JP4953769B2 - Cell culture device and cell culture cassette - Google Patents

Description

  The present invention relates to a cell culture apparatus for sequentially supplying a plurality of types of fluid substances containing a culture solution to cells and culturing the cells, and a cell culture cassette used for the cell culture apparatus.

  Conventionally, subculture has been performed for the purpose of cell cloning and cell mutation. There are two types of cells: suspension cells and adherent cells. In the subculture of adherent cells, the cells are washed with PBS (Phosphate Buffered Salts) and from the culture vessel with trypsin. Numerous operations are performed, such as an operation for separating the cells, an operation for separating trypsin and cells, and an operation for distributing cultured cells to a plurality of culture vessels.

Conventional cell culturing operations are performed by sucking and discharging with a pipette, storing the cells in a culture container, adding medium and serum, and then proliferating in an incubator (37 ° C., 95% humidity, 5% CO ₂ ). In addition, it was a very complicated operation to periodically change the medium by pipetting.

  Therefore, various culture apparatuses have been proposed for cell culture work. For example, a culture device that can circulate the medium in the culture vessel (Patent Documents 1 and 2), a culture device that can automatically replace the medium in the culture container (Patent Documents 3 and 4), a culture There have been proposed a culture apparatus (Patent Document 5) for reducing contamination at the time, a culture apparatus (Patent Document 6) for continuously culturing cells, and the like.

  25 includes an openable / closable chamber (9) for accommodating a plurality of culture containers (94), an openable / closable cooler (91) for accommodating a plurality of reagent containers (95), A plurality of tubes (93) extending between the cool box (91) and the chamber (9) and connecting the reagent container (95) in the cool box (91) and the culture container (94) in the chamber (9) to each other. ) And two pumps (92) (92) provided in the middle of the plurality of tubes (93). From the reagent container (95) to the culture container (94) via the tube (93) The cells are subcultured by supplying reagents (fluid substances) such as PBS, medium, and trypsin in a predetermined order.

  The chamber (9) is maintained at 37 ° C., which is necessary for cell culture, whereas the cold storage (91) is kept at 10 ° C. or lower in order to maintain the quality of reagents such as PBS, medium, and trypsin. Maintained.

JP-A-5-260958 JP-A-5-276922 Japanese Patent Laid-Open No. 8-4172956 JP 2002-262856 A JP 2006-141328 A JP-T-2001-502526

However, in the conventional cell culture apparatus shown in FIG. 25, when the reagent is supplied from the reagent container (95) in the cool box (91) to the culture container (94) in the chamber (9), the temperature is 10 ° C. or lower. If the reagent is supplied as it is into the culture vessel (94) and brought into contact with the cells, the cells are damaged greatly, and in some cases, the cells may be killed. Therefore, it is conceivable that the reagent is heated by a heater in the process from the reagent container (95) to the culture container (94).
In order to omit the heater, it is necessary to form a long path for the tube (93) and heat the reagent at room temperature. As a result, the tube (93) may be connected incorrectly, the tube (93) may be easily disconnected, the tube (93) may be easily damaged, and the risk of contamination may increase. There is a problem that a large amount of reagent remains in the inside and waste is increased.

  Even in a culture apparatus (Patent Document 5) for reducing contamination and a culture apparatus (Patent Document 6) for continuously culturing cells, a long tube is required for each container, and there is a similar problem.

  Further, in the conventional cell culture apparatus shown in FIG. 25, a setting operation for cell culture, that is, the reagent container (95) is accommodated in the cool box (91) and the culture container (94) is accommodated in the chamber (9). ) And connecting the reagent container (95) and the culture container (94) to each other by the tube (93) is very complicated.

  Therefore, an object of the present invention is to provide a cell culture apparatus and a cell culture cassette that can automate the cell culture work, have almost no risk of contamination, and can be very easily set for cell culture. It is to be.

The cell culture device according to the present invention sequentially supplies a plurality of types of reagents (fluid substances such as liquid) such as PBS, a medium, and trypsin to cells, and cultures the cells. It consists of a body (1) and a cell culture cassette (2) detachably mounted inside the housing (1).
The housing (1) has a temperature-controlled room (12), a cold room (14), and an intermediate room (13) formed between the temperature-controlled room (12) and the cold room (14). A space for accommodating the cell culture cassette (2) is formed across the chamber (12), the intermediate chamber (13), and the cold insulation chamber (14).
The cell culture cassette (2) includes one or a plurality of culture containers (21) for culturing the cells, a plurality of reagent containers (22) containing the fluid substance, the culture containers (21), and Each of the reagent containers (22) is detachably connected to a joint unit (3). The joint unit (3) includes one or more culture containers (21) and a plurality of reagent containers (22). Are connected to each other, and a fluid mechanism is provided at a proper position in the flow path system for transporting the flowable substance and changing the flow path.
Then, in a state where the cell culture cassette (2) is mounted in the casing (1), the whole culture container (21), the joint unit (3) and the whole reagent container (22) of the cell culture cassette (2). ) Are respectively housed in the temperature-controlled room (12), the intermediate room (13), and the cold room (14) of the casing (1).

  Here, the housing (1) has a cassette insertion slot (10) for accommodating the cell culture cassette (2) in the cassette accommodation space, and a cover (11) for closing the cassette insertion slot. It has.

  In the cell culture apparatus of the present invention, one or a plurality of culture containers (21) are connected to the joint unit (3) and a plurality of reagent containers (22) are connected when setting for cell culture. Assemble the cell culture cassette (2). As a result, one or a plurality of culture containers (21) and a plurality of reagent containers (22) are connected to each other via a flow path system and a fluid mechanism, and each reagent container ( The reagent can be supplied from 22) to the necessary culture vessel (21).

  When the assembled cell culture cassette (2) is mounted in the housing (1), the entire culture container (21), the joint unit (3) and the total reagent container (22) of the cell culture cassette (2) are respectively The casing (1) is housed in a temperature-controlled room (12), an intermediate room (13), and a cold room (14). By storing the reagent container (22) in the cold storage chamber (14), the reagent container (22) in the cold storage chamber (14) is kept at a predetermined temperature or less necessary for storage of the reagent. Further, by accommodating the culture container (21) in the temperature-controlled room (12), the culture container (21) in the temperature-controlled room (12) is kept in a temperature range necessary for cell culture.

Here, since the intermediate chamber (13) in which the joint unit (3) is accommodated is formed between the temperature-controlled room (12) and the cold room (14), heat is generated from the temperature-controlled room (12). Accordingly, the temperature in the intermediate chamber (13) rises toward the temperature in the temperature-controlled room (12).
As a result, the reagent in the reagent container (22) in the cold chamber (14) is heated to a temperature that does not damage the cells in the process of passing through the intermediate chamber (13), and then cultured in the temperature-controlled room (12). Supplied to the container (21).
Therefore, it is not necessary to form the flow path system in the intermediate chamber (13) excessively long or install a heater in the intermediate chamber (13).

In a specific configuration, the casing (1) includes a temperature control device for keeping the temperature-controlled room (12) in a certain temperature range, and a cooling device for cooling the cold storage room (14) to a certain temperature or less. And an air blower for causing the air in the temperature-controlled room (12) to flow toward the intermediate room (13) to bring the temperature of the intermediate room (13) close to the temperature of the temperature-controlled room (12).
According to the specific configuration, by causing the air in the temperature-controlled room (12) to flow toward the intermediate chamber (13), the heat of the air is transferred to the intermediate chamber (13), and the intermediate chamber (13) The temperature will approach the temperature of the temperature-controlled room (12).

In another specific configuration, with the cell culture cassette (2) attached to the casing (1), the temperature-controlled room (12), the intermediate chamber (13), and the cold storage chamber (14) of the casing (1). ) Are separated from each other by the cell culture cassette (2).
As a result, the flow of air between the temperature-controlled room (12) and the intermediate room (13) is blocked, and a temperature drop in the temperature-controlled room (12) is prevented, and the intermediate room (13) and the cold room (14) ) Is blocked, and temperature rise in the cold insulation chamber (14) is prevented.

In a specific configuration, the casing (1) includes a culture vessel (21) of a cell culture cassette (2) housed in a temperature-controlled room (12), and a connection portion with a joint unit (3). A rocking mechanism (5) for rocking up and down as a center is provided.
According to the specific configuration, the swinging mechanism (5) is operated and the rear part of the culture vessel (21) is moved up and down, so that the culture vessel (21) is tilted into the culture vessel (21). It is possible to facilitate the injection of the reagent and the discharge of the reagent out of the culture vessel (21).

In another specific configuration, the housing (1) includes a drive mechanism (39) for driving a fluid mechanism of the joint unit (3) of the cell culture cassette (2) housed in the intermediate chamber (13). Is deployed.
According to the specific configuration, there is no need to provide a drive mechanism on the joint unit (3) side, so that the cell culture cassette (2) can be reduced in weight and the configuration can be simplified. 2) Disposable.

In a more specific configuration, the housing (1) includes a microscopic observation device (8) for observing cells in the culture vessel (21) of the cell culture cassette (2) housed in the temperature-controlled room (12). ) Is deployed.
This makes it possible to observe the cells in the culture vessel (21) without removing the culture vessel (21) from the temperature-controlled room (12).

The cell culture cassette according to the present invention is for sequentially supplying a plurality of types of reagents (fluid substances) such as PBS, medium, trypsin, etc. to the cells and culturing the cells. One or a plurality of culture containers (21) for culturing the cells, a plurality of reagent containers (22) containing the fluid substance, and the culture containers (21) and the reagent containers (22) are detachably connected to each other. And a joint unit (3).
The joint unit (3) is provided with a flow path system for connecting the one or more culture vessels (21) and the plurality of reagent vessels (22) to each other, and is disposed at an appropriate position of the flow path system so as to be fluid. A fluid mechanism for transporting substances and changing the flow path, and the joint unit (3) supports the one or more culture vessels (21) and a plurality of reagent vessels (22), It can be carried by gripping the joint unit (3).

  Since the cell culture according to the present invention can be carried by holding the joint unit (3), it is very easy to set the joint unit (3) in the culture apparatus, and it can be set by one operator. .

_{In a specific configuration, the culture vessel (21) and the reagent vessel (22) are each connected to the joint unit (3) via an adapter (4), and the adapter (4) is a rubber stopper that engages with the vessel opening. Part (43) and an attachment part (42) removably connected to the joint unit (3), and the rubber plug part (43) is inserted into the opening of the container opening (44 ) And a cover portion (45) covering the container opening, and the flow passage (41) of the fluid substance is formed through the rubber plug portion (43) and the attachment portion (42). An air flow path (46) for discharging the air in the container or introducing air into the container is formed, and the air flow path (46) has a filter (40) that can prevent passage of bacteria and viruses. ) Is deployed. According to this specific configuration, the culture vessel (21) and the joint unit (3) and the reagent container (22) and the joint unit (3) can be connected with a complete biological hermetic structure. Yes, by performing the work in a safety cabinet, the assembly can be performed in a substantially complete aseptic condition.}

  More specifically, the joint unit (3) is provided with a grip for carrying the culture container (21) and the reagent container (22) together with the joint unit (3), or the joint unit (3) is a cassette base. (31) and the flow path box (32), and the adapter (4) can be provided with a grip portion, and the flow path box and the fluid mechanism can be provided in the flow path box (32).

  According to the cell culture apparatus and the cell culture cassette according to the present invention, the cell culture operation can be almost completely automated, there is almost no risk of contamination, and the setting operation for cell culture is extremely simple. Become.

Hereinafter, embodiments of the present invention will be specifically described with reference to the drawings.
The cell culture apparatus according to the present invention comprises a compact housing (1) as shown in FIG. 1, and the cassette insertion slot (10) is exposed by removing the cover (11) as shown in FIG. The cell culture cassette (2) can be inserted into the casing (1) from the mouth (10) as shown in FIG.
On the side of the housing (1), there are an input device (84) for inputting various data and instructions, and a display (83) for displaying a cell image taken by a microscopic observation device described later. Is deployed.

  As shown in FIG. 4, a constant temperature room (12), an intermediate room (13), and a cold room (14) are formed inside the housing (1). The intermediate room (13) is a constant temperature room (12). The cell culture cassette (2) is housed between the temperature-controlled room (12), the intermediate room (13), and the cold room (14) as shown in FIG.

  The temperature-controlled room (12) is surrounded by a peripheral wall (15) containing a heater (16), and the temperature inside the temperature-controlled room (12) is maintained at 37 ° C. by the heating of the heater (16). In the temperature-controlled room (12), an upper cross flow fan (7) and a lower cross flow fan (71) are arranged. As indicated by arrows in FIG. 3, the upper cross flow fan (7) An air flow along the front wall (15b) is formed from the ceiling wall (15a), and an air flow along the front wall (15b) is formed from the bottom wall (15c) by the lower cross flow fan (71). .

In the temperature-controlled room (12), a cell microscope observation device (8) for observing the cells in the culture vessel (21) is provided as shown in FIG. The microscope observation apparatus (8) includes a frame (80), an illumination unit (81) to be positioned above the culture vessel (21), and a microscope unit (82) to be positioned below the culture vessel (21). The illuminating part (81) and the microscopic part (82) are moved together in a biaxial direction along the horizontal plane.
This makes it possible to observe the cell culture state and cell detachment state at any position in all the culture vessels without changing the culture environment.

  The intermediate chamber (13) is provided with a lower cassette guide (18) and an upper cassette guide (19), and a shutter (17) is provided between the cassette guides (18) and (19) so as to be opened and closed. Yes. The shutter (17) is spring-biased in the closing direction as shown in FIG. 19, opens when the cell culture cassette (2) is inserted, and closes when the cell culture cassette (2) is removed.

  As shown in FIG. 4, the cold insulation chamber (14) is surrounded by a heat insulating material (14a), and the cool insulation chamber (14) has two coolers (72) and (72) made of Peltier elements at the bottom. The cooler (72) (72) keeps the inside of the cold insulation chamber (14) at 10 ° C. or less.

  As shown in FIG. 7, the cell culture cassette (2) is formed by connecting a plurality of culture containers (21) and a plurality of reagent containers (22) to the joint unit (3). One side of the joint unit (3) is connected to one culture vessel (21a) for primary culture and four culture vessels (21b) to (21e) for secondary culture, and the joint unit ( 3) On the other side, one reagent container (22a) for PBS, two reagent containers (22b) (22c) for medium, one reagent container (22d) for serum, one for trypsin The reagent container (22e) and one reagent container (22f) for disposal are connected.

As shown in FIG. 8, the joint unit (3) includes a cassette base (31) having a large number of through holes (31a) and a flow path box (32) to be installed on the cassette base (31). The flow path box (32) is provided with a male attachment portion (33a) corresponding to the number of culture vessels (21) and a male attachment portion (33b) corresponding to the number of reagent vessels (22). ) Is projected.
A pair of gripping portions (30) and (30) project from both side portions of the cassette base (31).

  On the other hand, the adapter (4) is attached to each of the culture container (21) and the reagent container (22), and the male attachment portions (33a) (33b) of the flow path box (32) are connected via the adapter (4). ) Is possible. In the flow path box (32), a flow path system for connecting a plurality of culture containers (21) and a plurality of reagent containers (22) to each other as described later, and an appropriate position of the flow path system are arranged. A fluid mechanism for transporting the reagent and changing the flow path is provided.

  As shown in FIG. 3, in a state in which the cell culture cassette (2) is accommodated in the housing (1), the entire culture container (21), the joint unit (3), and all the reagent containers (2) of the cell culture cassette (2) 22) are housed in the temperature-controlled room (12), the intermediate room (13), and the cold room (14) of the casing (1), respectively. The cell culture cassette (2) separates the temperature-controlled room (12), intermediate room (13), and cold storage room (14) of the housing (1) from each other.

FIG. 13 shows a system diagram of the cell culture apparatus according to the present invention. As shown in the figure, five culture containers (21) to (21) are connected to the flow path box (32) via an adapter (4a), and six reagent containers (22) are connected via an adapter (4b). To (22), and the flow path box (32) has a plurality of tubes for connecting the culture containers (21) to (21) and the reagent containers (22) to (22) to each other. A flow path system (38) consisting of two diaphragm pumps (34) (34) and 14 for transporting reagents and changing flow paths is provided in the flow path system (38). A single valve (35) is provided to constitute a fluid mechanism.
The plurality of tubes constituting the flow path system (38) are accommodated in the flow path box (32) in an orderly arrangement in a substantially fixed state.
Further, the flow path box (32), CO ₂ cylinder (not shown) is connected, and can be supplied CO ₂ to the culture vessel (21).

On the other hand, as shown in FIG. 3, a drive mechanism (39) for driving the fluid mechanism is disposed in the intermediate chamber (13) of the housing (1).
By driving the drive mechanism (39), the valve (35) shown in FIG. 13 is switched to form a single channel connecting any culture vessel (21) and any reagent vessel (22). By starting the pump (34) interposed between the reagent container (22) and the reagent container (22), the reagent can be transported from the culture container (21). In addition, a single flow path connecting the two culture vessels (21) and (22) is formed, and a pump (34) interposed in the flow channel is started to activate these culture vessels (21) and (21). Cell transfer between them becomes possible.

Specifically, as shown in FIGS. 11 (a) and 11 (b), the intermediate chamber (13) has an electromagnetic solenoid (in the vicinity of the valve (35) of the flow path box (32) toward the valve (35). 36) is provided, and the valve (35) is switched between the open state (FIG. 11 (a)) and the closed state (FIG. 11 (b)) by the on / off operation of the electromagnetic solenoid (36).
12 (a) and 12 (b), the intermediate chamber (13) has an electromagnetic solenoid (37) in the vicinity of the pump (34) in the flow path box (32) toward the pump (34). The pump (34) is alternately set to the suction state (FIG. 12 (a)) and the discharge state (FIG. 12 (b)) by the on / off operation of the electromagnetic solenoid (37), and pumping is performed. .

  5 shows a cross-sectional view of the cell culture cassette (2) at the position of the pump (34) in a state where the culture container (21) and the reagent container (22) are connected to the flow path box (32). FIG. 6 shows a cross-sectional view of the cell culture cassette (2) at the position of the valve (35). By housing the cell culture cassette (2) in the housing (1) as shown in FIG. 3, the valve (35) and the pump (34) of the flow path box (32) are placed as shown in FIGS. The electromagnetic solenoids (36) and (37) in the intermediate chamber (13) face each other.

  As shown in FIG. 9, an adapter (4b) for connecting the reagent container (22) to the joint unit (3) and a culture container (21) for connecting the culture container (21) to the joint unit (3) as shown in FIG. The adapter (4a) has basically the same structure, both of which engage with the opening (21a) of the culture vessel (21) or the opening (22a) of the reagent vessel (22). It has a plug part (43) and a female attachment part (42) removably connected to the male attachment part (33) of the joint unit (3).

  The rubber stopper (43) has a fitting part (44) that fits into the opening of the opening of the reagent container (22) or the culture container (21), and a cover part (45) that covers the container opening. And a reagent flow path (41) is formed through the rubber stopper (43) and the female attachment (42), and is used for discharging air from the container or introducing air into the container. An air flow path (46) is formed. A filter (40) capable of blocking the passage of bacteria and viruses is provided at the end of the air flow path (46).

  As shown in FIGS. 9 and 15, the adapter (4b) to be attached to the reagent container (22) has an L-shaped reagent container tube (48) to be accommodated in the reagent container (22). It is connected to the flow path (41). As shown in FIGS. 10 and 14, the adapter (4a) to be attached to the culture vessel (21) has an L-shaped culture vessel tube (47) to be accommodated in the culture vessel (21). Are connected to the flow path (41).

_{As shown in FIG. 16, by attaching the adapter (4a) to the culture vessel (21), the insertion portion (44) constituting the rubber plug portion (43) of the adapter (4a) is inserted into the container opening and the rubber plug. The cover part (45) constituting the part (43) covers the container opening to obtain a tightly connected state. Then, by fitting the female attachment part (42) of the adapter (4a) with the male attachment part (33) of the flow path box (32), the space between the flow path box (32) and the culture vessel (21) is obtained. They can be connected in a completely biologically sealed structure and can be assembled in a nearly perfect aseptic condition by performing the work in a safety cabinet.}

Similarly, by attaching the adapter (4b) to the reagent container (22), the fitting part (44) constituting the rubber plug part (43) of the adapter (4b) is fitted into the container opening part, and the rubber plug part ( The cover part (45) constituting 43) covers the container opening, and a tightly connected state is obtained.
Then, by fitting the female attachment part (42) of the adapter (4b) with the male attachment part (33) of the flow path box (32), the gap between the flow path box (32) and the reagent container (22) is obtained. They can be connected in a completely sealed structure and can be assembled in a substantially complete aseptic condition by performing the work in a safety cabinet.

  Further, at the back of the housing (1), as shown in FIGS. 17 and 18, the culture vessel (21) of the cell culture cassette (2) housed in the temperature-controlled room (12) is connected to the joint unit (3). It is easy to inject the reagent into the culture vessel (21) and to discharge the reagent out of the culture vessel (21), or to move the inside of the culture vessel (21). A rocking mechanism (5) for stirring the reagents is provided.

  Specifically, as shown in FIGS. 20 and 21, the slide guides (54) are attached to the pair of side surface guide plates (69) (69) disposed on both sides of the temperature-controlled room (12) so as to be movable up and down. The left rack gear (57) is fixed to the left slide guide (54), and the right rack gear (58) is fixed to the right slide guide (54). A lifting shaft (51) is horizontally installed between the rack gears (57) and (58). Here, the elevating shaft (51) is disposed at a position where the rear end of the culture vessel (21) in the temperature-controlled room (12) can be supported from below (see FIG. 3).

  The pair of side surface guide plates (69) and (69) is provided with a connecting shaft (52) extending horizontally, and a motor (50) is attached to the left side surface guide plate (69). ) Is connected to the connecting shaft (52) via the worm gear (53).

  The rotation of the motor (50) is transmitted to the connecting shaft (52) through the worm gear (53), and the left rack gear (57) and the right rack gear (58) are driven up and down by the rotation of the connecting shaft (52). Accordingly, the elevating shaft (51) moves up and down.

A sensor plate (56) is attached to each end of the lifting shaft (51), and a limit sensor (55) is attached to the left guide plate (69) opposite the left sensor plate (56). Thus, the lowering end and the rising end of the elevating shaft (51) are defined.
An origin sensor (59) is attached to the right side guide plate (69) in correspondence with the right sensor plate (56), thereby defining the origin position of the elevating shaft (51).

  According to the swing mechanism (5), the culture vessel (21) can be held horizontally by setting the elevating shaft (51) to the origin position. In addition, the lifting shaft (51) is raised to the rising end, and the culture vessel (21) is lifted at the rear end with the end on the joint unit (3) side as the center, thereby allowing the liquid in the culture vessel (21) to rise. Easy to discharge. Further, by raising and lowering the elevating shaft (51), the culture vessel (21) can be swung to stir the liquid in the culture vessel (21).

Furthermore, as shown in FIG. 17 and FIG. 18, the inner part of the housing (1) is vibrated to the culture vessel (21) of the cell culture cassette (2) housed in the temperature-controlled room (12). A detachment mechanism (6) is provided to promote cell detachment.
Specifically, as shown in FIGS. 22 and 23, the motor (60) is attached to the right side guide plate (69), and the worm gear (62) is fixed to the output shaft of the motor (60). ing. The worm gear (62) is connected to the cam (63) and rotationally drives the cam (63).

The right side guide plate (69) supports a hammer (61) via a leaf spring (64), and elastically deforms the leaf spring (64) by the rotation of the cam (63). As the leaf spring (64) returns to elasticity, the hammer (61) is moved vigorously toward the primary culture vessel (21), and the side surface of the culture vessel (21) is beaten.
As a result, vibration can be applied to the primary culture vessel (21) to promote cell detachment in the culture vessel (21).

FIG. 24 shows a series of cell culture steps using the above-described cell culture apparatus.
First, in step S1, adapters (4) are attached to all reagent containers (22) and all culture containers (21) containing reagents in the safety cabinet, and these containers (22) (21) are attached. The cell culture cassette (2) is assembled by connecting to the joint unit (3), and the cell culture cassette (2) is prepared.

In step S2, the cell culture cassette (2) is installed in the housing (1), and the primary culture of the cells is performed using the primary culture vessel (21).
Thereafter, in step S3, the medium in the primary culture vessel (21) is replaced. That is, after the medium in the primary culture vessel (21) is discarded and PBS is injected into the primary culture vessel (21), the culture vessel (21) is swung to perform washing. Subsequently, PBS is discarded from the primary culture container (21), serum is injected into the primary culture container (21), and further, a medium is injected, and then CO ₂ is supplied.

  In step S4, cell passage is performed. First, after discarding the medium from the primary culture container (21), the primary culture container (21) is washed with PBS, and the washed PBS is discarded. Subsequently, trypsin is injected into the primary culture container (21), and the primary culture container (21) is rocked to stir the liquid in the container. Thereafter, the detachment mechanism (6) is operated while observing the inside of the primary culture vessel (21) using the microscopic observation apparatus (8), and the cells are detached.

  Next, after injecting the medium into the primary culture container (21), the primary culture container (21) is shaken and stirred to inactivate trypsin. Thereafter, the cells in the primary culture container (21) are dispensed into four secondary culture containers (21). Subsequently, serum is injected into the secondary culture container (21), and further, the medium is injected, and then the flow path is washed with PBS.

In step S5, CO ₂ is supplied into the secondary culture vessel (21) to perform secondary culture.
Next, in step S6, the medium in the secondary culture vessel (21) is replaced. First, after the medium in the secondary culture container (21) is discarded, the secondary culture container (21) is washed with PBS. After that, the PBS in the secondary culture container (21) is discarded, then serum is injected into the secondary culture container (21), and further, the medium is injected, and then the secondary culture container.
(21) is swung and stirred. Finally, after supplying CO ₂ to the secondary culture vessel (21), the cells are observed in a timely manner by the microscopic observation device (8), and when it is determined that the cells are confluent, a series of culture operations are terminated.

_{According to the cell culture device of the present invention, all the culture operations can be automatically performed only by setting the cell culture cassette (2) in the casing (1). That is, after seeding the cells in the primary culture vessel (21), the medium for growth and CO2} supply, cell detachment for passage, _and dispensing into the secondary culture vessel (21), the secondary culture vessel The medium change and CO2 supply in (21) can be performed automatically. Therefore, there is almost no risk of contamination. Further, at the time of passage, the peeling operation can be completed in a short time (for example, about 30 seconds) using the peeling mechanism (6) while observing the peeling state with the microscopic observation device (8), so that it is given to the cells. Damage can be minimized.

  In assembling the cell culture cassette (2), the culture container (21) and the reagent container (22) can be connected to the joint unit (3) with one touch, and the operation is extremely simple. The assembled cell culture cassette (2) can be easily carried by gripping the grip portions (30) and (30) of the cassette base (31). Setting is possible. In addition, the reagent container (22) can be easily replaced and the reagent can be replenished.

In a state where the cell culture cassette (2) is accommodated in the housing (1), the culture vessel (21) is accommodated in the temperature-controlled room (12) and maintained at a temperature necessary for culture. The reagent container (22) is housed in the cold storage chamber (14), and is maintained at a temperature necessary for maintaining the quality of the reagent.
Here, since many through holes (31a) are opened in the cassette base (31) of the joint unit (3) (see FIG. 6), the reagent container (22) of the cell culture cassette (2) serves as a cold room. In the state accommodated in (14), the cool air from the coolers (72) and (72) passes through the numerous through holes (31a) of the cassette base (31), directly hits the reagent container (22), and the reagent The container (22) is cooled efficiently.

Further, as shown in FIG. 3, the temperature-controlled room (12) and the intermediate room (13) of the housing (1) are adjacent to each other, so that the heat of the temperature-controlled room (12) is transferred to the intermediate room (13), In addition to heating the intermediate chamber (13), air is circulated in the constant temperature chamber (12) by operating the upper cross flow fan (7) and the lower cross flow fan (71) installed in the constant temperature chamber (12). In addition, the temperature inside the temperature-controlled room (12) is maintained at a uniform temperature, and the air in the temperature-controlled room (12) flows along the wall with the intermediate room (13) to heat the intermediate room (13). The intermediate chamber (13) is maintained at substantially the same temperature as the temperature-controlled room (12).
As a result, the low-temperature reagent supplied from the reagent container (22) in the cold storage chamber (14) is heated while passing through the intermediate chamber (13), and its temperature approaches the temperature of the temperature-controlled room (12). become. Therefore, when the reagent is supplied to the culture container (21) in the temperature-controlled room (12), the cells in the culture container (21) are not damaged by contact with the reagent.

  The cell culture cassette (2) has a flow path system composed of a plurality of tubes connecting the reagent container (22) and the culture container (21) in the flow path box (32) of the joint unit (3). The flow path system is formed in the shortest path length and is compactly built in the flow path box (32) together with the pump (34) and the valve (35), and thus is caused by a long tube as in the prior art. There is no problem such as contamination, and the amount of reagent that is wasted after culturing is minimized. However, the time required for liquid feeding is also shortened.

It is a perspective view showing the appearance of the cell culture device concerning the present invention. It is a perspective view of the state which removed the cover from the housing | casing. It is sectional drawing of the cell culture apparatus of the state which accommodated the cell culture cassette. It is sectional drawing of the cell culture apparatus of the state which took out the cell culture cassette. It is sectional drawing of the cell culture cassette in the position of a pump. It is sectional drawing of the cell culture cassette in the position of a valve | bulb. It is a perspective view of a cell culture cassette. It is a top view of the decomposition | disassembly state of a cell culture cassette. It is sectional drawing of the decomposition | disassembly state by the side of the reagent container of a cell culture cassette. It is sectional drawing of the decomposition | disassembly state by the side of the culture container of a cell culture cassette. It is sectional drawing explaining operation | movement of the valve | bulb in a flow-path box. It is sectional drawing explaining operation | movement of the pump in a flow-path box. It is a systematic diagram of a flow path box. It is a perspective view of the adapter for culture vessels. It is a perspective view of the adapter for reagent containers. It is sectional drawing of the state which connected the culture container to the flow-path box through the adapter. It is a top view which shows the state which took out the cell culture cassette from the housing | casing. It is a top view which shows the state which accommodated the cell culture cassette in the housing | casing. It is a side view which shows the attachment state of a shutter. It is a top view of a rocking mechanism. It is a front view of a rocking mechanism. It is a top view of a peeling mechanism. It is a front view of a peeling mechanism. It is process drawing of a cell culture. It is a perspective view of the conventional cell culture apparatus.

Explanation of symbols

(1) Case
(11) Cover
(10) Cassette insertion slot
(12) Constant temperature room
(13) Intermediate room
(14) Cold room
(2) Cell culture cassette
(21) Culture vessel
(22) Reagent container
(3) Joint unit
(31) Cassette base
(32) Channel box
(33) Male attachment section
(34) Pump
(35) Valve
(36) Electromagnetic solenoid
(37) Electromagnetic solenoid
(38) Channel system
(39) Drive mechanism
(4) Adapter
(42) Female attachment
(43) Rubber stopper
(44) Insertion section
(45) Cover
(5) Oscillation mechanism
(6) Peeling mechanism
(7) Upper cross flow fan
(71) Lower cross flow fan
(72) Cooler
(8) Microscopic observation equipment
(81) Lighting unit
(82) Microscopic part

Claims (9)

  A cell culture apparatus for sequentially supplying a plurality of types of fluid substances including a culture solution to cells and culturing the cells. The case (1) and the inside of the case (1) are detachably mounted. The casing (1) is formed between the temperature-controlled room (12), the cold room (14), and the temperature-controlled room (12) and the cold room (14). And a space for accommodating the cell culture cassette (2) is formed across the temperature-controlled room (12), the intermediate room (13), and the cold storage room (14). The cell culture cassette (2) includes one or a plurality of culture containers (21) for culturing the cells, a plurality of reagent containers (22) containing the fluid substance, and the culture containers (21). And a joint unit (3) to which each of the reagent containers (22) is detachably connected. The joint unit (3) includes one or more culture containers (21) and a plurality of reagent containers (22). And And a fluid mechanism disposed at a suitable position in the flow path system for transporting the fluid substance and changing the flow path, and the cell culture cassette (2) is provided in the housing. When mounted in the body (1), the entire culture container (21), the joint unit (3) and the total reagent container (22) of the cell culture cassette (2) are each kept at a constant temperature in the casing (1). A cell culture device accommodated in a chamber (12), an intermediate chamber (13), and a cold storage chamber (14).   The casing (1) has a cassette insertion slot (10) for accommodating the cell culture cassette (2) in the cassette accommodation space and a cover (11) for closing the cassette insertion slot. The cell culture device according to claim 1.   The casing (1) includes a temperature adjusting device for keeping the temperature-controlled room (12) in a certain temperature range, a cooling device for cooling the cold-reserving chamber (14) to a certain temperature or less, and a temperature-controlled room ( 12. An air blower is provided for flowing the air in the interior chamber (13) toward the intermediate chamber (13) to bring the temperature of the intermediate chamber (13) close to the temperature of the temperature-controlled room (12). 2. The cell culture apparatus according to 2.   With the cell culture cassette (2) attached to the housing (1), the temperature-controlled room (12), the intermediate chamber (13), and the cold room (14) of the housing (1) include the cell culture cassette. The cell culture device according to any one of claims 1 to 3, which are partitioned from each other by (2).   In the case (1), the culture vessel (21) of the cell culture cassette (2) accommodated in the temperature-controlled room (12) is swung up and down around the joint with the joint unit (3). The cell culture device according to any one of claims 1 to 4, wherein a rocking mechanism (5) is provided.   The housing (1) is provided with a drive mechanism (39) for driving a fluid mechanism of a joint unit (3) of a cell culture cassette (2) accommodated in an intermediate chamber (13). The cell culture device according to claim 5.   The housing (1) is provided with a microscope observation device (8) for observing cells in the culture vessel (21) of the cell culture cassette (2) accommodated in the temperature-controlled room (12). The cell culture device according to any one of claims 1 to 6.   A cell culture cassette (2) for sequentially supplying a plurality of types of fluid substances containing a culture solution to cells and culturing the cells, wherein one or a plurality of culture containers for culturing the cells (21), a plurality of reagent containers (22) containing the flowable substance, and a joint unit (3) to which the culture container (21) and the reagent container (22) are detachably connected, The joint unit (3) is provided with a flow path system for connecting the one or more culture vessels (21) and the plurality of reagent vessels (22) to each other, and is disposed at an appropriate position of the flow path system so as to be fluid. A fluid mechanism for transporting substances and changing the flow path, and the joint unit (3) supports the one or more culture vessels (21) and a plurality of reagent vessels (22), A cell culture cassette that can be carried by holding the joint unit (3). _{The culture container (21) and the reagent container (22) are each connected to the joint unit (3) via an adapter (4), and the adapter (4) includes a rubber plug part (43) that engages with the container opening. And an attachment part (42) removably coupled to the joint unit (3), the rubber plug part (43) having a fitting part (44) fitted into the opening of the container opening, and a container opening A cover portion (45) covering the portion, and the flow passage (41) of the fluid substance is formed through the rubber plug portion (43) and the attachment portion (42), and the air in the container An air flow path (46) for discharging air or introducing air into the container is formed, and the air flow path (46) is provided with a filter (40) capable of blocking the passage of bacteria and viruses. The cell culture cassette according to claim 8.}

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