Cell culture device

Abstract

To provide a cell culture apparatus capable of shortening the time required for cell culturing process in a plurality of culture vessels.SOLUTION: The cell culture apparatus comprises: a rotary table on which a plurality of culture vessels is placed; a liquid aspirating device for aspirating the liquid in the culture vessels; a liquid injecting device for injecting liquid into the culture vessels; a liquid sucking / discharging device for sucking the liquid in the culture vessels and discharging the sucked liquid into the culture vessels; and an oscillating device that oscillates the culture vessels. On the upper surface of the rotary table, four or more vessel placement positions, on which the culture vessels are placed one by one, are set at equal intervals around the center of rotation of the rotary table. The liquid aspirating device, the liquid injecting device, the liquid sucking / discharging device, and the oscillating device are arranged in this order around the rotary table along the outer peripheral direction of the rotary table and arranged so that they are simultaneously positioned outside the vessel placement positions one by one when the rotary table is stationary in a predetermined state.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a cell culture device. [Background technology]

[0002] In the culture of cells such as iPS cells, processes such as injecting and discarding chemical solutions according to the culture protocol are required. In order to accommodate various protocols, a conventional cell culture device is known that has a mechanism in which a series of operations is completed for each culture vessel before operations are performed on the next vessel (see, for example, Patent Document 1). The device described in Patent Document 1 is equipped with units necessary for various cell cultures, and can transport culture vessels and centrifuge tubes as appropriate. This mechanism allows various processes such as the suction and discharge of chemical solutions to be performed on the culture vessel, and a series of protocols can be completed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2005-218413 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, in the configuration described in Patent Document 1, a series of cell culture processes such as medium replacement and subculture must be performed for each culture vessel to complete a certain amount of processing. Therefore, for example, during preparation work for a culture vessel or chemical solution, other culture vessels cannot be processed, and there is a problem that it takes a long time to process multiple culture vessels.

[0005] The present disclosure is intended to solve the above-mentioned problems, and aims to provide a cell culture device that can shorten the time required for cell culture processing of multiple culture vessels. [Means for solving the problem]

[0006] The cell culture apparatus of the present disclosure comprises a rotating table on whose upper surface a plurality of culture vessels are placed, a liquid suction device for aspirating liquid from within the culture vessel, a liquid injection device for injecting liquid into the culture vessel, a liquid suction and discharge device for aspirating liquid from within the culture vessel while discharging the aspirated liquid into the culture vessel, and a rocking device for rocking the culture vessel, and on the upper surface of the rotating table, four or more container mounting positions, each of which can be used to place one culture vessel, are set at equal intervals around the center of rotation of the rotating table, and the liquid suction device, liquid injection device, liquid suction and discharge device, and rocking device are arranged around the rotating table in this order along the outer periphery of the rotating table, and are simultaneously positioned outside one of the container mounting positions when the rotating table is stationary in a predetermined state. Effect of the Invention

[0007] According to the cell culture device of the present disclosure, the time required for cell culture processing of multiple culture vessels can be reduced. [Brief description of the drawings]

[0008] [Figure 1] Basic configuration of cell culture device [Diagram 2] Flowchart showing an example of a culture medium replacement operation [Diagram 3] Illustration of medium replacement operation [Figure 4] Illustration of medium replacement operation [Diagram 5] Illustration of medium replacement operation [Figure 6] Flowchart showing an example of a passaging operation [Figure 7] Flowchart of washing steps in passaging operation [Figure 8] Illustration of the cleaning process and the stripping solution addition process [Figure 9] Illustration of the cleaning process and the stripping solution addition process [Figure 10] Illustration of the cleaning process and the stripping solution addition process [Figure 11]Flowchart of the detachment solution addition process in the passaging operation [Figure 12] Flowchart of the recovery process in the passaging operation [Figure 13] Illustration of the recovery process [Figure 14] Illustration of the recovery process [Figure 15] Flowchart of centrifugation steps in passaging operation [Figure 16] Illustration of the centrifugation process [Figure 17] Illustration of the centrifugation process [Figure 18] Flowchart of the seeding process in the subculture operation [Figure 19] Illustration of the seeding process [Figure 20] Illustration of the seeding process [Figure 21] Illustration of the seeding process DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] [Embodiment Mode] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings.

[0010] <Configuration of cell culture device> First, a description will be given of the configuration of the cell culture device 1. Fig. 1 is a basic configuration diagram of the cell culture device.

[0011] As shown in FIG. 1, the cell culture device 1 includes an equipment cabinet 11, a rotating table 12, a lid opening device 13, an observation device 14, a liquid suction device 15, a liquid injection device 16, a liquid suction / discharge device 17, a lid closing device 18, a rocking device 19, a transfer device 20, a centrifuge device 21, a container loading / unloading section 22, a chemical liquid refrigeration device 23, and a control device 24.

[0012] The equipment cabinet 11 is a body in which various devices constituting the cell culture device 1 can be arranged.

[0013] The turntable 12 is a circular plate-shaped table mounted on the device cabinet 11. The turntable 12 rotates clockwise or counterclockwise in FIG. 1 around the center of the turntable 12 as a rotation center C by driving a table driving mechanism. A plurality of placement positions are set on the upper surface of the turntable 12. The plurality of placement positions are set at equal intervals around the rotation center C of the turntable 12. In this embodiment, a configuration in which eight placement positions are set is illustrated. Each placement position is set at 45° intervals around the rotation center C of the turntable 12. Each placement position includes a container placement position and a lid placement position.

[0014] The container placement positions are set on the outer periphery of the turntable 12. One culture container 31 is placed on each container placement position. With this configuration, when performing cell culture processing, the culture containers 31 placed on each container placement position simultaneously face various devices installed around the turntable 12, and it is possible to perform work on each culture container 31 simultaneously. The lid placement positions are set on the inner periphery of the turntable 12. One container lid 32 is placed on each lid placement position. It is preferable that the lid placement positions are set on a straight line passing through the container placement position and the rotation center C of the turntable 12, and in this embodiment, they are set between the container placement position and the rotation center C of the turntable 12. With this configuration, the culture containers 31 and the container lids 32 of the culture containers 31 are placed on the turntable 12 in one-to-one correspondence, and contamination between the culture containers 31 can be reduced. Each vessel mounting position and each lid mounting position may be provided with a positioning portion for positioning the culture vessel 31 and the vessel lid 32. Examples of the positioning portion include recesses formed on the upper surface of the turntable 12 into which the culture vessel 31 and the vessel lid 32 are fitted, and protrusions protruding from the upper surface of the turntable 12 and coming into contact with the outer edges of the culture vessel 31 and the vessel lid 32.

[0015] Around the turntable 12, the same number of work positions as the placement positions are set. Each work position is set so as to be located outside each placement position when the turntable 12 is stationary in a predetermined state. At each work position, various devices required for cell culture processing, specifically, a lid opening device 13, an observation device 14, a liquid suction device 15, a liquid injection device 16, a liquid suction and discharge device 17, a lid closing device 18, a rocking device 19, and a delivery device 20 are arranged in this order along the outer periphery of the turntable 12. Hereinafter, the work position where the liquid suction device 15 is arranged may be referred to as the first work position, the work position where the liquid injection device 16 is arranged may be referred to as the second work position, the work position where the liquid suction and discharge device 17 is arranged may be referred to as the third work position, and the work position where the rocking device 19 is arranged may be referred to as the fourth work position. The order of operations for the culture vessel 31 is always the first operation position, the second operation position, the third operation position, and the fourth operation position, and is never the reverse order. The positions of the lid opening device 13, the observation device 14, the liquid suction device 15, the liquid injection device 16, the liquid suction and discharge device 17, the lid closing device 18, the rocking device 19, and the delivery device 20 may be shifted clockwise or counterclockwise from the positions shown in FIG.

[0016] The lid opening device 13 mechanically grasps and opens the vessel lid 32 of the culture vessel 31, and places the vessel lid 32 in the lid placement position.

[0017] The observation device 14 is a device for observing cells grown in the culture vessel 31, and moves a phase-contrast microscope to a position to be observed in the culture vessel 31, adjusts the focus, and then captures an image.

[0018] The liquid aspirator 15 is a device for aspirating and discarding liquid, such as culture medium, in the culture vessel 31, and continuously aspirates the liquid in the culture vessel 31 by a pump and stores it in a waste bottle. The liquid aspirator 15 inserts a pipette into a tube 213 in the centrifugal separator 21 from a first aspirating and discharging port 214A (described later) of the centrifugal separator 21 located at the center of the turntable 12, and aspirates the liquid in the tube 213. That is, the liquid aspirator 15 can drive an arm that performs aspirating from the vessel mounting position of the turntable 12 to the first aspirating and discharging port 214A. The liquid aspirator 15 also has a function of tilting the culture vessel 31 so that the part of the culture vessel 31 closer to the rotation center C of the turntable 12 is higher or lower than the part of the culture vessel 31 farther from the rotation center C. This function allows the liquid aspirator 15 to aspirate the liquid in the culture vessel 31 without leaving any liquid behind. The tube 213 is an example of a centrifugal separator vessel.

[0019] The liquid injection device 16 is a device for injecting liquid, such as a culture medium, into the culture vessel 31, and sweeps a required amount of liquid from the liquid refrigeration device 23 with a pump and injects it into the culture vessel 31. The liquid injection device 16 also inserts a pipette into a tube 213 in the centrifugal separator 21 from a second suction and discharge port 214B (described later) of the centrifugal separator 21, and injects liquid into the tube 213. That is, the liquid injection device 16 can drive an arm that performs injection from the vessel mounting position of the turntable 12 to the second suction and discharge port 214B. The liquid injection device 16 also has a function of tilting the culture vessel 31 in the same way as the liquid suction device 15. With this function, the liquid injection device 16 can inject liquid while making it follow the wall surface of the culture vessel 31, and can suppress the generation of bubbles during liquid injection.

[0020] The liquid suction and discharge device 17 is a device for sucking and discharging the liquid in the culture vessel 31, and sucks a certain amount of liquid in the culture vessel 31 with a pump and discharges the sucked liquid. By repeating this operation, the liquid can be stirred. The liquid suction and discharge device 17 also inserts a pipette into a tube 213 in the centrifugal separator 21 from a third suction and discharge port 214C described later of the centrifugal separator 21, and sucks and discharges the liquid in the tube 213. That is, the liquid suction and discharge device 17 can drive an arm that performs suction and discharge from the container placement position of the turntable 12 to the position of the third suction and discharge port 214C. The liquid suction and discharge device 17 also has a function of tilting the culture vessel 31 in the same way as the liquid suction device 15. With this function, the liquid suction and discharge device 17 can suck the liquid in the culture vessel 31 without leaving any liquid in it in the same way as the liquid suction device 15, and can suppress the generation of bubbles when discharging the liquid in the same way as the liquid injection device 16.

[0021] The lid fastening device 18 mechanically grips the vessel lid 32 placed in the lid placement position and attaches it to the culture vessel 31.

[0022] The rocking device 19 has a mechanism for moving the culture vessel 31 up and down and tilting it back and forth and left and right, and by repeatedly and continuously tilting the culture vessel 31 back and forth and left and right, the culture vessel 31 is rocked and the liquid inside the culture vessel 31 is allowed to mix.

[0023] The transfer device 20 is a device for transferring the culture vessel 31 and the vessel lid 32 between the incubator 25, which stores the culture vessel 31 with the vessel lid 32 closed, the turntable 12, and the vessel loading / unloading section 22, and transfers the culture vessel 31 by mechanically grasping it. The transfer device 20 is an example of a transfer device that transfers the culture vessel 31 to a vessel mounting position on the turntable 12.

[0024] The centrifugal separator 21 is disposed in a hollow portion of the turntable 12. The centrifugal separator 21 includes a rotation mechanism 211 and a cover 212.

[0025] The rotation mechanism 211 rotates about a rotation axis set coaxially with the rotation center C of the turntable 12. One tube 213 is mounted in the rotation mechanism 211. The tube 213 is mounted in a position such that it can be located directly below any one of the first to third intake and exhaust ports 214A to 214C.

[0026] The cover 212 is fixed to the equipment cabinet 11 so as to cover the upper surface and side surfaces of the rotation mechanism 211. First to third intake and exhaust ports 214A to 214C are formed in the portion of the cover 212 covering the upper surface of the rotation mechanism 211. The first to third intake and exhaust ports 214A to 214C are arranged so as to be aligned at equal intervals around the rotation center of the rotation mechanism 211. The first, second, and third intake and exhaust ports 214A, 214B, and 214C are formed on straight lines connecting the first, second, and third work positions and the rotation center C of the turntable 12, respectively. Three lids 215 for opening and closing the first to third intake and exhaust ports 214A to 214C are arranged on the cover 212. The tube 213 loaded in the rotation mechanism 211 is stopped just below any one of the first to third intake and exhaust ports 214A to 214C, and the lid 215 is opened, whereby liquid can be injected into the tube 213 or liquid can be sucked out from the tube 213. Furthermore, by closing the lid 215, the inside of the tube 213 can be maintained as a closed space during centrifugation. The liquid in the tube 213 is centrifuged by the centrifugal force generated when the rotation mechanism 211 rotates. The centrifuge device 21 is mainly used for separating cells from a cell suspension. The lid 215 may be configured to rotate up and down around an axis extending in the horizontal direction, or may be configured to slide in the horizontal direction.

[0027] The container loading / unloading section 22 is configured so that a user can load and unload the culture containers 31. The container loading / unloading section 22 is, for example, a rack that can store the culture containers 31 in an aligned state.

[0028] The chemical liquid refrigeration device 23 is a device for temporarily keeping a chemical liquid such as a culture medium cool, and can store the chemical liquid at a low temperature to prevent deterioration of the chemical liquid. The chemical liquid refrigeration device 23 is also connected to the liquid injection device 16, and can send a required amount of liquid to the liquid injection device 16 via a tube pump or the like.

[0029] The control device 24 is a device for comprehensively controlling the operation of various devices in accordance with the contents of the cell culture work, and issues operation commands to the various devices at appropriate timing in accordance with a pre-programmed operation flow.

[0030] The cell culture device 1 performs medium replacement and subculture. The culture vessel 31 stored in the vessel loading / unloading section 22 is temporarily stored in the incubator 25 and kept at a constant temperature until medium replacement or subculture is performed. The culture vessel 31 taken out of the incubator 25 for medium replacement or other operations is placed on the vessel placement position of the turntable 12 by the delivery device 20. When the turntable 12 rotates clockwise in FIG. 1, the culture vessel 31 moves in front of the lid opening device 13, the observation device 14, the liquid suction device 15, the liquid injection device 16, the liquid suction / discharge device 17, the lid closing device 18, and the rocking device 19 in this order, and after various processes are performed by these devices, it is stored again from the turntable 12 to the incubator 25 by the delivery device 20. In this way, by arranging the liquid suction device 15, liquid injection device 16, liquid suction and discharge device 17, and rocking device 19 in this order along the outer periphery of the turntable 12, efficient culture medium replacement and passaging operations can be performed.

[0031] <Operation of cell culture device> Next, a description will be given of the operation of the cell culture device 1. The operation described below is performed under the control of the control device 24.

[0032] (Culture medium replacement operation) First, a medium exchange operation, which is an example of the first mode, will be described. Fig. 2 is a flow chart showing an example of the medium exchange operation. Figs. 3, 4, and 5 are explanatory diagrams of the medium exchange operation. In the following, the medium exchange operation will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Figs. 3, 4, and 5.

[0033] First, as shown in FIG. 2, the transfer device 20 removes the culture container 31A with the container lid 32A closed from the incubator 25, and transfers it to a container placement position located in front of the transfer device 20 on the turntable 12, as shown in the upper diagram of FIG. 3 (step S1).

[0034] As the turntable 12 rotates, the culture vessel 31A placed on the turntable 12 in step S1 moves in front of the lid opening device 13, and then the lid opening device 13 performs a lid opening process in which the vessel lid 32A of the culture vessel 31A is opened and the vessel lid 32A is placed in the lid placement position, as shown in the central diagram of Figure 3 (step S2).

[0035] As shown in the lower diagram of FIG. 3, the turntable 12 is further rotated to move the culture vessel 31A, which has been subjected to the lid opening process, in front of the observation device 14, and then the observation device 14 observes the cells in the culture vessel 31A (step S3).

[0036] As shown in the upper diagram of Figure 4, after further rotation of the turntable 12 moves the culture vessel 31A where cell observation has been performed in front of the liquid suction device 15, the liquid suction device 15 aspirates and discards the old culture medium in the culture vessel 31A (step S4).

[0037] As shown in the central diagram of Figure 4, further rotation of the turntable 12 moves the culture vessel 31A from which the old culture medium has been aspirated in front of the liquid injection device 16, and then the liquid injection device 16 injects new culture medium into the culture vessel 31A (step S5).

[0038] 4, the culture vessel 31A into which the new culture medium has been poured moves in front of the liquid suction and discharge device 17 by further rotation of the turntable 12 (step S6). The liquid suction and discharge device 17 waits without performing any process on the culture vessel 31A.

[0039] After a predetermined time has elapsed without any processing being performed by the liquid suction and discharge device 17, further rotation of the turntable 12 moves the culture vessel 31A in front of the lid closing device 18, and the lid closing device 18 performs a lid closing process to attach the vessel lid 32A to the culture vessel 31A, as shown in the upper diagram of Figure 5 (step S7).

[0040] As shown in the central diagram of Figure 5, further rotation of the turntable 12 moves the culture vessel 31A, which has been subjected to the lid closing process, in front of the rocking device 19, and then the rocking device 19 rocks the culture vessel 31A to allow the culture medium to become familiar with the culture vessel 31A (step S8).

[0041] As shown in the lower diagram of FIG. 5, the turntable 12 is further rotated to move the swung culture vessel 31A in front of the transfer device 20, and then the transfer device 20 places the culture vessel 31A back into the incubator 25 (step S9).

[0042] In the above steps S1 to S9, it is important to stop the turntable 12 and perform the culture medium replacement work so that the culture vessel 31 is positioned in front of the liquid suction device 15, the liquid injection device 16, and the rocking device 19. The stop time of the turntable 12 is set according to the process that requires the longest time among the processing times of each device, but it is preferable to adjust the processing time of each device in advance so that there is no significant difference in the processing time of each device. By adjusting the processing time in this way, all culture vessels 31 can be processed in a constant operation time by rotating the turntable 12 at regular intervals.

[0043] By performing the above operations, a series of culture medium replacement operations can be performed by rotating the turntable 12 once. By sequentially and consecutively removing multiple culture vessels 31 from the incubator 25 and placing them sequentially on the turntable 12 and rotating it, it is possible to continuously replace the culture medium in all of the culture vessels 31.

[0044] (Passaging operation) Next, a passage operation, which is an example of the second mode, will be described below. Fig. 6 is a flow chart showing an example of the passage operation.

[0045] First, as shown in FIG. 6, the cell culture device 1 aspirates and discards the old medium in the culture vessel 31, and injects a cleaning liquid such as PBS (phosphate buffered saline) into the culture vessel 31 (step S11: cleaning step). Next, the cell culture device 1 aspirates and discards the cleaning liquid from the culture vessel 31, and injects a detachment liquid for detaching the cells (step S12: detachment liquid addition step). After this, the cell culture device 1 waits for a predetermined time until the detachment liquid detaches the cells (step S13: detachment step). Next, the cell culture device 1 aspirates a suspension mixed with the detached cells from the culture vessel 31 containing the detachment liquid, and recovers it in the tube 213 preloaded in the centrifugal separator 21 (step S14: recovery step). After this, the cell culture device 1 centrifuges the suspension in the tube 213, removes the supernatant, injects a new culture medium, and stirs it to prepare a suspension of the new culture medium (step S15: centrifugation step). Finally, the cell culture device 1 seeds the prepared new culture medium suspension into a new culture vessel 31, thereby preparing a culture vessel 31 containing the seeded cells and the culture medium (step S16: seeding step).

[0046] Each of steps S11 to S16 will be described in detail below.

[0047] (Cleaning process (step S11)) First, the washing step will be described. Fig. 7 is a flow chart of the washing step in the passaging operation. Figs. 8, 9, and 10 are explanatory diagrams of the washing step and the peeling solution adding step. In the following, the washing step will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Figs. 8, 9, and 10.

[0048] The washing process includes seven steps as shown in Fig. 7. In the washing process, as in the culture medium replacement operation, the turntable 12 rotates by a fixed angle at fixed time intervals.

[0049] First, the cell culture device 1 performs operations similar to steps S1 and S2 of the culture medium replacement operation to remove the culture container 31A containing cells from the incubator 25 and transfer it to the container placement position located in front of the transfer device 20 (step S111) as shown in the upper diagram of Figure 8, and then performs a lid opening process (step S112) as shown in the center diagram of Figure 8.

[0050] As the turntable 12 rotates further, the culture vessel 31A that has been subjected to the lid-opening process moves in front of the observation device 14, and the observation device 14 waits without performing any process on the culture vessel 31A. Then, as shown in the lower diagram of Fig. 8, as the turntable 12 rotates further, the culture vessel 31A that has been subjected to the lid-opening process moves in front of the liquid suction device 15, and the liquid suction device 15 suctions and discards the old culture medium in the culture vessel 31A (step S113).

[0051] As shown in the upper diagram of Figure 9, after further rotation of the turntable 12 moves the culture vessel 31A from which the old culture medium has been aspirated in front of the liquid injection device 16, the liquid injection device 16 injects cleaning liquid into the culture vessel 31A (step S114).

[0052] With further rotation of the turntable 12, the culture vessel 31A into which the cleaning fluid has been poured moves in front of the liquid suction and discharge device 17, and then the liquid suction and discharge device 17 waits without performing any processing on the culture vessel 31A. Thereafter, with further rotation of the turntable 12, the culture vessel 31A moves in front of the lid closing device 18, and the lid closing device 18 performs the same lid closing processing as in step S7 (step S115), as shown in the center diagram of FIG.

[0053] As shown in the lower diagram of Figure 9, further rotation of the turntable 12 moves the culture vessel 31A, which has been subjected to the lid closing process, in front of the rocking device 19, and then the rocking device 19 rocks the culture vessel 31A to spread (acclimate) the cleaning solution over a wide area within the culture vessel 31A (step S116).

[0054] 10, after the swung culture vessel 31A moves in front of the delivery device 20 by further rotation of the turntable 12, the delivery device 20 stores the culture vessel 31A in the incubator 25 again (step S117). When processing the culture vessels 31 in a number exceeding the number that can be placed on the turntable 12 at one time, they are temporarily stored in the incubator 25 in this manner. In this embodiment, since a maximum of eight culture vessels 31 can be placed on the turntable 12, when nine culture vessels 31 are to be processed, they are temporarily stored in the incubator 25. When the number of culture vessels 31 is seven or less, they can be shifted to the stripping solution adding step without returning them to the incubator 25.

[0055] (Removal solution addition process (step S12)) Next, the detachment liquid adding step will be described. Fig. 11 is a flow chart of the detachment liquid adding step in the passaging operation. In the following, the detachment liquid adding step will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Figs. 8, 9, and 10.

[0056] The stripping liquid adding process includes seven steps, as shown in Fig. 11. In the stripping liquid adding process, as in the culture medium replacing operation, the turntable 12 rotates by a fixed angle at fixed time intervals.

[0057] First, the cell culture device 1 performs operations similar to steps S111 and S112 of the cleaning process to remove the culture container 31A containing cleaning solution from the incubator 25 and transfer it to the container placement position located in front of the transfer device 20 (step S121) as shown in the upper diagram of Figure 8, and then performs a lid opening process (step S122) as shown in the center diagram of Figure 8.

[0058] As the turntable 12 rotates further, the culture vessel 31A that has been subjected to the lid-opening process moves in front of the observation device 14, and then the observation device 14 waits in the same manner as in the washing step. As shown in the lower diagram of Fig. 8, as the turntable 12 rotates further, the culture vessel 31A that has been subjected to the lid-opening process moves in front of the liquid suction device 15, and then the liquid suction device 15 suctions and discards the washing liquid in the culture vessel 31A (step S123).

[0059] As shown in the upper diagram of Figure 9, after further rotation of the turntable 12 moves the culture vessel 31A from which the old culture medium has been aspirated in front of the liquid injection device 16, the liquid injection device 16 injects the stripping liquid into the culture vessel 31A (step S124).

[0060] As the turntable 12 rotates further, the culture vessel 31A into which the washing liquid has been poured moves in front of the liquid suction and discharge device 17, and then, as in the washing step, the culture vessel 31A waits in front of the liquid suction and discharge device 17. Thereafter, as the turntable 12 rotates further, the culture vessel 31A moves in front of the lid closing device 18, and the lid closing device 18 performs the same lid closing process as in step S7 (step S125), as shown in the center diagram of FIG.

[0061] As shown in the lower diagram of Figure 9, further rotation of the turntable 12 moves the culture vessel 31A, which has undergone the lid closing process, in front of the rocking device 19, and then the rocking device 19 rocks the culture vessel 31A to spread the stripping solution over a wide area within the culture vessel 31A (step S126).

[0062] 10, after the swung culture vessel 31A moves in front of the delivery device 20 by further rotation of the turntable 12, the delivery device 20 stores the culture vessel 31A in the incubator 25 again (step S127). When a waiting time occurs for the detachment of cells with the detachment solution, or when more culture vessels 31 than can be placed on the turntable 12 at one time are to be processed, the culture vessels 31 are temporarily stored in the incubator 25 as in the washing step. When no waiting time is required for the detachment of cells and the number of culture vessels 31 is seven or less, the culture vessels 31 can be moved to the recovery step without being returned to the incubator 25.

[0063] (Peeling process (step S13)) Next, the detachment step will be described. The detachment step is a standby step required for the detachment liquid injected in the detachment liquid adding step to detach the cells, and the cell culture device 1 simply stores and warms the culture container 31 in the incubator 25, and does not perform any special processing.

[0064] (Recovery process (step S14)) Next, the recovery step will be described. Fig. 12 is a flow chart of the recovery step in the passaging operation. Fig. 13 and Fig. 14 are explanatory diagrams of the recovery step. In the following, the washing step will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Fig. 13 and Fig. 14.

[0065] The recovery process includes six steps, as shown in Fig. 12. In the recovery process, as in the culture medium replacement operation, the turntable 12 rotates by a fixed angle at fixed time intervals.

[0066] First, the cell culture device 1 performs operations similar to steps S111 and S112 of the cleaning process to remove the culture container 31A containing the stripping solution from the incubator 25 and transfer it to the container placement position located in front of the transfer device 20 (step S131) ​​as shown in the upper diagram of Figure 13, and then performs a lid opening process (step S132) as shown in the center diagram of Figure 13.

[0067] Thereafter, the turntable 12 is further rotated twice by a fixed angle, and the observation device 14 and the liquid suction device 15 are placed on standby. As shown in the lower diagram of Fig. 13, after the culture vessel 31A containing the stripping liquid is moved in front of the liquid injection device 16, the liquid injection device 16 injects the culture medium into the culture vessel 31A (step S133).

[0068] As shown in the upper diagram of FIG. 14, after the culture vessel 31A into which the culture medium has been poured is moved in front of the liquid suction and discharge device 17 by further rotation of the turntable 12, the centrifugal separator 21 positions the tube 213 containing the cells 42 directly under the third suction and discharge port 214C. After the cover 215 of the third suction and discharge port 214C is opened, the liquid suction and discharge device 17 aspirates the suspension containing the detachment liquid, cells, and culture medium contained in the culture vessel 31, and discharges it into the tube 213 to recover it (step S134). If the cells are not sufficiently detached, the liquid suction and discharge device 17 can promote the detachment of the cells by discharging the suspension once aspirated onto the cell adhesion surface on the culture vessel 31. The liquid suction and discharge device 17 repeats this process multiple times as necessary, and recovers the suspension containing the detached cells into the tube 213.

[0069] Thereafter, when the incubation vessel 31A is moved in front of the capping device 18 by further rotation of the turntable 12, the capping device 18 performs the same capping process as in step S7 (step S135), as shown in the center diagram of FIG.

[0070] As the turntable 12 rotates further, the culture vessel 31A that has been subjected to the lid closing process moves in front of the rocking device 19, and the rocking device 19 waits for the culture vessel 31A. Then, as shown in the lower diagram of Fig. 14, as the turntable 12 rotates further, the culture vessel 31A moves in front of the delivery device 20, and the delivery device 20 transfers the culture vessel 31A to the vessel loading / unloading section 22 (step S136). The culture vessel 31 transferred to the vessel loading / unloading section 22 is not needed for the subsequent steps, and is therefore discarded by the user after the series of operations is completed.

[0071] As described above, through the washing step (step S11), the detachment solution adding step (step S12), the detachment step (step S13), and the recovery step (step S14), the culture vessel 31 is rotated a total of three times on the turntable 12 to perform the processing, whereby the cells can be detached and recovered.

[0072] (Centrifugal separation step (step S15)) Next, the centrifugation step will be described. Fig. 15 is a flow chart of the centrifugation step in the passaging operation. Fig. 16 and Fig. 17 are explanatory diagrams of the centrifugation step. In the following, the centrifugation step will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Fig. 17.

[0073] The centrifugation process is a process for purifying the suspension containing the cells recovered in the recovery process, and includes five steps as shown in Fig. 15. Moreover, the centrifugation process does not involve the turntable 12, and is a process in which the tube 213 in the centrifuge device 21, the liquid suction device 15 at the first working position, the liquid injection device 16 at the second working position, and the liquid suction and discharge device 17 at the third working position work together to perform processing. The liquid suction device 15, the liquid injection device 16, and the liquid suction and discharge device 17 each perform processing on the tube 213 located directly below the first to third suction and discharge ports 214A to 214C.

[0074] First, the centrifuge device 21 performs a centrifugation process on the suspension recovered in the recovery process (step S141). By the centrifugation process in step S141, the suspension 40 in the tube 213 shown in the leftmost diagram of Fig. 16 is separated into two layers, a supernatant 41 and cells 42, as shown in the second diagram from the left in Fig. 16.

[0075] Next, the cell culture device 1 performs a process of removing the supernatant 41 (step S142). In the process of removing the supernatant, the centrifugal separator 21 positions the tube 213 containing the separated supernatant 41 and the cells 42 directly below the first suction and discharge port 214A as shown in the upper diagram of FIG. 17. After the cover 215 of the first suction and discharge port 214A is opened, the liquid suction device 15 moves the pipette to aspirate and discard the supernatant 41 in the tube 213 as shown in the center diagram of FIG. 16. At this time, since the tube 213 is positioned directly below the first suction and discharge port 214A, the liquid suction device 15 can move the pipette in the radial direction of the turntable 12 to minimize the moving distance of the pipette, and can quickly aspirate and discard the supernatant 41.

[0076] Next, the cell culture device 1 performs an injection process of the culture medium 43 (step S143). The centrifuge device 21 positions the tube 213 containing the cells 42 directly below the second suction and discharge port 214B as shown in the center diagram of FIG. 17. After the cover 215 of the second suction and discharge port 214B is opened, the liquid injection device 16 moves the pipette to inject the culture medium 43 into the tube 213 as shown in the second diagram from the right in FIG. 16. At this time, since the tube 213 is positioned directly below the second suction and discharge port 214B, the liquid injection device 16 can minimize the moving distance of the pipette and can rapidly inject the culture medium 43.

[0077] Next, the cell culture device 1 performs the stirring process (step S144). The centrifugal separator 21 positions the tube 213 containing the cells 42 directly below the third suction and discharge port 214C as shown in the lower diagram of FIG. 17. After the cover 215 of the third suction and discharge port 214C is opened, the liquid suction and discharge device 17 moves the pipette and stirs the inside of the tube 213 by pipetting, to prepare a cell suspension 44 as shown in the rightmost diagram of FIG. 16. At this time, since the tube 213 is positioned directly below the third suction and discharge port 214C, the liquid suction and discharge device 17 can minimize the moving distance of the pipette and can perform the stirring process quickly. After this, the cell suspension 44 is successively seeded into new culture containers 31.

[0078] Next, the cell culture device 1 determines the volume of the cell suspension 44 to be seeded (step S145). The volume can be determined using a preset fixed value or by performing a cell count process using a portion of the cell suspension 44.

[0079] (Seeding step (step S16)) Next, the seeding step will be described. Fig. 18 is a flow chart of the seeding step in the subculture operation. Fig. 19, Fig. 20, and Fig. 21 are explanatory diagrams of the seeding step. In the following, the seeding step will be described with attention to the hatched culture vessel 31A and vessel cover 32A in Fig. 19, Fig. 20, and Fig. 21.

[0080] The seeding process includes eight steps. In the seeding process, as in the culture medium replacement operation, the turntable 12 rotates by a fixed angle at fixed time intervals.

[0081] First, the cell culture device 1 performs operations similar to steps S111 and S112 of the cleaning process to remove the culture container 31A for seeding from the incubator 25 and transfer it to the container placement position located in front of the transfer device 20 (step S151) as shown in the upper diagram of Figure 19, and then performs a lid opening process (step S152) as shown in the center diagram of Figure 19.

[0082] Thereafter, the turntable 12 is further rotated by a certain angle, and the container is put on standby in the observation device 14. As shown in the lower diagram of Fig. 19, after the culture container 31A for seeding is moved in front of the liquid suction device 15, the liquid suction device 15 suctions and discards the coating liquid and the like that has been placed in advance in the culture container 31A (step S153).

[0083] As shown in the upper diagram of FIG. 20, when the turntable 12 is further rotated to move the culture vessel 31A in front of the liquid injection device 16, the liquid injection device 16 injects the culture medium into the culture vessel 31A (step S154).

[0084] As shown in the center of FIG. 20, when the turntable 12 is further rotated and the culture vessel 31A moves in front of the liquid suction and discharge device 17, the liquid suction and discharge device 17 stirs the cell suspension 44 in the tube 213 in the centrifugal separator 21, then aspirates a certain amount and discharges it into the culture vessel 31 to perform seeding (step S155). At this time, the liquid volume of the cell suspension 44 to be seeded is the liquid volume determined in step S145 of the centrifugation process, for example, a fixed value or a liquid volume determined by a process of counting the number of cells in the cell suspension 44. By the centrifugal separator 21 positioning the tube 213 directly below the third suction and discharge port 214C in advance, the liquid suction and discharge device 17 can minimize the moving distance of the pipette, and can quickly pipette and aspirate the cell suspension 44.

[0085] Thereafter, when the incubation vessel 31A moves in front of the capping device 18 by further rotation of the turntable 12, the capping device 18 performs the same capping process as in step S7 (step S156), as shown in the lower diagram of FIG. 20.

[0086] As shown in the upper diagram of Figure 21, the turntable 12 is further rotated to move the culture vessel 31A, which has been subjected to the lid closing process, in front of the rocking device 19, and then the rocking device 19 rocks the culture vessel 31A to thoroughly mix it with the injected culture medium and cell suspension 44 (S157).

[0087] As shown in the lower diagram of FIG. 21, the turntable 12 is further rotated to move the swung incubation vessel 31A in front of the transfer device 20, and then the transfer device 20 stores the incubation vessel 31A in the incubator 25 again (step S158).

[0088] As described above, by rotating the turntable 12 through steps S11 to S16 and stopping it at positions corresponding to the first working position, the second working position, the third working position, and the fourth working position, respectively, the passaging operation can be performed continuously and quickly on multiple culture vessels 31.

[0089] [Variations] Needless to say, the present disclosure is not limited to the embodiments described above, and various modifications can be made without departing from the spirit of the present disclosure. Furthermore, the above embodiments and the modifications shown below may be combined in any way as long as they function normally.

[0090] Although the lid fastening device 18 is disposed between the liquid suction / discharge device 17 and the rocking device 19, for example, the lid fastening device 18 may be disposed above the liquid suction / discharge device 17 or the rocking device 19.

[0091] The container lid 32 may be placed between the culture containers 31 adjacent to each other in the rotation direction of the turntable 12. The container lid 32 may not be placed on the turntable 12, and the cell culture device 1 may perform processing on the culture container 31 with the container lid 32 open. Good too.

[0092] The liquid in the tube 213 may be stirred not by the liquid suction / discharge device 17 but by a device dedicated to stirring.

[0093] The centrifuge device 21 may be disposed next to the turntable 12 or outside the cell culture device 1 .

[0094] Although three intake and discharge ports (first to third intake and discharge ports 214A to 214C) are formed in the centrifugal separator 21, a single intake and discharge port may be formed by connecting the first to third intake and discharge ports 214A to 214C. Also, for example, the second and third intake and discharge ports 214B and 214C may not be formed, and the liquid injector 16 and the liquid intake and discharge device 17 may move the pipette to the first intake and discharge port 214A to perform processing. [Industrial Applicability]

[0095] The cell culture device of the present disclosure is capable of processing multiple culture vessels simultaneously in parallel, and therefore can shorten the total processing time required for medium exchange and subculturing of many culture vessels, and can provide a cost-effective cell culture environment by miniaturizing the device housing and improving the area productivity in mass production of cells. Therefore, it can be applied to the automation of medium exchange and subculturing in the culture of iPS cells, etc. [Explanation of symbols]

[0096] 1 Cell culture device 11 Equipment Cabinet 12 Rotating table 13 Lid opening device 14 Observation equipment 15 Liquid suction device 16 Liquid injection device 17 Liquid suction / drainage device 18 Lid Closing Device 19 Rocking device 20 Delivery device 21 Centrifugal separator 22 Container loading and unloading section 23 Liquid medicine refrigeration equipment 24 Control device 25 Incubator 31,31A Culture vessel 32,32A container lid 40 Suspension 41 Supernatant 42 cells 43 Culture medium 44 Cell Suspension 211 Rotation Mechanism 212 Cover 213 Tube 214A First intake and exhaust port 214B Second intake and exhaust port 214C 3rd intake / exhaust port 215 Lid C Rotation center

Claims

1. A rotating table on which a plurality of culture vessels are placed; A liquid aspirating device that aspirates liquid in the culture vessel; A liquid injection device that injects liquid into the culture vessel; A liquid suction and discharge device that aspirates the liquid in the culture vessel while discharging the aspirated liquid into the culture vessel; A rocking device for rocking the culture vessel, four or more container mounting positions, each of which can accommodate one of the culture containers, are set at equal intervals around a rotation center of the turntable on an upper surface of the turntable; the liquid suction device, the liquid injection device, the liquid suction and discharge device, and the rocking device are arranged around the turntable in this order along the outer circumferential direction of the turntable, and are simultaneously positioned outside one of the container placement positions when the turntable is stationary in a predetermined state. Cell culture equipment.

2. A control unit is further provided. The control unit is When an instruction to control the medium exchange operation is obtained, activating the liquid suction device, the liquid injection device, and the rocking device; When an instruction to control by passaging operation is obtained, activating the liquid suction device, the liquid injection device, the liquid suction / discharge device, and the rocking device; The cell culture device according to claim 1 .

3. The turntable has an upper surface on which lids of the culture vessels are placed, and the number of lid placement positions is the same as the number of vessel placement positions. The cell culture device according to claim 1 or 2.

4. The lid placement position is set on a straight line passing through the container placement position and the rotation center of the rotary table. The cell culture device according to claim 3 .

5. The apparatus further includes a centrifuge having a rotation mechanism in which a centrifuge container capable of storing liquid is mounted, a rotation center of the rotation mechanism is located coaxially with a rotation center of the rotary table; The liquid suction and discharge device moves liquid between the culture container and the centrifugation container. The cell culture device according to claim 1 .

6. The centrifugal separator is provided with an intake / discharge port for exposing an opening of the centrifugal separator container to the outside, and a lid for opening and closing the intake / discharge port. The cell culture device according to claim 5 .

7. The centrifugal separator is provided with three of the intake and exhaust ports, the three suction and discharge ports are provided on three straight lines connecting the liquid suction device, the liquid injection device, and the liquid suction and discharge device, respectively, with a rotation center of the rotation mechanism; The cell culture device according to claim 6.

8. A transfer device for transferring the culture vessel to the vessel mounting position is further provided, Five or more container mounting positions are set on an upper surface of the turntable at equal intervals around a rotation center of the turntable, the liquid suction device, the liquid injection device, the liquid suction and discharge device, the rocking device, and the transfer device are arranged around the turntable in this order along the outer circumferential direction of the turntable, and are simultaneously positioned outside one of the container placement positions when the turntable is stationary in a predetermined state. The cell culture device according to claim 1 .

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