container U30 are taken out from the tray 44, and these are used as culture containers.
- the tray 44 is inclined so that the side on which the port U11 of the medium container U10 is provided is lowered as shown in FIG. It is preferable to make it a state. By doing in this way, when a culture medium is supplied to the culture containers U20 and U30 from the culture medium container U10, it can suppress that a culture medium remains in the culture medium container U10.
- the cell culture unit U stored in the cell culture device 1 is not limited to the above-described example.
- a cell that is to be cultured (cultured cell), a culture vessel that is supplied with a medium prepared for culturing the cell and performs cell culture, and a medium vessel for supplying the culture vessel with the medium are provided. It only has to be.
- the number of connected medium containers is not particularly limited, and the cell culture unit U can be configured by connecting one or more medium containers to a transfer tube.
- the cell culture unit U can also be configured by connecting a collection container for collecting the contents (cell suspension) of the culture container after cell culture to a transfer tube.
- the culture container accommodation shelf If the part 32 and the culture medium container storage shelf part 42 are paired and provided separately in the incubation tank 3 and the cold storage tank 4, the specific structure of each tank 3 and 4 will not be ask
- the cell culture device 1 may be provided with a casing that forms the incubation tank 3 and a casing that forms the cold storage tank 4 independently, and is configured by connecting these casings. It is good.
- the present invention can be suitably used in regenerative medicine, immunotherapy, antibody drug production, etc., in which cells are cultured in large quantities in a closed system.
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Abstract
Description
このような細胞培養において、培地を培養容器に供給するための培地容器と、細胞培養に用いるための培養容器とをチューブにより接続して細胞培養ユニットを構成し、この細胞培養ユニットを用いて閉鎖系で細胞培養を行うことにより、コンタミネーションのリスクを回避して、細胞を増殖させることが行われている。
また、例えば、特許文献1には、この種の培養ユニットを多段に格納して細胞培養を行う培養装置が開示されている。
このような構成とすることで、培養容器と培地容器とを備える複数の細胞培養ユニットを多段に格納して、細胞培養ユニットごとに細胞培養を個別に行うにあたり、全ての細胞培養ユニットについて、隣接する細胞培養ユニットと干渉することなく、計測対象の細胞培養ユニットにおける細胞培養の進行状況を一つの計測ユニットで計測することができる。
このような構成とすることで、培養容器と培地容器とを備える複数の細胞培養ユニットを多段に格納して、細胞培養ユニットごとに細胞培養を個別に行うにあたり、培養容器と培地容器のそれぞれに適した温度環境下で細胞培養を効率よく行うことができる。
閉鎖系を維持したまま培養容器に入り込んだ気泡を取り除くには、例えば、培養容器とともに閉鎖系を形成する培地容器などに、当該気泡を流入させることが考えられる。しかしながら、気泡を取り除く操作を終えるタイミングを誤ってしまうと、気泡が培養容器に残ってしまったり、培養容器の内容物まで培地容器などに流入させてしまったりすることになる。
このような構成とすることで、培養容器と培地容器とを備える細胞培養ユニットを格納して、この細胞培養ユニットにおいて細胞培養を行うにあたり、細胞培養の過程で培養容器に入り込んだ気泡を取り除く操作をする際に、その操作を終了するタイミングを判断して、自動的に又は手動で操作を終了することができる。
このような構成とすることで、培養容器と培地容器とを備える細胞培養ユニットを格納して、この細胞培養ユニットにおいて細胞培養を行うにあたり、細胞培養の過程で培養容器に入り込んだ気泡を容易に取り除くとともに、培養容器の内容物を取り出す際に、内容物の残留を抑制することができる。
第二の培養容器U30は、第一の培養容器での細胞培養の後に、第一の培養容器における細胞懸濁液を移し換えて細胞培養を行うための容器であり、例えば、細胞を増殖させるための細胞培養に用いる増幅培養容器などとして好適に用いることができる。
このように、第一の培養容器U20を活性化培養容器として用いるとともに、第二の培養容器U30を増幅培養容器として用いることで、リンパ球などの浮遊系細胞を好適に増殖させることが可能となる。
培地容器U10は、送液用(送入及び送出を含む)のポートU11を備え、このポートU11において移送チューブU40に接続されている。
ポートU21は、図3に示すように、第一の培養容器U20の周縁部における移送チューブU40側であって、かつ第二の培養容器U30に対して最も反対側に設けることが好ましい。このようにすれば、第一の培養容器U20から内容物を送出するにあたり、すなわち、第一の培養容器U20から細胞懸濁液を第二の培養容器U30に移送するにあたり、第一の培養容器U20をこのポートU21が下になるように傾けることで、細胞懸濁液が第一の培養容器U20に残留することを抑制することが可能となる。
すなわち、細胞培養ユニットUには、ポンプは1つのみ備えられており、第二の培養容器U30は、第二の培養容器U30と移送チューブU40とを接続する培養容器接続チューブU42により、移送チューブU40におけるポンプU41の両側で、移送チューブU40に接続された構成となっている。
容器間で送液を行わない時は、各容器からの流路は、クリップなどの閉塞部材U44によって閉塞されている。すなわち、図3の例では、各容器とポンプU41間の流路はクリップ止めされている。そして、容器間で送液を行うにあたって、送液を行う2つの容器についての閉塞部材U44のみ閉塞を解除して流路を開放し、ポンプU41による送液を行う。
次に、ポンプU41を作動させ、このポンプU41によって、図3に示すように、培地容器U10から第一の培養容器U20へ培地を移送する(同図矢印(1))。そして、第一の培養容器U20において、所定の期間、細胞培養が行われる。
第一の培養容器U20における細胞培養の後、ポンプU41によって、第一の培養容器U20から第二の培養容器U30へ細胞懸濁液を移送し(同図矢印(2))、次いで培地容器U10から第二の培養容器U30へ培地を移送する(同図矢印(3))。
また、培地容器U10から第二の培養容器U30への培地の移送は、ポンプU41の第一の培養容器U20側において移送チューブU40に接続された培養容器接続チューブU42を経由して行う。
各ポートは、移送チューブU40側に配置することが好ましく、収容部を、チューブの取り付け部分に向かって次第に狭くなるようにすることも好ましい。
また、移送チューブU40におけるチューブ間の接続は、ルアーコネクターやその他の連結手段により行うことができる。さらに、移送チューブU40において、例えばピンチバルブや二方活栓、三方活栓等の流路開閉手段を設けても良い。
なお、図4に示す例では、培養容器収容棚部32には、その手前側に細胞培養ユニットUを構成するポンプU41、閉塞部材U44を取り付けているが、移送チューブU40などの図示は省略してある。
なお、図5は、培地容器収容棚部42を手前に引き出した状態を平面視して示しており、培地容器収容棚部42には、その手前側に細胞培養ユニットUを構成する閉塞部材U44を取り付けているが、培地容器U10に設けられたポートU11及び移送チューブU40の図示は省略してある。
なお、図2では、対になった培養容器収容棚部32と培地容器収容棚部42とを手前に引き出した状態を示しているが、作図上、これらに収容される細胞培養ユニットUや、その他の付属機器などの図示は省略している。
支持フレーム30,40を筐体2内から取り出した後、これらは台車に載せて移動するようにしてもよいが、支持フレーム30,40には、これらを容易に移動できるようにキャスターなどの移動手段を設けてもよい。
さらに、本実施形態において、細胞培養ユニットUは、第一の培養容器U20と第二の培養容器U30とを備えるが、それぞれの培養容器U20,U30における細胞培養の進行状況を計測できるように、計測ユニット5は、図8に矢印で示すように、水平方向にも移動できるようにすることができる。
図8に示す例では、計測ユニット5を水平方向に移動できるようにガイド部材50a、50bに保持し、かかるガイド部材50a、50bを支柱50に上下移動可能に取り付けているが、具体的な機構はこれに限定されない。
このようにすることで、全ての細胞培養ユニットUについて、隣接する細胞培養ユニットUと干渉することなく、計測対象の細胞培養ユニットUにおける細胞培養の進行状況を一つの計測ユニット5で計測することができる。
なお、図6~図8に示す例では、培養容器収容棚部32には、その手前側に細胞培養ユニットUを構成するポンプU41と閉塞部材U44を取り付けているが、第一の培養容器U20に設けられたポートU21,U22、第二の培養容器U30に設けられたポートU31,U32,U33,U34、移送チューブU40及び培養容器接続チューブU42の図示は省略してある。
なお、本実施形態では、このような揺動アーム34に支持された揺動プレート33を傾斜手段として備えるが、上記したように培養容器U20,U30を傾かせることができれば、所定の軸周りに回動して傾斜するように構成されたプレート上に培養容器U20,U30を据えつけて、これを傾斜手段としてもよい。
なお、図4では、撹拌ローラの図示を省略してある。
また、本実施形態では、培地容器収容棚部42が重量検知手段43を備えているが、培養容器収容棚部32が重量検知手段を備えるようにして、第一の培養容器U20又は第二の培養容器U30の内容物が流出する際の重量減少を検知して、気泡の移送操作を自動的に又は手動で停止するようにしても、培養容器U20,U30の内容物が培地容器U10に流入してしまうのを抑止することができる。
このようにすることで、例えば、細胞培養ユニットUを細胞培養装置1に格納する作業をするに際して、第一の培養容器U20及び第二の培養容器U30も一緒にトレー44に載せて運ぶことができる。これにより、培地容器収容棚部42にトレー44を載置して培地容器U10を収容した後は、トレー44から第一の培養容器U20及び第二の培養容器U30を取り出して、これらを培養容器収容棚部32に据えつけることができ、これらの一連の作業を効率よく行うことができる。
また、トレー44を介して培地容器収容棚部42に培地容器U10を収容するにあたり、図10に示すように、培地容器U10のポートU11が設けられている側が低くなるように、トレー44を傾けた状態にするのが好ましい。このようにすることで、培地容器U10から培地を培養容器U20,U30に供給する際に、培地容器U10に培地が残留することを抑制することができる。
また、培地容器の接続個数は特に限定されず、1つ又は2つ以上の培地容器を移送チューブに接続して、細胞培養ユニットUを構成することができる。さらに、細胞培養を終えた培養容器の内容物(細胞懸濁液)を回収する回収容器を移送チューブに接続して、細胞培養ユニットUを構成することもできる。
2 筐体
20 断熱壁
3 インキュベート槽
30 インキュベート槽側支持フレーム
31 スライドレール
32 培養容器収容棚部
33 揺動プレート
34 揺動アーム
4 保冷槽
40 保冷槽側支持フレーム
41 スライドレール
42 培地容器収容棚部
43 重量検知手段
44 トレー
5 計測ユニット
U 細胞培養ユニット
U10 培地容器
U20 第一の培養容器(培養容器)
U30 第二の培養容器(培養容器)
U40 移送チューブ
U41 ポンプ
Claims (10)
- 細胞とともに培地を供給して細胞培養を行う培養容器と、前記培養容器に培地を供給する培地容器とを少なくとも備え、前記培養容器と前記培地容器とが移送チューブで接続された複数の細胞培養ユニットを筐体内に多段に格納して、前記細胞培養ユニットごとに細胞培養を個別に行う細胞培養装置であって、
高さ方向に沿って複数のスライドレールが多段に列設された支持フレームが前記筐体内に設置され、前記スライドレールのそれぞれに、前記培養容器が収容される培養容器収容棚部をスライド可能に取り付けて、
前記筐体の背板部と前記培養容器収容棚部との間に、撮像部を備える計測ユニットが上下に移動可能に設置され、
前記計測ユニットが、計測対象となる細胞培養ユニットの計測位置に到達すると、当該細胞培養ユニットが備える前記培養容器の被計測部位が前記撮像部の撮像範囲に入るように、当該細胞培養ユニットが備える前記培養容器を収容する前記培養容器収容棚部が前記計測ユニットに向かって移動することを特徴とする細胞培養装置。 - 前記計測ユニットが水平方向にも移動可能に設置された請求項1に記載の細胞培養装置。
- 前記培養容器が格納されるインキュベート槽と、前記培地容器が格納される保冷槽とを備え、
前記インキュベート槽と前記保冷槽とのそれぞれに、高さ方向に沿って複数のスライドレールが多段に列設されたインキュベート槽側支持フレームと保冷槽側支持フレームとを設置して、
前記インキュベート槽側支持フレームに多段に列設された前記スライドレールのそれぞれに、前記培養容器収容棚部をスライド可能に取り付けるとともに、前記保冷槽側支持フレームに多段に列設された前記スライドレールのそれぞれに、前記培地容器を収容する培地容器収容棚部をスライド可能に取り付けて、
前記インキュベート槽と前記保冷槽とに、前記培養容器収容棚部と前記培地容器収容棚部とを対をなして別々に設けた請求項1又は2に記載の細胞培養装置。 - 前記筐体内が断熱壁によって、前記インキュベート槽と前記保冷槽とに仕切られた二槽構造となっている請求項3に記載の細胞培養装置。
- 前記インキュベート槽と前記保冷槽とのそれぞれに、前記インキュベート槽側支持フレームと前記保冷槽側支持フレームとを出し入れ自在に設置した請求項3又は4に記載の細胞培養装置。
- 前記培養容器収容棚部又は前記培地容器収容棚部が、重量検知手段を備え、
細胞培養の過程で前記培養容器内に入り込んだ気泡を取り除くにあたり、当該気泡を、前記移送チューブを通して前記培地容器に移送するとともに、前記培養容器収容棚部又は前記培地容器収容棚部が備える重量検知手段が、前記培養容器又は前記培地容器の重量変化を検知して、気泡の移送操作を終了する請求項3~5のいずれか一項に記載の細胞培養装置。 - 前記移送チューブに取り付けられたポンプを作動させて気泡の移送操作をし、前記ポンプを停止して気泡の移送操作を終了した後に、前記ポンプを逆作動させることによって、前記移送チューブ内の内容物が前記培養容器に戻される請求項6に記載の細胞培養装置。
- 前記培養容器収容棚部が、前記培養容器が備える液送用のポートが下位又は上位に位置するように、前記培養容器を傾かせるための傾斜手段を備える請求項1~7のいずれか一項に記載の細胞培養装置。
- 前記傾斜手段が、揺動アームに支持された揺動プレートである請求項8に記載の細胞培養装置。
- 前記培地容器をトレーに載置して、前記トレーを介して前記培地容器収容棚部に前記培地容器を収容する請求項3~9のいずれか一項に記載の細胞培養装置。
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