JP7478546B2 - Cultivation system, culture device, and multi-layer culture vessel operation device - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act Announced at "Regenerative Medicine JAPAN 2018" held at Pacifico Yokohama from October 10 to 12, 2018

Application of Article 30, Paragraph 2 of the Patent Act Announced at the 5th Interphex Osaka held at Intex Osaka from February 20th to 22nd, 2019

The present invention relates to a culture system, a culture method, and a multi-layer culture vessel operation device that can culture cells using a multi-layer culture vessel that contains multiple trays, and that can also operate the multi-layer culture vessel in the same space as the cell culture.

Conventionally, a technology for cell culture using a multi-layer culture vessel in which multiple trays are stacked is known in order to culture a large amount of cells efficiently in a small size (see, for example, Patent Document 1). In addition, when culturing cells using such a multi-layer culture vessel, a device is known that performs a handling operation to hold and rotate the multi-layer culture vessel when introducing culture medium or the like into or recovering it from the multi-layer culture vessel in order to reduce the burden on the operator (see Patent Document 2).

JP 2016-103984 A JP 2015-505472 A

When cell culture is performed using a multi-layer culture vessel, a series of operations are performed, including manipulating the multi-layer culture vessel to fill it with culture medium, placing the multi-layer culture vessel in an incubator to perform cell culture, manipulating the multi-layer culture vessel after cell culture to drain the culture medium from the multi-layer culture vessel, manipulating the multi-layer culture vessel to fill it with a detachment liquid such as trypsin for cell detachment, rocking the multi-layer culture vessel to perform cell detachment, and manipulating the multi-layer culture vessel after cell detachment to recover the detachment liquid such as trypsin containing the cells from the multi-layer culture vessel.
However, such work places a heavy burden on the operator, and therefore, as in the above-mentioned Patent Document 2, an apparatus for handling the multi-layer culture vessel is known, but a certain amount of space is required for the handling operation, and from the viewpoint of cost, the handling operation is often performed outside the incubator. However, when filling and discharging the culture medium or the detachment solution such as trypsin into the multi-layer culture vessel, when observing the cells in the multi-layer culture vessel under a microscope, when the multi-layer culture vessel is taken out of the incubator, there is a risk of contamination due to the influence on the cell culture caused by the drop in temperature and the operation of switching the liquid supply pipe connected to the multi-layer culture device, etc., so there was a demand for an apparatus that can perform the operation of the multi-layer culture vessel and the cell culture in a series in the same space while the multi-layer culture vessel is still in the incubator.

The present invention aims to provide a culture system, a culture device, and a multi-layer culture vessel operating device that can reduce the burden on the operator by performing operation of the multi-layer culture vessel, cell observation, and cell culture in the same space, while preventing the adverse effects of temperature changes and effectively preventing contamination.

The culture system of the present invention has a housing that houses a multi-layer culture vessel having multiple trays built in it in its internal space, and an operating unit that operates the multi-layer culture vessel while it is still housed in the internal space, and the multi-layer culture vessel is connected to a fluid supply tube, and fluid material can be introduced into the multi-layer culture vessel from outside the housing via the fluid supply tube, or fluid can be discharged from within the multi-layer culture vessel to the outside of the housing via the fluid supply tube.
In the above culture system, the operation unit can be configured to have a rotation unit that rotates or rocks the multi-layer culture vessel.
In the above culture system, the operation unit can be configured to have an opening/closing unit that opens and closes a communication passage between the liquid transfer tube and the multi-layer culture vessel.
In the above-mentioned culture system, an air filter can be provided between the inside and outside of the multi-layer culture vessel, and the operating unit can be configured to have an opening/closing unit that opens and closes the communication passage between the air filter and the multi-layer culture vessel.
In the above-mentioned culture system, the housing has an insertion portion through which the fluid supply tube passes through the housing, and can be configured to introduce fluid material from outside the housing into the multi-layer culture vessel via the insertion portion and the fluid supply tube, or to discharge fluid from within the multi-layer culture vessel to the outside of the housing.
In the above culture system, the liquid supply tube can be configured to be switchably connected to a plurality of containers or devices present outside the housing.
In the above-mentioned culture system, a plurality of individual fluid supply pipes each connected to a plurality of containers are connected to a common fluid supply pipe connected to the multi-layer culture container, and a valve is installed on the individual fluid supply pipe or at the junction of the individual fluid supply pipe and the common fluid supply pipe, and the communication state between the individual fluid supply pipe and the common fluid supply pipe can be controlled by controlling the valve.
The above culture system can be configured to further include an observation device below the multi-layer culture vessel for capturing an image of the inside of the multi-layer culture vessel.

The culture device of the present invention has a housing that houses a multi-layer culture vessel having multiple trays built in it in its internal space, and an operating unit that operates the multi-layer culture vessel while it is still housed in the internal space, and the multi-layer culture vessel is connected to a fluid supply tube, so that fluid material can be introduced into the multi-layer culture vessel from outside the housing via the fluid supply tube, or fluid can be discharged from within the multi-layer culture vessel to the outside of the housing via the fluid supply tube.
In the above culture device, the operation unit can be configured to have a rotation unit that rotates or rocks the multi-layer culture vessel.
In the above culture device, the operation unit can be configured to have an opening/closing unit that opens and closes a communication passage between the liquid transfer tube and the multi-layer culture vessel.

The multi-layer culture vessel operating device of the present invention has an operating unit for operating a multi-layer culture vessel incorporating a plurality of trays, and is housed within a housing, wherein the multi-layer culture vessel is connected to a fluid supply tube within the housing, and the operating unit is capable of operating the multi-layer culture vessel while the multi-layer culture vessel remains housed within the housing, and by operating the multi-layer culture vessel with the operating unit, it is possible to introduce fluid material from outside the housing into the multi-layer culture vessel via the fluid supply tube, or to discharge fluid from within the multi-layer culture vessel to the outside of the housing via the fluid supply tube.
In the above multi-layer culture vessel manipulation device, the manipulation section can be configured to have a rotation section that rotates or rocks the multi-layer culture vessel.
In the above-mentioned multi-layer culture vessel manipulation device, the rotating unit can be configured to have a first rotation axis and a second rotation axis as rotation axes for rotating or rocking the multi-layer culture vessel, and the rotating unit can be configured so that the first rotation axis and the second rotation axis are rotation axes passing through the multi-layer culture vessel.
In the above multi-layer culture vessel operating device, the operating section can be configured to have an opening/closing section that opens and closes a communication passage between the liquid supply pipe and the multi-layer culture vessel.
The above-mentioned multi-layer culture vessel operating device can be configured to further have a liquid level sensor that detects the liquid level of the fluid material in the multi-layer culture vessel when the fluid material is introduced into the multi-layer culture vessel from outside the housing via the liquid delivery pipe.
In the above-mentioned multi-layer culture vessel operating device, the liquid level sensor can be configured to have a first liquid level sensor that detects whether the fluid material has reached a first water level indicating that a predetermined amount of the fluid material has been introduced into the multi-layer culture vessel, or a second liquid level sensor that detects whether the fluid material has reached a second water level that is lower than the first water level and indicates that the volume of the fluid material is approaching a predetermined amount.

According to the present invention, the operation of the multi-layer culture vessel and cell culture can be performed consecutively in the same space, which reduces the burden on the operator, prevents adverse effects due to temperature changes, and effectively prevents contamination.

1 is a block diagram showing a configuration of a culture system according to a first embodiment. FIG. FIG. 1 is a perspective view showing a culture device according to a first embodiment. FIG. 2 is a diagram for explaining a multi-layer culture vessel according to the first embodiment. FIG. 13 is a diagram for explaining a method of distributing fluid materials to each tray in a multi-layer culture vessel. 11A and 11B are diagrams for explaining an example of the structure of an opening/closing unit and a clamp. 1 is a flowchart showing a cell culture method according to a first embodiment. 1A to 1C are diagrams for explaining a rotation operation of the multilayer culture vessel according to the first embodiment. FIG. 11 is a perspective view of a culture system according to a second embodiment. FIG. 11 is a perspective view showing the configuration inside the housing of the culture system according to the second embodiment. FIG. 11 is a perspective view showing the internal configuration of the housing of the culture system according to the second embodiment, as viewed from below.

Embodiments of the culture system, culture device, and multi-layer culture vessel operating device according to the present invention will be described with reference to the drawings.

First Embodiment
Fig. 1 is a block diagram showing the configuration of a culture system 1 according to the first embodiment, and Fig. 2 is a perspective view showing a culture device 10 according to the first embodiment. As shown in Fig. 1, the culture system 1 according to the first embodiment includes a culture device 10 that accommodates a multilayer culture vessel 12, liquid delivery pumps 20 and 70 that deliver a culture solution or a detachment solution such as trypsin, a flowmeter 30 that measures the flow rate of the culture solution or the detachment solution such as trypsin, containers 40, 50, and 80 for supplying or recovering the culture solution or the detachment solution such as trypsin, and a centrifugal separator 60.

As shown in FIG. 1, the culture system 1 has each component connected via liquid supply pipes 3a-3g, and the flow paths (liquid supply pipes 3a-3g) through which the culture solution or the detachment solution such as trypsin is supplied can be switched by valves 2a-2d. Note that the valves 2a-2d may be switched manually by an operator, or automatically by a machine.

As shown in Figs. 1 and 2, the culture device 10 has a housing 11, a multi-layer culture vessel 12, a liquid supply pipe 13, an operation unit 14, an insertion unit 15, a temperature adjustment unit 16, a gas concentration adjustment unit 171, a pH adjustment unit 172, a control unit 18, and an input unit 19. As shown in Fig. 2, the culture device 10 accommodates the multi-layer culture vessel 12 in the internal space S of the housing 11. The housing 11 can be opened and closed, but is used in a sealed state when culturing cells. The size of the internal space S of the housing 11 is not particularly limited, but is appropriately designed according to the size of the multi-layer culture vessel 12 so that the multi-layer culture vessel 12 can be rotated or rocked by the operation unit 14, as described below.

Here, the multi-layer culture vessel 12 according to the first embodiment will be described. FIG. 3(A) is a perspective view for explaining the multi-layer culture vessel 12 according to the first embodiment, and is a schematic diagram of the multi-layer culture vessel 12 viewed from the left side to the right side shown in FIG. 2. FIG. 3(B) is a schematic cross-sectional view of the multi-layer culture vessel 12 along IIIB-IIIB in FIG. 3(A), and FIG. 3(C) is a schematic cross-sectional view of the multi-layer culture vessel 12 along IIIC-IIIC in FIG. 3(A). In FIG. 3(A), the wall portion 1211 of the tray 121 is omitted for convenience of explanation. The multi-layer culture vessel 12 is configured such that a plurality of trays 121 are stacked as shown in FIG. 3(A) to (C) in order to efficiently culture cells. In addition, holes are opened in two of the four corners of each tray 121 of the multi-layer culture vessel 12 as shown in FIG. 3(A). As a result, in the multi-layer culture vessel 12, as shown in FIG. 3(A), a plurality of trays 121 are spatially connected to each other, and the multi-layer culture vessel 12 is spatially connected to the liquid supply tube 13 via the connection part 122 and to the air filter 133 via the connection part 123. Furthermore, a clamp 131 is provided in a portion of the liquid supply tube 13 close to the connection part 122 of the multi-layer culture vessel 12, and by opening the clamp 131, a culture medium or a detachment liquid such as trypsin can be introduced into the multi-layer culture vessel 12 through the liquid supply tube 13, or a detachment liquid such as culture medium or trypsin can be discharged from the multi-layer culture vessel 12. Furthermore, the multi-layer culture vessel 12 is connected to the air filter 133 via the connection part 123. The air filter 133 is a filter that allows air to pass but blocks the intrusion of bacteria into the multi-layer culture vessel 12, and is disposed between the inside and outside of the multi-layer culture vessel 12. In this embodiment, a clamp 132 is also present between the air filter 133 and the connection part 123, and air can be taken in and discharged from the multi-layer culture vessel 12 by controlling the opening and closing of the clamp 132.

When cell culture is performed using the multi-layer culture vessel 12, for example, the multi-layer culture vessel 12 is rotated counterclockwise by about 90° so that the multi-layer culture vessel 12 changes from the state shown in FIG. 3(C) to the state shown in FIG. 4(A). Then, the clamps 131 and 132 are opened, and the air in the multi-layer culture vessel 12 can be discharged to the outside through the air filter 133, and a culture medium in which cells are suspended is introduced into the multi-layer culture vessel 12 through the liquid supply tube 13. Next, the clamps 132 are closed, and the multi-layer culture vessel 12 is rotated clockwise by about 180°, and then the multi-layer culture vessel 12 is returned to an upright position as shown in FIG. 4(B), and the culture medium is distributed to each tray 121 of the multi-layer culture vessel 12. Then, the clamps 131 are closed, and cell culture is performed in each tray 121. During cell culture, the clamps 132 are also kept closed (depending on the type of the multi-layer culture vessel 12, culture can also be performed with the clamps 132 open). FIG. 4 is a diagram for explaining a method of distributing fluid materials to each tray 121 in the multi-layer culture vessel 12. Similarly to FIG. 3(C), FIG. 4(A) is a schematic cross-sectional view of the multi-layer culture vessel 12 taken along IIIC-IIIC in FIG. 3(A), and FIG. 4(B) is a schematic cross-sectional view of the multi-layer culture vessel 12 taken along IIIB-IIIB in FIG. 3(A), similar to FIG. 3(B).

After cell culture, a culture medium discharge process is performed to discharge the culture medium from the multilayer culture vessel 12, a trypsin or other detachment liquid introduction process is performed to introduce a trypsin or other detachment liquid into the multilayer culture vessel 12 to detach the cells attached to the bottom surface of each tray 121 of the multilayer culture vessel 12, and a trypsin or other detachment liquid recovery process is performed to recover the trypsin or other detachment liquid containing the detached cells from the multilayer culture vessel 12. The treatment of these fluid materials (including culture medium before culture and detachment liquid such as trypsin liquid; the same applies below) and fluids (culture medium after culture and detachment liquid such as trypsin containing the detached cells) also requires the introduction or discharge of fluid materials by rotating or rocking the multilayer culture vessel 12, and the opening and closing of the clamps 131 and 132.

When an operator operates the multi-layer culture vessel 12 manually, the multi-layer culture vessel 12 containing the culture medium and the peeling liquid such as trypsin is heavy, and the burden on the operator increases. In addition, manual operation may cause inconsistencies in the work, or the operator may touch the multi-layer culture vessel 12 unnecessarily, damaging the multi-layer culture vessel and making it impossible to continue cell culture. In addition, a certain amount of space is required for the rotation and rocking of the multi-layer culture vessel 12, so that outside the culture device (for example, a commercially available dedicated culture device), a liquid supply tube is attached to the multi-layer culture vessel 12, the multi-layer culture vessel 12 is operated to fill the multi-layer culture vessel 12 with culture medium, and then the multi-layer culture vessel 12 is placed in the culture device, and the multi-layer culture vessel 12 is removed from the culture device, the liquid supply tube attached to the multi-layer culture vessel 12 is replaced, and the multi-layer culture vessel 12 is operated. However, the installation or replacement of the liquid supply tube to the multi-layer culture vessel 12 outside the culture device may change the temperature inside the multi-layer culture vessel 12, which may have a negative effect.

In order to solve such problems, in the culture device 10 according to this embodiment, the multilayer culture vessel 12 is accommodated in the internal space S of the housing 11, and without removing the multilayer culture vessel 12 from the internal space S, the culture medium or the peeling liquid such as trypsin can be introduced into the multilayer culture vessel 12 through the liquid supply tube 13 while the multilayer culture vessel 12 is accommodated in the internal space S, and the culture medium or the peeling liquid such as trypsin can be discharged from the multilayer culture vessel 12 through the liquid supply tube 13. Specifically, in the culture device 10 according to this embodiment, the multilayer culture vessel 12 is accommodated in the internal space S of the housing 11, and the liquid supply tube 13 is attached to the connection part 122 of the multilayer culture vessel 12 in the internal space S, and the liquid supply tube 13 and the multilayer culture vessel 12 are connected to each other. In addition, the liquid supply tube 13 is connected to the liquid supply tube 3a outside the housing 11 through the insertion part 15 provided in the wall part of the housing 11. The insertion portion 15 is not particularly limited as long as it has a structure that connects the inside and outside of the housing 11, and can be, for example, a hole with the same diameter as the outer periphery of the liquid supply tube 13. In this case, by inserting the liquid supply tube 13 into the insertion portion 15, the part of the liquid supply tube 13 outside the housing 11 forms the liquid supply tube 3a.

The culture device 10 also has an operation unit 14 for operating the multi-layer culture vessel 12. The operation unit 14 has a rotating unit 141 capable of holding the multi-layer culture vessel 12 and a driving unit 142 for rotating the rotating unit 141, and performs a rotation operation for rotating the rotating unit 141 around two axes of rotation axes X1 and X2 as shown in FIG. 2. The rotation axis X1 is a rotation axis extending in the X-axis direction as shown in FIG. 2, and this allows the rotating unit 141 and the multi-layer culture vessel 12 held by the rotating unit 141 to rotate in the roll direction R. The rotation axis X2 is a rotation axis extending in the Y-axis direction, and this allows the rotating unit 141 and the multi-layer culture vessel 12 held by the rotating unit 141 to rotate in the pitch direction P. In addition, in the rotation operation, the rotation in the roll direction R is possible within a range of less than ±180°, and in this embodiment, the rotating unit 141 can be rotated in the roll direction R within a range of ±120°. Rotation in the pitch direction P is also possible within a range of less than ±180°, and in this embodiment, the rotating unit 141 can be rotated in the roll direction R within a range of ±30°. In this embodiment, the driving unit 142 includes a first driving unit 1421 (electric motor and/or air cylinder) that rotates the rotating unit 141 about the rotation axis X1, and a second driving unit 1422 (electric motor and/or air cylinder) that rotates the rotating unit 141 about the rotation axis X2, thereby allowing the rotating unit 141 to rotate on two axes.

Furthermore, the driving unit 142 of the operation unit 14 can also perform a rocking operation of rotating the rotating unit 141 (multilayer culture vessel 12) back and forth around the rotation axis X1 or the rotation axis X2. For example, the driving unit 142 can perform a rocking operation around the rotation axis X1 by rotating the rotating unit 141 (multilayer culture vessel 12) back and forth around the rotation axis X1 in the roll direction R in the range of ±120°. In addition, the driving unit 142 can perform a rocking operation around the rotation axis X2 by rotating the rotating unit 141 (multilayer culture vessel 12) back and forth around the rotation axis X2 in the pitch direction P in the range of ±30° so that the upper part of the rotating unit 141 (multilayer culture vessel 12) is tilted forward (X-axis negative direction) and then the lower part of the rotating unit 141 (multilayer culture vessel 12) is tilted forward (X-axis negative direction).

The operation unit 14 also has an opening/closing unit 143 that opens and closes the clamps 131 and 132 of the liquid supply tube 13. Here, FIG. 5 is a diagram for explaining an example of the structure of the opening/closing unit 143 and the clamp 131. The opening/closing unit 143 can be configured to push a pressing member such as a piston toward the liquid supply tube 13 side by a driving unit such as an air cylinder, an electric cylinder, or a solenoid, and close the flow path of the liquid supply tube 13 by sandwiching the liquid supply tube 13 between the pushed-out piston and the fixed part of the clamp 131. In this case, the liquid supply tube 13 can be opened by pushing back the pushing-out pressing member such as a piston to the opposite side of the liquid supply tube 13 by a driving unit such as an air cylinder, an electric cylinder, or a solenoid, thereby opening the flow path of the liquid supply tube 13. Note that the opening/closing unit 143 is not limited to the above structure, and a known structure can be applied. In addition, a plurality of structures that perform the same operation as the clamp 131 and the opening/closing unit 143 can be provided and used for venting. In this embodiment, the multilayer culture vessel 12 is rotated by the rotating unit 141, and the liquid supply tube 13 and the clamp 131 also rotate accordingly, so the opening/closing unit 143 is also provided so as to be rotatable by the rotating unit 141. The opening/closing unit 143 for opening and closing the clamp 132 can also have the same mechanism as the opening/closing unit 143 for opening and closing the clamp 131.

The temperature adjustment unit 16 adjusts the temperature in the internal space S of the housing 11. The culture device 10 according to this embodiment has an input unit 19 on the outside of the housing 11, and an operator can operate the input unit 19 to set the temperature of the internal space S of the housing 11. The set temperature input by the input unit 19 is transmitted to the temperature adjustment unit 16 via the control unit 18. When the temperature is set by the input unit 19, the temperature adjustment unit 16 adjusts the temperature in the internal space S so that the temperature in the internal space S becomes the set temperature.

The gas concentration adjustment unit 171 is equipped with a gas concentration sensor that measures the concentration of gases such as carbon dioxide in the internal space S of the housing 11. In addition, the gas concentration adjustment unit 171 is connected to a gas supply unit provided outside the housing 11, and introduces gases such as carbon dioxide into the internal space S based on the measurement results of the gas concentration sensor, and adjusts the gas concentration in the internal space S to the gas concentration set by the operator via the input unit 19. This allows a gas such as carbon dioxide at an appropriate concentration to be supplied into the multilayer culture vessel 12. In addition, instead of a configuration in which the gas supply unit is provided outside the housing 11, the gas concentration adjustment unit 171 can also be configured to include a gas supply unit (a gas supply unit is provided inside the housing 11).

The pH adjustment unit 172 adjusts the pH of the culture solution etc. filled in the multi-layer culture vessel 12. For example, the pH adjustment unit 172 is equipped with a pH sensor that measures the pH of the fluid material filled in the multi-layer culture vessel 12, and is connected to a gas supply unit (not shown) provided outside the housing 11, and can adjust the pH inside the multi-layer culture vessel 12 to the pH set by the operator via the input unit 19 based on the measurement result of the pH sensor.

The input unit 19 receives instructions input by the operator and transmits the input instructions to the control unit 18. The input unit 19 may be a button such as a switch, or a touch panel that also serves as a display.

The control unit 18 controls the operation of the operation unit 14, the temperature adjustment unit 16, and the pH adjustment unit 172 based on instructions from the operator inputted from the input unit 19. The control unit 18 can also control the opening and closing operations of the valves 2a to 2d, the operation of the opening and closing unit 143, the operation of the liquid delivery pumps 20 and 70, and the operation of the centrifuge device 60 based on instructions from the operator inputted from the input unit 19.

Next, the configuration other than the culture device 10 will be described. The liquid delivery pump 20 is connected to the liquid delivery pipe 13 and the multi-layer culture vessel 12 housed inside the culture device 10 via the liquid delivery pipe 3a. The liquid delivery pump 20 is also connected to the vessels 40, 50 and the centrifuge device 60 via the liquid delivery pipes 3b to 3e. The liquid delivery pump 20 delivers the culture liquid or the detachment liquid such as trypsin from the vessels 40, 50 to the multi-layer culture vessel 12, and delivers the culture liquid or the detachment liquid such as trypsin from the multi-layer culture vessel 12 to the vessels 40, 50. The amount of the culture liquid or the detachment liquid such as trypsin delivered by the liquid delivery pump 20 is measured by the flow meter 30, and the liquid delivery pump 20 can automatically or manually stop the liquid delivery based on the measurement result of the flow meter 30.

For cell culture, the container 40 is prepared by filling it with a culture solution in which cells are suspended. A liquid supply pipe 3c is attached to the container 40, and the container 40 and the multi-layer culture container 12 can be connected or disconnected by opening and closing a valve 2a provided near the container 40. For example, when cell culture is performed, the valve 2a is opened, and the culture solution can be introduced from the container 40 to the multi-layer culture container 12 by the liquid supply pump 20. After cell culture, the valve 2a is opened, and the culture solution can be sent from the multi-layer culture container 12 to the container 40 by the liquid supply pump 20, and collected in the container 40.

In addition, for cell recovery, the container 50 is prepared by filling it with a detachment liquid such as trypsin. A liquid supply pipe 3d is attached to the container 50, and the container 50 and the multi-layer culture container 12 can be connected or disconnected by opening and closing a valve 2b provided near the container 50. For example, when recovering cells, the valve 2b is opened, and the liquid supply pump 20 can introduce a detachment liquid such as trypsin from the container 50 into the multi-layer culture container 12.

The centrifuge 60 centrifuges the trypsin or other detachment solution containing cultured cells after the cell detachment process to separate the cultured cells from the trypsin or other detachment solution. In this embodiment, a liquid supply pipe 3e is attached to the centrifuge 60, and the centrifuge 60 and the multi-layer culture vessel 12 can be connected or disconnected by opening and closing a valve 2c provided near the centrifuge 60. After the cell detachment process, the valve 2c is opened to allow the liquid supply pump 20 to introduce the trypsin or other detachment solution containing cultured cells from the multi-layer culture vessel 12 to the centrifuge 60. The centrifuge 60 may be configured to automatically control the operation by the control unit 18, or may be configured to be operated manually by an operator.

In this embodiment, the centrifuge 60 is connected to the liquid delivery pump 70 and the container 80 via the liquid delivery pipes 3f and 3g. After centrifugal separation and washing of the trypsin or other detachment solution in the centrifuge 60, the valve 2c is closed and the valve 2d is opened, so that the cells separated and recovered from the trypsin or other detachment solution in the centrifuge can be recovered in the container 80 by the liquid delivery pump 70.

Next, a cell culture method using the culture system 1 according to the first embodiment will be described. FIG. 6 is a flowchart showing the cell culture method according to the first embodiment. FIG. 7 is a diagram for explaining the rotation operation of the multi-layer culture vessel 12 according to the first embodiment. For ease of explanation, FIG. 7 shows only the multi-layer culture vessel 12 and the operation unit 14, and omits the other components.

6, first, in step S101, a process of introducing a culture medium into the multi-layer culture vessel 12 is performed. Specifically, first, the operator prepares a vessel 40 containing a culture medium in which cells are suspended, attaches a liquid supply tube 3c to the vessel 40, and opens the valve 2a. Next, the operator operates the input unit 19 to open the clamp 131 of the liquid supply tube 13 by the opening/closing unit 143, thereby communicating the vessel 40 with the multi-layer culture vessel 12. In addition, the operator opens the clamp 132 so that the air in the multi-layer culture vessel 12 can be discharged from the air filter 133. Then, the operator operates the input unit 19 from the state shown in FIG. 7(A) or the state shown in FIG. 3(C), and causes the operation unit 14 of the culture device 10 to perform a rotation operation of rotating the multi-layer culture vessel 12 by about 90° so that the connection part 122 of the multi-layer culture vessel 12 is located on the lower side and the connection part 123 is located on the upper side, as shown in FIG. 7(B) or FIG. 4(A). The operator also operates the liquid supply pump 20 to introduce the culture medium from the container 40 into the multi-layer culture vessel 12. The amount of culture medium introduced from the container 40 into the multi-layer culture vessel 12 is measured by the flow meter 30, and the liquid supply pump 20 ends its operation when the culture medium supply is completed. When the culture medium supply is completed, the clamp 132 is closed to protect the air filter 133 from the culture medium. After the culture medium is introduced into the multi-layer culture vessel 12, the operator operates the input unit 19 and rotates the multi-layer culture vessel 12 by about 180° as shown in FIG. 7(C) by the operation unit 14 so that the connection part 122 of the multi-layer culture vessel 12 is located on the upper side and the connection part 123 is located on the lower side, and then, as shown in FIG. 7(D), stands the multi-layer culture vessel 12 upright, so that the culture medium in which the cells are suspended can be distributed to each tray 121 as shown in FIG. 4(B). Then, a process of closing the valve 2a and the clamp 131 is performed.

In step S102, cell culture is performed. For example, an operator can use the input unit 19 to set in advance the temperature of the internal space S of the housing 11, and the temperature adjustment unit 16 adjusts the temperature of the internal space S of the housing 11 to the temperature set by the operator. Then, the operator can culture the cells by statically culturing or shaking-culturing the multi-layer culture vessel 12 containing a culture solution in which cells are suspended, in the internal space S of the housing 11 for a predetermined period of time.

In step S103, the culture medium is collected. For example, the operator operates the input unit 19 to open the clamps 131 and 132 using the operation unit 14 and rotate the multi-layer culture vessel 12 as shown in FIG. 7(B) or FIG. 4(A). The operator also opens the valve 2a to connect the vessel 40 to the multi-layer culture vessel 12, and then causes the liquid supply pump 20 to send the culture medium from the multi-layer culture vessel 12 to the vessel 40. As a result, the culture medium is collected in the vessel 40, and the cultured cells remain attached to the tray 121 of the multi-layer culture vessel 12.

In step S104, a process of introducing a detachment liquid such as trypsin into the multi-layer culture vessel 12 is performed to detach the cells adhering to the tray 121. Specifically, the operator closes the valve 2a while the clamps 131 and 132 are open, and opens the valve 2b near the vessel 50 containing the detachment liquid such as trypsin, thereby communicating the vessel 50 with the multi-layer culture vessel 12. Then, the operator uses the liquid supply pump 20 to introduce the detachment liquid such as trypsin into the multi-layer culture vessel 12 through the liquid supply pipes 3d, 3b, and 3a and the liquid supply pipe 13. When introducing the detachment liquid such as trypsin into the multi-layer culture vessel 12, the operator operates the input unit 19 to rotate the rotation unit 141 of the drive unit 142 of the operation unit 14 to perform a rotation operation to rotate the multi-layer culture vessel 12, as in step S101, as shown in Figures 7 (A) to (D).

In step S105, a cell detachment process is performed. For example, the operator can operate the input unit 19 to input an instruction to cause the operation unit 14 to perform an operation of rocking the multi-layer culture vessel 12. In addition, in the cell detachment process, the operation program is programmed so that the multi-layer culture vessel 12 is rocked back and forth in a first direction (e.g., rightward) and a second direction (e.g., leftward) around the first rotation axis X1 or the second rotation axis X2 by the rotating unit 141. In addition, this operation program has a stop mode that stops the movement of the multi-layer culture vessel 12 for a specified time when switching from a rotation operation in the first direction to a rotation operation in the second direction and when switching from a rotation operation in the second direction to a rotation operation in the first direction. By providing a stop mode, even if the rocking motion is performed at a speed faster than the movement of the liquid in the container, the rocking motion can be stopped for a specified period of time when changing the direction of the rotation motion, so that the liquid in the container can reliably apply shear force to the cells adhering to the tray 121, promoting the detachment of the cells from the tray 121. In order to effectively perform cell detachment processing, it is important to rock the container at high speed, but it is possible to eliminate the problem of delays in the movement of the liquid (time lag) that occur when the container is rocked at high speed.

In step S106, centrifugation is performed. Specifically, the operator operates the input unit 19 to rotate the multilayer culture vessel 12 so that the connection part 122 is on the lower side and open the clamps 131 and 132 on the operation unit 14. The operator also operates the input unit 19 to open the valve 2c near the centrifuge device 60 to communicate the multilayer culture vessel 12 with the centrifuge device 60. Then, the operator uses the liquid delivery pump 20 to introduce the detachment liquid such as trypsin containing the detached cells through the liquid delivery tube 13 and the liquid delivery tubes 3a, 3b, and 3e into the centrifuge device 60. Furthermore, the operator operates the centrifuge device 60 to centrifuge the detachment liquid such as trypsin containing the cells, and separate the cultured cells from the detachment liquid such as trypsin.

In step S107, the centrifuge 60 can be a device configured with a service tank, a continuous centrifuge, a culture medium supply tank, a cell collection bag, a waste liquid collection bag, a pump, etc. A process of collecting cultured cells is performed. For example, the operator can collect the cultured cells precipitated by the centrifuge 60. The operator can also close valve 2c and open valve 2d to connect the centrifuge 60 to the container 80, and use the liquid delivery pump 70 to transfer the culture medium in which the separated cells are suspended from the centrifuge 60 to the container 80 for collection.

As described above, the culture system 1 according to the first embodiment has a housing 11 that houses a multi-layer culture vessel 12 having a plurality of built-in trays 121 in an internal space S, a temperature adjustment unit 16 that adjusts the temperature of the internal space S, and an operation unit 14 that operates the multi-layer culture vessel 12 while it is housed in the internal space S. The multi-layer culture vessel 12 is in communication with the liquid supply tube 13, and a culture medium or a detachment liquid such as trypsin can be introduced into the multi-layer culture vessel 12 from the outside of the housing 11 through the liquid supply tube 13, or the culture medium can be discharged from the inside of the multi-layer culture vessel 12 to the outside of the housing 11 through the liquid supply tube 13. This allows the operation of the multi-layer culture vessel 12 and cell culture to be performed in succession in the same space, which eliminates the need for the operator to take out the multi-layer culture vessel 12 from the incubator and house the multi-layer culture vessel 12 in the incubator every time the multi-layer culture vessel 12 is operated, thereby reducing the burden on the operator. In addition, since it is not necessary to replace the liquid supply tube 13 outside the incubator, it is possible to effectively prevent temperature changes that have a negative effect on cell culture.
Furthermore, the above series of operations can also be carried out automatically without the intervention of an operator.

In addition, in the culture system 1 according to this embodiment, by setting up multiple systems, it is possible to perform cell culture, cell detachment, and recovery of cultured cells using multiple multi-layer culture vessels 12, further reducing the burden on the operator.

Second Embodiment
Next, the culture system 1a according to the second embodiment will be described. FIG. 8 is a perspective view of the culture system 1a according to the second embodiment, and FIG. 9 is a perspective view showing the configuration inside the housing 11a of the culture system 1a according to the second embodiment. The culture system 1a according to the second embodiment has the same configuration as the culture system 1 according to the first embodiment, except that it has an observation device 101, has two liquid level sensors 102 and 103, and the rotation axis X2 of the rotation unit 141a is configured to pass through the multi-layer culture vessel 12, and operates in the same manner as the culture system 1 according to the first embodiment. In addition, in FIG. 8 and FIG. 9, for convenience of explanation, the field of view of the observation device 101 and the liquid level sensors 102 and 103 is illustrated (the same applies to FIG. 10 described later).

Specifically, in the culture system 1a according to the second embodiment, as shown in FIG. 8, the space S in the housing 11a is divided into two parts, an upper storage section 111 and a lower storage section 112, by a partition plate 113. The upper storage section 111 stores the culture device 10a including the multi-layer culture vessel 12, and the lower storage section 112 stores the observation device 101. The partition plate 113 is partially or entirely made of a light-transmitting material (light-transmitting resin or glass), and the observation device 101 stored in the lower storage section 112 can observe the inside of the multi-layer culture vessel 12 stored in the upper storage section 111 at the position of the light-transmitting material.

9, the observation device 101 has a camera 1011, a camera first driving unit 1012, a camera second driving unit 1013, and a lens 1014. The camera 1011 is, for example, a CCD camera or a CMOS camera, and is arranged on the lower side of the multi-layer culture vessel 12 via the lens 1014, and is installed so as to capture an image of the inner bottom surface of the tray 121 of the multi-layer culture vessel 12 from the lower side of the multi-layer culture vessel 12. The captured image (including a moving image) captured by the observation device 101 is transmitted to a display device (not shown) and displayed to an operator on the display device. In the embodiment, the multi-layer culture vessel 12 is held by a rotating unit 141a, and as shown in FIG. 10, a plurality of notches 1412 are formed on the bottom surface 1411 of the rotating unit 141a. The camera 1011 can capture an image of the inner bottom surface of the tray 121 in the multi-layer culture vessel 12 via the notch 1412. FIG. 10 is a perspective view of the internal configuration of the housing 11a of the culture system 1a according to the second embodiment, seen from below, showing the culture system 1a at an observation position for observing cells in the multilayer culture vessel 12 with the observation device 101.

In this embodiment, the camera 1011 is connected to the camera first drive unit 1012, and the camera 1011 can be moved linearly in a predetermined first direction. The camera second drive unit 1013 is connected to the camera first drive unit 1012, and the camera 1011 can be moved linearly together with the camera first drive unit 1012 in a second direction perpendicular to the first direction. That is, the camera 1011 can be freely moved in a two-dimensional direction by the camera first drive unit 1012 and the camera second drive unit 1013. Therefore, the operator can observe the culture state from the notch 1412 at the desired position. The position of the camera 1011 in the two-dimensional direction can be specified by the operator through the input unit 19. In addition, a lighting device (not shown) may be installed on the opposite side of the camera 1011 across the multi-layer culture vessel 12. In this case, by aligning the optical axis of the lighting device with the optical axis of the camera 1011, the illuminance of the multilayer culture vessel 12 can be increased and the state of the cultured cells can be imaged with appropriate brightness.

In addition, the culture system 1a according to the second embodiment has a first liquid level sensor 102 and a second liquid level sensor 103, as shown in Figs. 8 and 9. The first liquid level sensor 102 and the second liquid level sensor 103 are sensors for determining whether an appropriate amount (a predetermined amount required for culture) of fluid material, such as culture medium or a stripping liquid such as trypsin, has been introduced into the multi-layer culture vessel 12 when the fluid material is introduced into the multi-layer culture vessel 12. The first liquid level sensor 102 and the second liquid level sensor 103 have a camera such as a CCD or CMOS, and detect that an appropriate amount of the fluid material has been introduced into the multi-layer culture vessel 12 based on an image (or a moving image) captured by the camera, and output a signal to the control unit 18 to stop the introduction of the fluid material into the multi-layer culture vessel 12.

Specifically, the first liquid level sensor 102 and the second liquid level sensor 103 are fixed to the inner wall of the housing 11a and are installed so as to capture the liquid level of the fluid material introduced into the multi-layer culture vessel 12. When introducing the fluid material into the multi-layer culture vessel 12, the fluid material is introduced into the multi-layer culture vessel 12 with the multi-layer culture vessel 12 tilted at approximately 90 degrees by the rotating unit 141a. When the fluid material is introduced into the multi-layer culture vessel 12 in this manner, the second liquid level sensor 103 captures the vicinity of a second water level slightly below the first water level at which the fluid material is an appropriate amount in the multi-layer culture vessel 12. When the liquid level of the fluid material reaches the second water level, the second liquid level sensor 103 detects that the culture medium or the detachment liquid such as trypsin is close to the appropriate amount and transmits a signal to the control unit 18. When the control unit 18 receives a signal from the second liquid level sensor 103, it determines that the fluid material is close to the appropriate amount and controls the operation of the liquid supply pump 20 to reduce the inflow amount of the fluid material.

When the fluid material is being introduced into the multi-layer culture vessel 12, the first liquid level sensor 102 captures an image of the vicinity of the first water level at which the fluid material is at an appropriate amount. When the water level of the fluid material reaches the first water level at which the fluid material is at an appropriate amount, the first liquid level sensor 102 detects that the amount of fluid material is appropriate and transmits a signal to the control unit 18. When the control unit 18 receives a signal from the first liquid level sensor 102, it determines that the amount of fluid material has reached an appropriate amount, and controls the opening and closing operations of the valves 2a to 2d, the operation of the opening and closing unit 143, and the operation of the liquid supply pump 20 to stop the inflow of the fluid material.

Furthermore, in the second embodiment, the configuration of the rotating unit 141a is different from the configuration of the rotating unit 141 according to the first embodiment. That is, in the rotating unit 141 according to the first embodiment, as shown in FIG. 2, the rotation axis X2 that rotates the multilayer culture vessel 12 in the pitch direction P passes through the rotating unit 141, and the multilayer culture vessel 12 rotates in the pitch direction P around this rotation axis X2.

In contrast, in the second embodiment, as shown in Figs. 9 and 10, the rotating unit 141a includes a first driving unit 1421 that rotates the multilayer culture vessel 12 in the roll direction R and a second driving unit 1422 that rotates the multilayer culture vessel 12 in the pitch direction P, and the second driving unit 1422 is installed so that the rotation axis X2 of the second driving unit 1422 passes through the multilayer culture vessel 12. In other words, as shown in Figs. 9 and 10, the second driving unit 1422 is disposed on the side of the multilayer culture vessel 12, thereby allowing the multilayer culture vessel 12 to be rotated in the pitch direction P by the rotation axis X2 passing through the multilayer culture vessel 12. As a result, the second driving unit 1422 according to the second embodiment makes it possible to rotate the multilayer culture vessel 12 at a wider angle. That is, in the culture system 1 according to the first embodiment, the second drive unit 1422 can only rotate the multilayer culture vessel 12 in the pitch direction P around the rotation axis X2 within a range of ±30°, but in the culture system 1a according to the second embodiment, the second drive unit 1422 can rotate the multilayer culture vessel 12 in the pitch direction P (specifically, the clockwise pitch direction P in FIG. 9) around the rotation axis X2 by 90° or more, and as a result, it is also possible to rock the multilayer culture vessel 12 in a state in which the multilayer culture vessel 12 is rotated 90°. Note that, as in the first embodiment, the first drive unit 1421 rotates the multilayer culture vessel 12 in the roll direction R around the rotation axis X1 passing through the multilayer culture vessel 12.

As described above, the culture system 1a according to the second embodiment has an observation device 101 below the multi-layer culture vessel 12, which captures an image of the inside from the bottom surface of the tray 121 of the multi-layer culture vessel 12. This allows the operator to properly grasp the culture state of the cells in the multi-layer culture vessel 12 without removing the multi-layer culture vessel 12 from the housing 11a. In addition, in this embodiment, the bottom surface 1411 of the rotating part 141a has multiple notches 1412, and the observation device 101 can observe the state of the cells on the inside bottom surface of the tray 121 of the multi-layer culture vessel 12 through the notches 1412.

The culture system 1a according to the second embodiment also has a second liquid level sensor 103 that detects when the fluid material, such as the culture medium, is close to the appropriate amount, and a first liquid level sensor 102 that detects when the fluid material has reached the appropriate amount. As a result, in the culture system 1a according to the second embodiment, when the fluid material has been introduced to the appropriate amount in the multi-layer culture vessel 12, the introduction of the fluid material can be automatically stopped.

Furthermore, in the culture system 1a according to the second embodiment, the second drive unit 1422 is installed so that the rotation axis X2 of the second drive unit 1422 that rotates the multilayer culture vessel 12 in the pitch direction P passes through the multilayer culture vessel 12. In the first embodiment, the rotation axis X2 of the pitch direction P of the multilayer culture vessel 12 is separated from the multilayer culture vessel 12, so the space required for rotation is larger by that distance, and the size of the entire culture system 1 is also larger. However, in the second embodiment, the distance between the rotation axis X2 that rotates the multilayer culture vessel 12 in the pitch direction P and the multilayer culture vessel 12 is short, so the space required for rotation of the multilayer culture vessel 12 can be reduced, and the size of the entire culture system 1 can be reduced accordingly.

The above describes preferred embodiments of the present invention, but the technical scope of the present invention is not limited to the above description of the embodiments. Various modifications and improvements can be made to the above embodiments, and such modifications or improvements are also included in the technical scope of the present invention.

For example, in the above-described embodiment, the culture device 10 and the culture system 1 including the culture device 10 are exemplified as embodiments of the culture device and culture system according to the present invention, but as a multi-layer culture vessel operation device according to the present invention, it is also possible to provide a device having an operation unit 14 used in the culture device 10, as shown in FIG. 7.

In addition, in the above-described embodiment, the culture device 10 is illustrated as having a temperature adjustment unit 16, a gas concentration adjustment unit 171, and a pH adjustment unit 172, but is not limited to this configuration, and may be configured to have one or two of the temperature adjustment unit 16, the gas concentration adjustment unit 171, and the pH adjustment unit 172.

Furthermore, in the above-mentioned embodiment, as shown in FIG. 1, the valves 2a to 2c are installed on the individual liquid supply pipes (liquid supply pipes 3c to 3e) that are connected to the containers 40, 50, and 80, respectively, and the valves 2a to 2c are controlled to control the communication state between the individual liquid supply pipes (liquid supply pipes 3c to 3g) and the common liquid supply pipe (liquid supply pipes 3a, 3b, 13) to an open or closed state. However, this is not limited to the above configuration, and for example, a valve can be installed at the junction of the individual liquid supply pipes (liquid supply pipes 3c to 3g) and the common liquid supply pipe (liquid supply pipes 3a, 3b), and the communication state between each individual liquid supply pipe (liquid supply pipe 3c to 3g) and the common liquid supply pipe (liquid supply pipe 3a, 3b, 13) can be controlled by controlling the valve.

In the above-mentioned embodiment, a configuration using a camera sensor as the liquid level sensor 102, 103 is exemplified, but the present invention is not limited to this configuration, and an ultrasonic, capacitive, or pressure type liquid level sensor can also be used, among which a capacitive liquid level sensor is preferably used. In addition, when an ultrasonic or capacitive liquid level sensor is used, the liquid level sensor can be attached to a predetermined position (a position where the first water level and the second water level can be measured) of the rotating part 141, so that even if the multi-layer culture vessel 12 is replaced, it is possible to determine whether the fluid material has reached the first water level or the second water level in the multi-layer culture vessel 12. In addition, in the above-mentioned embodiment, for convenience of explanation, a configuration in which the liquid level sensors 102, 103 determine whether the culture medium or the detachment liquid such as trypsin has reached the first water level or the second water level is exemplified, but a configuration in which the culture medium and the detachment liquid such as trypsin are injected to different water levels can be used. In this case, for example, the liquid level of the culture medium can be monitored by the first liquid level sensor 102 and the second liquid level sensor 103, and the liquid level of the detachment liquid such as trypsin can be monitored by a third liquid level sensor and a fourth liquid level sensor different from the first liquid level sensor 102 and the second liquid level sensor 103. That is, the fourth liquid level sensor can be used to determine whether the detachment liquid such as trypsin has reached a fourth liquid level slightly below the third liquid level at which the detachment liquid such as trypsin is at an appropriate amount in the multi-layer culture vessel 12, and the third liquid level sensor can be used to determine whether the detachment liquid such as trypsin has reached the third liquid level. Furthermore, the multi-layer culture vessel 12 can be further configured to include a liquid level sensor for recovering fluid material from the multi-layer culture vessel 12 and determining whether the multi-layer culture vessel 12 is empty.

DESCRIPTION OF SYMBOLS 1, 1a... Cultivation system 10, 10a... Cultivation device 11, 11a... Housing 111... Upper storage section 112... Lower storage section 113... Partition plate 12... Multi-layer culture vessel 121... Tray 122, 123... Connection section 13... Liquid supply pipe 131, 132... Clamp 133... Air filter 14... Operation section 141, 141a... Rotation section
1411...Bottom
1412...notch 142...drive section
1421...First drive unit
1422: Second drive unit 143: Opening/closing unit 15: Insertion unit 16: Temperature adjustment unit 171: Gas concentration adjustment unit 172: pH adjustment unit 18: Control unit 19: Input unit 101: Observation device 1011: Camera 1012: Camera first drive unit 1013: Camera second drive unit 1014: Lens 102: First liquid level sensor 103: Second liquid level sensor 20: Liquid delivery pump 30: Flow meter 40: Container (for supplying and recovering culture liquid)
50: Container (for supplying a detachment solution such as trypsin)
60: Centrifugal separator 70: Liquid pump 80: Container (for recovering cells)
2a to 2d: Valves 3a to 3g: Liquid supply pipes

Claims (17)

A culture device having a housing that accommodates a multi-layer culture vessel having a plurality of trays in an internal space , and an operation unit that operates the multi-layer culture vessel while it is accommodated in the internal space;
A culture system having a pump for introducing a fluid material into the multi-layer culture vessel or discharging a fluid material from the multi-layer culture vessel,
The operation unit has a rotation unit that rotates or rocks the multilayer culture vessel in the housing,
The multi-layer culture vessel is connected to a fluid supply pipe, and a fluid material can be introduced into the multi-layer culture vessel from outside the housing via the fluid supply pipe, or a fluid can be discharged from within the multi-layer culture vessel to the outside of the housing via the fluid supply pipe. The culture system according to claim 1 , wherein the liquid supply pipe communicating with the multilayer culture vessel passes through a single insertion portion formed in the housing and is led out to the outside of the housing. The culture system according to claim 1 or 2, wherein the operation unit has an opening/closing unit that opens and closes the communication passage between the liquid supply pipe and the multi-layer culture vessel. An air filter is provided between the inside and the outside of the multilayer culture vessel,
The culture system according to claim 1 , wherein the operation unit has an opening/closing unit that opens and closes a communication passage between the air filter and the multi-layer culture vessel. The culture system according to any one of claims 1 to 4, wherein the housing has an insertion part through which the liquid supply tube penetrates the housing, and a fluid material is introduced from outside the housing into the multi-layer culture vessel via the insertion part and the liquid supply tube, or a fluid is discharged from inside the multi-layer culture vessel to outside the housing. The culture system according to any one of claims 1 to 5, wherein the liquid supply tube is switchably connected to a plurality of containers or devices present outside the housing. The culture system according to claim 6, wherein a plurality of individual liquid supply pipes each connected to a plurality of containers are connected to a common liquid supply pipe connected to the multi-layer culture container, a valve is installed on the individual liquid supply pipe or at a junction of the individual liquid supply pipe and the common liquid supply pipe, and the state of communication between the individual liquid supply pipe and the common liquid supply pipe can be controlled by controlling the valve. The culture system according to any one of claims 1 to 7, further comprising an observation device below the multi-layer culture vessel for imaging the inside of the tray of the multi-layer culture vessel. A housing that accommodates a multi-layer culture vessel having a plurality of trays in an internal space;
An operation unit that operates the multilayer culture vessel while it is housed in the internal space,
The operation unit has a rotation unit that rotates or rocks the multilayer culture vessel in the housing,
The multi-layer culture vessel is connected to a fluid supply pipe, and a culture device capable of introducing fluid material from outside the housing into the multi-layer culture vessel via the fluid supply pipe, or discharging fluid from within the multi-layer culture vessel to the outside of the housing via the fluid supply pipe. The culture device according to claim 9 , wherein the liquid supply pipe communicating with the multilayer culture vessel passes through a single insertion portion formed in the housing and is led out to the outside of the housing. The culture device according to claim 9 or 10, wherein the operation unit has an opening/closing unit that opens and closes the communication passage between the liquid supply pipe and the multi-layer culture vessel. A multi-layer culture vessel operation device having an operation unit for operating a multi-layer culture vessel having a plurality of trays built therein and housed in a housing,
The multilayer culture vessel is in communication with a liquid supply pipe within the housing,
The operation unit can operate the multi-layer culture vessel while the multi-layer culture vessel is housed in the housing,
The operation unit has a rotation unit that rotates or rocks the multilayer culture vessel in the housing,
A multi-layer culture vessel operating device that enables the introduction of fluid material from outside the housing into the multi-layer culture vessel via the fluid supply tube, or the discharge of fluid from within the multi-layer culture vessel to the outside of the housing via the fluid supply tube, by operating the multi-layer culture vessel using the operating unit. The multi-layer culture vessel manipulation device according to claim 12 , wherein the liquid supply pipe communicating with the multi-layer culture vessel passes through a single insertion portion formed in the housing and is led out to the outside of the housing. The rotating unit has a first rotating shaft and a second rotating shaft as rotating shafts for rotating or rocking the multilayer culture vessel,
The multi-layer culture vessel manipulation device according to claim 13 , wherein the rotating unit is configured so that the first rotation axis and the second rotation axis are rotation axes passing through the multi-layer culture vessel. The multi-layer culture vessel operating device according to any one of claims 12 to 14, wherein the operating unit has an opening/closing unit that opens and closes a communication passage between the liquid supply pipe and the multi-layer culture vessel. The multi-layer culture vessel operating device according to any one of claims 12 to 15, further comprising a liquid level sensor that detects the liquid level of the fluid material in the multi-layer culture vessel when the fluid material is introduced into the multi-layer culture vessel from outside the housing through the liquid delivery tube. The multi-layer culture vessel operating device according to claim 16, wherein the liquid level sensor includes a first liquid level sensor that detects whether the fluid material has reached a first water level indicating that a predetermined amount of the fluid material has been introduced into the multi-layer culture vessel, or a second liquid level sensor that detects whether the fluid material has reached a second water level that is lower than the first water level and indicates that the fluid material is approaching a predetermined amount.