Automated culture device and automated culture system

Abstract

To provide an automated culture device and an automated culture system that can reduce the size of the housing of the device and operate a plurality of devices simultaneously while saving space.SOLUTION: Provided are: an automated culture device capable of accommodating a culture vessel, the automated culture device comprising a temperature maintaining mechanism for maintaining the culture vessel at a predetermined temperature, a gas supply unit for supplying humidified CO2 gas, and a ventilation adapter attached to the culture vessel and for supplying humidified CO2 gas from the gas supply unit to the culture vessel; and an automated culture system comprising a centralized management computer for controlling a plurality of automated culture devices.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an automatic culture device for culturing cells or tissues, and more particularly to an automatic culture device that can be made small, and an automatic culture system using a plurality of such devices. [Background technology]

[0002] Medical treatments using regenerated human cells and cells whose functions have been modified by gene transfer have the potential to overcome diseases that have been difficult to cure until now, and so there are growing expectations for their widespread use.

[0003] The most common treatment target is cancer, followed by various organs. Autologous transplants using the patient's own cells have a low risk of rejection, and this is thought to be the reason for the high demand from the perspective of improving the patient's quality of life (QOL).

[0004] The most common cell type is immune cells, accounting for more than 40% of the total. The main target disease is cancer, and specific cell types include T cells, NK cells, and NKT cells. In particular, the practical application of CAR-T (chimeric antigen receptor-T) cell therapy, in which T cells, which are immune cells, are extracted from the patient, processed by gene transfer into a form capable of attacking cancer cells, and then returned to the patient by injection, is currently at the forefront of development.

[0005] In the process of manufacturing cells for transplantation, biological samples taken from the patient or others are separated and purified, and then processed by amplification, gene introduction, etc.

[0006] This process is carried out in a Cell Processing Center (CPC) in accordance with Standard Operating Procedures (SOP) that meet the Good Manufacturing Practice (GMP), the standard for manufacturing and quality control of pharmaceuticals, etc. Therefore, operating a CPC requires significant costs and personnel with specialized culture techniques.

[0007] In addition, since the manufacturing process is mainly manual, there is a limit to the increase in production volume. Low productivity and high manufacturing costs are impeding the spread of regenerative medicine, and there is a demand for automation of the culture process, which is particularly labor-intensive and costly. Automation of the culture process will enable labor savings, cost reduction, and mass production.

[0008] An example of an automatic culture device is a device that automatically handles a closed flow path having a closed space, as shown in Patent Document 1. In the closed flow path, a closed culture vessel is constantly connected by a flow path tube or the like, and cells are cultured inside the closed culture vessel.

[0009] The closed flow path allows the movement of liquids and gases contained therein by operating valves, pumps, etc. installed outside the closed flow path. This allows the automated culture device to automatically perform cell seeding, medium replacement, microscope observation, etc. while maintaining the closed nature of the culture space. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] JP 2007-312668 A Summary of the Invention [Problem to be solved by the invention]

[0011] In the technology described in Patent Document 1, culture is possible in a closed culture vessel, but in order to carry out culture, the culture vessel is held in a CO2 incubator in which a constant CO2 gas concentration is maintained inside. Gas exchange is carried out by dissolving gas in the space above the medium in the culture vessel into the medium, or by releasing gas in the medium into the space.

[0012] However, CO2 incubators are large in order to ensure airtightness in order to keep the internal space filled with gas, and therefore it is difficult to miniaturize an automatic culture device using this configuration.

[0013] In addition, because the inside of a CO2 incubator is humidified, it is an environment in which metal parts are prone to rust, making it difficult to implement mechanical elements such as a rocking mechanism for the culture vessel used in culture operations or a camera mechanism for observing cells.

[0014] In addition, CO2 incubators pose the risk of microbial growth due to humidification, making their use difficult in facilities for manufacturing cells for transplantation, which require high levels of cleanliness.

[0015] An object of the present invention is to provide an automatic culture device that can be made compact and save space, and that allows multiple devices to be operated simultaneously, and an automatic culture system that uses multiple such devices. [Means for solving the problem]

[0016] In order to achieve the above object, the present invention is configured as follows. The automatic culture device is capable of accommodating a culture vessel, and is characterized by comprising a temperature maintaining mechanism for maintaining the culture vessel at a predetermined temperature, a gas supply unit for supplying humidified CO2 gas, and a ventilation adapter that is attached to the culture vessel and supplies humidified CO2 gas from the gas supply unit to the culture vessel. Effect of the Invention

[0017] According to the present invention, it is possible to provide an automatic culture device that can be made compact and save space, and that can operate multiple devices simultaneously, and an automatic culture system using multiple such devices. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram showing one configuration of an automatic culture device according to a first embodiment. [Diagram 2] FIG. 2 is a side view showing one configuration of the automatic culture device according to the first embodiment. [Figure 3A] FIG. 2 is a diagram showing an example of a ventilation adaptor according to the first embodiment. [Figure 3B]FIG. 2 is a diagram showing an example of a ventilation adaptor according to the first embodiment. [Figure 3C] FIG. 2 is a diagram showing an example of a ventilation adaptor according to the first embodiment. [Figure 4] FIG. 2 is a top view of the ventilation adapter in the first embodiment. [Diagram 5] FIG. 1 shows the results of gas exchange in a culture vessel using a ventilation adapter in Example 1. [Figure 6A] FIG. 2 is a diagram showing an example of a flow path circuit including a closed culture vessel according to Example 1. [Figure 6B] FIG. 2 is a diagram showing an example of a procedure for cell seeding according to Example 1. [Figure 6C] FIG. 4 is a diagram showing an example of a gas exchange procedure according to the first embodiment. [Figure 6D] FIG. 2 is a diagram showing an example of a procedure for adding a medium according to Example 1. [Figure 6E] FIG. 2 is a diagram showing an example of a procedure for changing a medium according to Example 1. [Figure 6F] FIG. 2 is a diagram showing an example of a procedure for sampling a supernatant according to Example 1. [Figure 6G] FIG. 2 is a diagram showing an example of a cell recovery procedure according to Example 1. [Figure 7] FIG. 4 is a diagram showing the state of inclination of the culture vessel during cell recovery in Example 1. [Figure 8] FIG. 2 is a diagram showing a flow of operation of the automatic culture device according to the first embodiment. [Figure 9] FIG. 11 is a diagram showing an example of a ventilation adaptor for a flask culturing vessel according to Example 2. [Figure 10] FIG. 1 is a diagram showing an example of an automatic culture system in which a plurality of automatic culture devices according to Example 1 are connected in parallel and controlled by a central management PC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, an embodiment of the present invention will be described with reference to the drawings, etc. The following description shows a specific example of the contents of the present invention, and the present invention is not limited to these descriptions. Various changes and modifications are possible by those skilled in the art within the scope of the technical ideas disclosed in this specification.

[0020] Furthermore, in all the drawings for explaining the present invention, components having the same functions are given the same reference numerals, and repeated explanations thereof may be omitted. EXAMPLES

[0021] The components of the automatic culture device of this embodiment, which performs culture using a closed culture vessel, will be described with reference to FIG.

[0022] The automatic culture device 100 includes a culture vessel 1, a ventilation adapter 7, a gas supply unit 9, a pump for supplying liquid or gas, flow paths connecting these, valves for opening and closing the flow paths, a control unit 38 for controlling the gas supply unit, the pump, and the valves, a rocking mechanism 30 for rocking the culture vessel 1, and an incubator 35 as a temperature holding mechanism for accommodating the culture vessel, the ventilation adapter, the rocking mechanism, and the like and controlling the temperature. The culture vessel 1 is a culture vessel with a ventilation surface 4 having a gas-permeable membrane disposed on the bottom surface, and cells 2 are held and cultured together with a medium 3. A pressure adjustment tube 5 and a vent filter 6 are connected to the culture vessel 1, allowing gas to enter and exit the culture vessel while preventing the intrusion of bacteria and viruses from the outside.

[0023] In addition, the culture vessel 1 is attached to a ventilation adaptor 7, so that gas supply and exchange required for culture can be performed for the culture vessel 1. The gas supply unit 9 is composed of a gas cylinder 10 that holds a predetermined gas concentration, a gas flow control unit (mass flow controller: MF) 11, a pressure sensor 12, and a humidification bottle 13 that is a humidification unit, and is connected upstream of the ventilation adaptor 7. A CO2 sensor 14 and a CO2 gas vent filter 15 are connected downstream of the ventilation adaptor 7, and the gas is discharged to the atmosphere outside the device. The CO2 sensor 14 can be used to monitor whether gas exchange in the ventilation adaptor is being performed appropriately, and can also be used to predict the culture state.

[0024] The liquid or gas is delivered by pumps 16 and 17, and a suitable pump is a tube pump that generates pressure by squeezing a rubber tube with a rotating roller. The pump 16 is configured to deliver the culture medium to the culture vessel 1, and is connected to a liquid delivery pipe 18 of the culture vessel 1 at one end and a supply pipe of a culture medium bottle 20 via an electromagnetic valve 19 at the other end.

[0025] An electromagnetic valve is suitable as a valve for opening and closing the rubber tube that constitutes the flow path. When an electric current is applied to the closed state of the rubber tube sandwiched between the valve actuated by a spring force, the electromagnetic valve operates to control the rubber tube to be open.

[0026] The pump 17 is configured to deliver a cell suspension to the culture vessel 1 for cell seeding, to discharge the medium 3 from the culture vessel 1, and to recover the proliferated cells.

[0027] The pump 17 is connected to a suction tube 21 of the culture vessel 1 through a tube, and the other end is connected to a cell seeding bottle 22 through an electromagnetic valve 19 and to a supernatant recovery bag 23 and a supernatant analysis bag 24 through another electromagnetic valve 19 .

[0028] The pump 17 is also connected to a cell recovery pipe 25 of the culture vessel 1 through a tube, and the other end is connected to a cell recovery bottle 26 via an electromagnetic valve 19 .

[0029] The weight of the culture medium bottle 20 is measured by a weight sensor 27, and the weights of the cell seeding bottle 22 and the cell recovery bottle 26 are measured by a weight sensor 28.

[0030] As shown in Figures 1 and 2, the medium bottle, cell seeding bottle, cell collection bottle, supernatant collection bag, supernatant analysis bag, flow paths connecting each component, pumps, solenoid valves, and weight sensors are provided in the fluid control unit 29, which is the main body of the device outside the incubator 35.

[0031] The oscillating mechanism 30 consists of an oscillating stage 31 that holds the ventilation adapter 7, a link mechanism 32 that supports the oscillating stage from three directions, oscillating shafts 33 connected to the link mechanisms 32, and an oscillating stage fixing mechanism 34 that is fixed inside the incubator.

[0032] The rocking operation of the culture vessel can be performed in the left-right direction on the paper by lowering the right rocking axis downward and simultaneously raising the left rocking axis upward, without moving the central rocking axis. If this is controlled, the rocking stage will move with an inclination and the culture vessel can be tilted. If the movement of the left and right axes is then reversed, the culture vessel can be tilted in the opposite direction.

[0033] By operating these continuously and moving the oscillation axis in the depth direction of the paper, the culture vessel can be tilted back and forth, so that the cells 2 and medium 3 inside the culture vessel 1 can be agitated.

[0034] The incubator 35 is an example of a temperature maintaining mechanism, and is a so-called dry incubator consisting of a thermostatic unit 36 ​​and an opening / closing door 37. The incubator 35 can accommodate the culture vessel 1, the rocking mechanism 30, and the humidifying bottle 13, and can maintain the temperature inside the incubator at a suitable temperature for cell culture. By adopting a small dry incubator instead of a large CO2 incubator and having a configuration that can supply gas required for culture, the device can be made smaller, and multiple devices can be operated simultaneously in a small space. The control unit 38 can control the operation of the gas supply unit 9, pumps 16, 17, and solenoid valve 19, and the operation of the rocking mechanism 30. By automatically controlling these mechanized elements at predetermined timing, during cell seeding, a cell suspension is sent from the cell seeding bottle 22 to the culture vessel 1, during gas exchange, humidified gas from the humidification bottle 13 is supplied to the ventilation adapter 7, during culture medium addition, culture medium is sent from the culture medium bottle 20 to the culture vessel 1, during culture medium replacement, the culture medium 3 in the culture vessel 1 is discharged into the supernatant collection bag 23 and then supplied to the culture vessel 1, and during supernatant sampling, a portion of the culture medium in the culture vessel can be sent to the supernatant analysis bag 24.

[0035] When recovering cells, the medium in the culture vessel 1 is discharged into the supernatant recovery bag 23, and then the rocking mechanism 30 is operated to stir the cell suspension, which can then be sent to the cell recovery bottle 26.

[0036] 2 is a right side view of the device described in Example 1. Side covers 40 are provided on the left and right sides of the device, and a bottle unit door 41 that can be opened and closed in the horizontal and vertical directions is provided on the front of the device.

[0037] The side cover 40 shields the movement of gas in the entire space in which the culture medium bottle 20, cell seeding bottle 22 and cell recovery bottle 26 are placed on the weight sensors 27, 28 and weighed, and acts as a windshield to ensure stable weighing.

[0038] The bottle unit door 41 is in an open state when in a horizontal position, and has the effect of serving as a stand on which bottles and tubes can be temporarily placed when installing a flow path consisting of bottles and tubes. After the flow path tubes are connected to pumps and valves, it can be closed vertically and used as a windshield. The bottle unit door 41 and the opening / closing door 37 of the incubator 35 described above open and close in different directions, so that the two doors can be opened and closed without interfering with each other, making it easy to access the device during work.

[0039] The components of the ventilation adapter 7 according to Example 1 will be described with reference to Figures 3A, 3B, and 3C. In Figure 3A, the ventilation adapter 7 is placed on a rocking stage 31, and is connected to an air supply pipe 42 connected to the above-mentioned humidification bottle 13, and an exhaust pipe 43 connected to a CO2 sensor. The culture vessel 1 is tightly attached to the ventilation adapter 7 on a horizontal stand (not shown), by being pressed against the rubber packing 44 mainly due to the weight of the culture vessel 1 and the elasticity of the rubber packing 44. At this time, a predetermined gas space 8 is formed below the culture vessel 1 and inside the ventilation adapter 7.

[0040] 3A shows an example of a ventilation adapter in which CO2 gas is supplied from the outer circumferential side of the ventilation adapter 7 and exhausted from the outer circumferential side. This type of ventilation adapter has the advantage that it can be made small in height.

[0041] We will now explain the contact state between the ventilation adapter 7 and the rubber packing 44. Humidified CO2 gas is held in the gas space 8 of the ventilation adapter 7, and in order to reduce leakage of this humidified CO2 gas from the gap between the ventilation adapter 7 and the rubber packing 44, a highly airtight structure is desirable.

[0042] By continuously supplying humidified CO2 gas to the internal space of the ventilation adapter 7, the internal space of the ventilation adapter 7 is maintained at a pressure slightly higher than atmospheric pressure. In other words, the internal space of the ventilation adapter 7 is at a positive pressure compared to atmospheric pressure. This reduces the risk of bacteria in the air entering the internal space of the ventilation adapter 7.

[0043] In addition, the volume of the internal space of the ventilation adaptor 7 is preferably as small as possible, provided that the volume is large enough to supply humidified CO2 gas uniformly to the gas permeable membrane of the culture vessel. If the volume of the internal space is large, it takes a long time to fill the internal space with humidified CO2 gas.

[0044] On the other hand, if the internal space is too small, there is a greater concern that the humidified CO2 gas may not be supplied uniformly. Therefore, it is preferable that the internal space is as thin as possible vertically and has an area in the horizontal direction that is equal to or slightly larger than the area of ​​the gas permeable membrane of the culture vessel. The space shape is circular if the vessel is cylindrical, and square if the vessel is square.

[0045] FIG. 3B shows a ventilation adapter that supplies CO2 gas from the bottom and exhausts it from the bottom. FIG. 3B shows the state before the installation of the culture vessel 1, rubber packing 44, and ventilation adapter 7, and FIG. 3C shows the state during use. This type of ventilation adapter has the feature that the internal space of the ventilation adapter 7 can be made smaller than that of the type shown in FIG. 3A. Also, in FIG. 3B, the cross-sectional shape of the rubber packing 44 is shaped along the outer periphery of the culture vessel in order to increase the airtightness of the rubber packing 44 and the culture vessel. Even if the inner circumference of the shape is smaller than the outer periphery of the culture vessel, the rubber packing 44 is closely attached to the ventilation adapter 7 by utilizing the elasticity of the rubber packing. The shape of the ventilation adapter can be appropriately selected according to the required specifications of the culture device (size of the culture vessel, culture speed, etc.). In addition to the shapes shown in FIGS. 3A and 3B, it is also possible to supply CO2 gas from the outer periphery and provide an exhaust pipe on the bottom.

[0046] 4 is a top view of the ventilation adaptor 7 in the shape of FIG. 3A. As described above, the internal space of the ventilation adaptor 7 is a circle slightly larger than the circular gas permeable membrane provided in the culture vessel when viewed from above. In this figure, there is one air supply tube 42 for humidified CO2 gas, but the air supply tube may be divided into multiple tubes so that the humidified CO2 gas can be supplied to the internal space as uniformly as possible, or the opening may be one large opening. In addition, the air supply tube 42 is not limited to being connected to the side surface of the ventilation adaptor 7, but may be connected from the bottom surface.

[0047] FIG. 5 shows the results of comparing the time change in the CO2 gas concentration in the gas phase inside the culture vessel in Example 1, using two measurement methods: a ventilation method using the ventilation adapter 7 and a ventilation method using a 5% CO2 incubator.

[0048] To measure the ventilation method using the ventilation adapter 7, a CO2 sensor (VISSLA GM70 handheld CO2 meter) (not shown) was installed inside the closed culture vessel 1, and the vent filter 6 was temporarily closed to create a vessel where gas passes only through the ventilation surface (gas permeable membrane) 4. The volume of the gas space 8 was set to 300 cc, and a 5% CO2-air mixed gas was continuously supplied from the air supply pipe 42 at a flow rate of 50 cc / min.

[0049] To measure the ventilation method using a CO2 incubator, a CO2 sensor was placed inside the incubator, which was constantly maintained at 5% CO2-air, and at the same time, a separate CO2 sensor was placed inside the closed system culture vessel 1, creating a vessel in which gas only passes through the ventilation surface (gas permeable membrane) 4. The measurements were taken over a 15-hour period when each was kept at 37°C.

[0050] As a result, the CO2 concentration reached 5% in 800 minutes using both the CO2 incubator ventilation method and the ventilation adapter ventilation method.

[0051] Ventilation using the ventilation adapter can achieve ventilation equivalent to that using a CO2 incubator by adjusting the amount of gas sent, taking into account that it takes time for the gas concentration in the gas space 8 to increase.

[0052] Fig. 6A is a diagram showing an example of a flow path circuit 45 including a closed culture vessel according to Example 1. The same elements as those in the embodiment shown in Fig. 1 are given the same numbers, and for the solenoid valve 19, numbers 46 to 54 are given to unique solenoid valves-1 to -9, respectively.

[0053] The rubber tube connected to the supernatant collection bag 23 is equipped with a manual valve 55 that can manually open and close the tube, and the rubber tube connected to the supernatant analysis bag 24 is equipped with a manual valve 56. Reference numeral 59 denotes a joint part, which is a part that allows the connection and disconnection of pipes by means of a male-female joint. Reference numeral 60 denotes a branching part, which is the branching point of tubes connected using a T-shaped connector.

[0054] The flow path circuit 45 shown here is detachable as a whole from the device body shown in Example 1, except for the solenoid valve 19 in the pumps 16 and 17, which are mechanical elements, the weight sensor (electronic balance) 28, the gas flow control unit 11, and the pressure sensor 12, and can be applied to cell culture by sterilizing only the flow path circuit.

[0055] Fig. 8 is a diagram showing a flowchart of the overall operation of cell culture in the cell culture device 100 shown in Fig. 6A. After "START", a flow path is installed in the cell culture device 100 (S01), and then a cell seeding bottle 22 that holds a separately prepared cell suspension, a culture medium bottle 20 that holds a culture medium, and a cell recovery bottle 26 are connected to the flow path (S02).

[0056] Fig. 6B shows the cell seeding process according to Example 1. In the initial state, the pump is stopped and the rollers are stopped by pinching the rubber tube, so the pumps 16 and 17 are in a closed state as valves. The solenoid valve is in a closed state by pinching the rubber tube. A predetermined amount of cell suspension is held in the cell seeding bottle 22 and placed on the weight sensor 28, and the culture vessel 1 is placed horizontally on the ventilation adapter 7 with the inside empty. When cell seeding begins, the solenoid valves 47, 48, 54 and 51 are opened, and the suction tube 21 of the culture vessel 1 and the pipeline to the CO2 gas vent filter 15 are opened.

[0057] Next, the pump 17 is operated to supply gas from one side of the pipeline of the cell seeding bottle 22 to pressurize the cell suspension inside, and the cell suspension passes through the pipeline and is sent to the culture vessel 1 through the suction tube 21 (S03). The flow of the liquid at this time is shown by a solid line, and the flow of the gas is shown by a dashed line (same below).

[0058] Next, when a predetermined amount of the cell suspension has been transferred, solenoid valve 47 is closed and solenoid valve 49 is opened, and at the same time pump 17 is stopped. At this time, the cell suspension is temporarily stopped in the pipeline by closing solenoid valve 47, and the gas that had been pressurizing cell seeding bottle 22 is discharged from branching part 61 to the vent filter that is open to the outside, and the movement of the liquid stops.

[0059] Next, by closing solenoid valves 48 and 49 and opening solenoid valve 46 and operating pump 17, the cell suspension in the pipeline closer to the culture vessel than branch 62 is sent to culture vessel 1. After that, pump 17 is stopped and solenoid valves 47, 48 and 49 are opened, solenoid valves 48 and 49 return the cell suspension in the pipeline to cell seeding bottle 22 due to the difference in height, and the liquid in the pipeline disappears, all solenoid valves are closed, and the cell seeding process is completed (S03).

[0060] 6C shows the process of gas exchange into the culture vessel 1 in Example 1. In the initial state, the solenoid valve is in a closed state with the rubber tube closed. Water is held in the humidification bottle 13, and the opening of one long tube is provided at the bottom of the vessel. A gas flow control unit 11 and a gas cylinder 10 are connected to one end of this.

[0061] The opening of the short tube in the humidifying bottle 13 is provided at the top inside the container, and one end of this is connected to the ventilation adapter. When the gas flow control unit 11 is operated, gas controlled at a predetermined air supply rate is supplied to the inside of the humidifying bottle, humidified, and then supplied from the humidifying bottle. Next, the gas concentration in the gas space 8 of the ventilation adapter 7 increases and is maintained at a predetermined gas concentration, so that gas exchange to the culture container 1 continues (S04).

[0062] 6D shows the process of adding culture medium to culture vessel 1 in Example 1. In the initial state, the pump is stopped and the solenoid valve is in a closed state with the rubber tube closed. Culture medium is held in culture medium bottle 20, which is attached to weight sensor 27. First, solenoid valve 53 is opened, and the liquid supply pipe 18 of culture vessel 1 and the piping to culture medium bottle 20 are opened. Next, when pump 16 is operated, the culture medium passes through the piping and is sent to culture vessel 1 through liquid supply pipe 18.

[0063] Next, when a predetermined amount of culture medium has been transferred, the solenoid valve 51 is opened and at the same time the pump 16 is stopped. At this time, the flow of culture medium is temporarily stopped in the pipeline by stopping the pump 16, and the culture medium in the pipeline close to the culture medium bottle 20 returns to the inside of the culture medium bottle 20 due to the difference in head as outside air enters through the vent filter opened to the outside from the branching part 63.

[0064] Next, by closing the solenoid valve 53 and operating the pump 16, the medium in the pipeline closer to the culture vessel than the branching point 63 is sent to the culture vessel 1. After that, by stopping the pump 16, the liquid in the pipeline can be removed, all the solenoid valves are closed, and the culture medium adding process is completed (S05).

[0065] 6E shows the process of discharging the culture medium during culture medium replacement in the culture vessel 1 in Example 1. In the initial state, the pump is stopped and the solenoid valve is in a closed state with the rubber tube closed. Culture medium is held in the culture vessel 1, and the supernatant collection bag 23 is installed empty. First, solenoid valve 46, solenoid valve 52, and manual valve 55 are opened, and the piping from the suction tube 21 of the culture vessel 1 to the supernatant collection bag 23 is opened. Next, when pump 17 is operated, the culture medium is sent from the culture vessel 1 through the suction tube 21 and reaches the supernatant collection bag 23.

[0066] Next, when a predetermined amount of culture medium has been transferred, the solenoid valve 51 is opened and at the same time the pump 16 is stopped. At this time, the flow of culture medium is temporarily stopped in the pipeline by stopping the pump 16, and the culture medium in the pipeline close to the culture medium bottle 20 returns to the inside of the culture medium bottle 20 due to the difference in height as outside air enters through the vent filter opened to the outside from the branching part 62.

[0067] Next, when solenoid valve 49 is opened, the medium in the pipeline closer to culture vessel 1 than branching point 64 is sent to culture vessel 1 by the difference in height. After that, solenoid valve 46 is closed and pump 17 is operated, outside air is introduced through the vent filter and the medium in the pipeline reaches supernatant collection bag 23. All solenoid valves are closed and the medium discharge process is completed (S06). Next, medium replacement can be performed by similarly performing the process of adding medium to culture vessel 1 described using Figure 6D (S07). After medium replacement, cell culture can be further promoted by rocking culture vessel 1 with rocking mechanism 30 to mix new medium with the cells.

[0068] 6F shows the process of sampling the supernatant from the culture vessel 1 in Example 1. In the initial state, the pump is stopped and the solenoid valve is in a closed state with the rubber tube closed. The culture medium is held in the culture vessel 1, and the supernatant analysis bag 24 is installed empty. First, the solenoid valve 46, the solenoid valve 52, and the manual valve 56 are opened, and the piping from the suction tube 21 of the culture vessel 1 to the supernatant collection bag 23 is opened. Next, when the pump 17 is operated, the culture medium is sent from the culture vessel 1 through the suction tube 21 and reaches the supernatant collection bag 23.

[0069] Next, when a predetermined amount of culture medium has been transferred, the solenoid valve 51 is opened and at the same time the pump 16 is stopped. At this time, the flow of culture medium is temporarily stopped in the pipeline by stopping the pump 16, and the culture medium in the pipeline close to the culture medium bottle 20 returns to the inside of the culture medium bottle 20 due to the difference in height as outside air enters through the vent filter opened to the outside from the branching part 62.

[0070] Next, when the solenoid valve 49 is opened, the culture medium in the pipeline closer to the culture vessel 1 than the branching point 64 is sent to the culture vessel 1 by the difference in height. After that, when the solenoid valve 46 is closed and the pump 17 is operated, outside air is introduced through the vent filter and the culture medium in the pipeline reaches the supernatant analysis bag 24. All the solenoid valves are closed and the supernatant sampling process is completed (S08).

[0071] The cell recovery process from the culture vessel 1 in Example 1 will be described with reference to Figures 6E and 6G. After the cells have sufficiently proliferated, they sink to the bottom of the culture vessel and float there. As in the supernatant recovery process shown in Figure 6E, the supernatant is recovered from the culture vessel 1 through the same flow path circuit, and the supernatant recovery is continued until the culture medium 3 is below the height of the opening of the suction tube 21. This makes it possible to increase the ratio of the culture medium 3 and the cells present in the culture vessel 1. Next, the culture vessel is rocked by the rocking mechanism 30, and the culture medium and cells are stirred to form a cell suspension.

[0072] Next, the cell recovery process from the culture vessel 1 will be described with reference to Figures 6G and 6A. In the initial state, the pump is stopped and the solenoid valve is in a closed state by closing the rubber tube. The culture vessel 1 holds a cell suspension 65, and the cell recovery bottle 26 is installed empty.

[0073] The inclination of the culture vessel during cell recovery will be described with reference to FIG. 7. First, the rocking unit 70 of the rocking mechanism 30 is tilted toward the direction in which the recovery pipe 25 exists, so that the cell suspension is collected on the wall side of the culture vessel 1 where the recovery pipe 25 exists. The inclination angle of the rocking unit 70 may be greater than 0 degrees and less than 90 degrees as long as the cell suspension can be collected on the wall side of the culture vessel 1. The rocking unit 70 must be capable of tilting in a certain direction, and may be capable of tilting in multiple directions. When the culture vessel 1 is placed on the rocking mechanism, the culture vessel 1 is placed in a direction in which it can be tilted toward the direction in which the opening of the recovery pipe 25 exists. At that time, a mark specifying the installation direction of the culture vessel 1 may be written on the ventilation adapter 7 fixed to the rocking unit 70.

[0074] Next, solenoid valves 50 and 51 are opened, and the conduits from recovery pipe 25 of culture vessel 1 to cell recovery bottle 26 and to CO2 gas vent filter 15 open to the outside air are opened. Next, when pump 17 is operated, cell suspension 65 is pumped from culture vessel 1 through recovery pipe 25 and reaches cell recovery bottle 26.

[0075] Next, when the entire amount of cell suspension 65 has been transferred to cell collection bottle 26, solenoid valve 49 is opened and pump 17 is stopped at the same time. At this time, outside air enters through the vent filter that is open to the outside, and the inside of cell collection bottle 26 becomes normal pressure through branch 66, and the liquid transfer stops. All solenoid valves are closed and the cell collection process ends (S09).

[0076] After the cells are collected, the waste liquid bag is removed from the flow path (S10), and the flow path is removed from the automatic culture device (S11), completing all the steps of the automatic culture.

[0077] As described above, the automatic culture device according to the present embodiment can perform culture while miniaturizing the device. In addition, it is possible to suppress the risk of rust on mechanical elements such as the rocking mechanism of the culture vessel used in the culture operation and the camera observation mechanism of the cells, and to implement these. In addition, the risk of microbial generation throughout the entire incubator can be suppressed by partially supplying humidified gas to the culture vessel by the ventilation adapter. EXAMPLES

[0078] FIG. 9 is a diagram showing an example of a ventilation adaptor for a flask culturing vessel according to the second embodiment.

[0079] The components of the ventilation adaptor 7 according to Example 2 will be described with reference to Fig. 9. The culture vessel 1 is a flask-type culture vessel, and is mainly used for cell culture with cells adhering to the bottom surface of the vessel. Two screw ports 101 are usually provided on the ceiling surface of the main body, and each is closed by a ventilation cap 103 with a gas-permeable membrane 102 provided at the opening.

[0080] In this embodiment, a method is shown in which the ventilation cap of one screw port is left as it is and gas exchange is performed using a ventilation adapter 7, and a ported cap 104 with a liquid supply tube is installed on the other screw port, and automatic liquid supply is performed using an automatic culture device. More specifically, the culture vessel 1 is installed on a rocking stage 31, and connected to an air supply tube 42 connected to the humidification bottle 13 and an exhaust tube 43 connected to a CO2 sensor. One ventilation cap 103 in the culture vessel 1 is closely fitted with a ventilation adapter 7. At this time, a predetermined gas space 8 is created inside the ventilation cap 103 and the ventilation adapter 7.

[0081] The ported cap 104 is provided with a liquid delivery tube 18, a suction tube 21, and a cell recovery tube 25, and these three ports are connected in the same manner as the automatic culture device (Figure 1) and flow path circuit (Figure 6A) described in Example 1 above, and the above-mentioned cell seeding process, gas exchange process, medium addition process, medium exchange process, supernatant sampling process, and cell recovery process can be performed. EXAMPLES

[0082] FIG. 10 is a diagram showing an example of an automatic culture system in which a plurality of automatic culture devices according to the first embodiment are installed, connected to a network, and controlled by a central management PC.

[0083] Four sets of automatic culture devices, each consisting of an incubator 35 and a fluid control unit 29, are installed on a stand 105. In this embodiment, a stand on which four sets of devices are installed, two sets on each of the upper and lower stages, is shown, but the number of devices installed per stage of the stand in this embodiment can be arbitrary, making it easy to increase the number of devices integrated in the limited CPC facility space. From the viewpoint of accurate process management and aseptic operation, it is also possible to fully automate the entire process of installing and removing the flow channels by a robot, and the number of devices integrated can be manufactured beyond the reach of human hands.

[0084] Each control unit 38 is attached to the front of the door 37 of the incubator 35, and each is connected to the central control PC 106 via a network device. The central control PC 106 has a function of monitoring the state of each of the automatic culture devices, a function of managing updates and modifications of the operation programs, and a function of transmitting information when an abnormality occurs in the operation of the device or when the operation is terminated.

[0085] By adopting the configuration described in this embodiment, it is possible to provide an automatic culture device that saves space and allows multiple devices to be operated simultaneously, and an automatic culture system that uses multiple such devices. [Explanation of symbols]

[0086] 1 culture vessel, 2 cells, 3 culture medium, 4 ventilation surface, 5 pressure adjustment tube, 6 vent filter, 7 ventilation adapter, 8 gas space, 9 gas supply unit, 10 gas cylinder, 11 flow control unit, 12 pressure sensor, 13 humidification bottle, 14 CO2 sensor, 15 CO2 gas vent filter, 16, 17 pump, 18 liquid delivery tube, 19 solenoid valve, 20 culture medium bottle, 21 suction tube, 22 cell seeding bottle, 23 supernatant collection bag, 24 supernatant analysis bag, 25 cell collection tube, 26 cell collection bottle, 28 weight sensor, 29 fluid control unit, 30 rocking mechanism

Claims

1. An automatic culture device capable of accommodating a culture vessel for accommodating a culture subject, A temperature maintaining mechanism for maintaining the culture vessel at a predetermined temperature; a gas supply unit for supplying CO2 gas necessary for cultivation; A ventilation adapter is attached to the culture vessel and supplies and exchanges gases necessary for culture to the culture vessel, The culture vessel has a gas permeable portion at least a part of which is made of a material having gas permeability, The ventilation adapter is connected to an air supply pipe connected to the gas supply unit and an exhaust pipe that releases gas from the culture vessel into the atmosphere, and is attached opposite the gas permeable unit of the culture vessel to exchange humidified CO2 gas inside the culture vessel, characterized in that the automatic culture device.

2. The automatic culture apparatus according to claim 1, The automatic culture device is characterized in that the ventilation adapter is constructed separately from the culture vessel and is detachable from the culture vessel.

3. The automatic culture apparatus according to claim 1, The automatic culture device is characterized in that the ventilation adaptor is structured so that the culture vessel is placed above the ventilation adaptor and is attached to the ventilation adaptor by pushing down the culture vessel.

4. The automatic culture apparatus according to claim 1, The automatic culture device is characterized in that the ventilation adapter is attached by being pushed down toward the culture vessel from above the culture vessel.

5. The automatic culture apparatus according to claim 1, An automatic culture device, characterized in that a predetermined gas space is formed below the culture vessel and inside the ventilation adapter.

6. In the automatic culture apparatus according to claim 1, An automatic culture apparatus, characterized in that the air supply pipe and the exhaust pipe are connected to a side wall surface of the ventilation adapter.

7. In the automatic culture apparatus according to claim 1, The automatic culture apparatus is characterized in that the air supply pipe and the exhaust pipe are connected to a bottom surface of the ventilation adapter.

8. The automatic culture apparatus according to claim 1, The temperature maintenance mechanism comprises at least the ventilation adapter and a housing that accommodates the culture container, and is characterized in that it is equipped with a temperature control mechanism that maintains the temperature inside the housing at the predetermined temperature.

9. The automatic culture apparatus according to claim 8, The automatic culture device is characterized in that the gas released from the exhaust pipe of the ventilation adapter is exhausted to the outside of the housing.

10. The automatic culture apparatus according to claim 9, An automatic culture apparatus comprising: a gas sensor disposed in a flow path of the exhaust pipe for measuring the amount of gas discharged from the ventilation adapter.

11. The automatic culture apparatus according to claim 8, An automatic culture device characterized in that a rocking mechanism for rocking the culture vessel is provided inside the casing.

12. The automatic culture apparatus according to claim 8, The automatic culture apparatus is characterized in that the housing is provided with a humidification unit as part of the gas supply unit for humidifying the supplied gas.

13. The automatic culture apparatus according to claim 8, The automatic culture device is characterized by being provided with a fluid control unit having, outside the housing of the temperature maintenance mechanism, a first container capable of containing a supply medium, etc., a second container capable of containing waste liquid, etc., and a pump that enables fluid manipulation.

14. A plurality of the automatic culture devices according to any one of claims 1 to 13 are provided, An automatic culture system comprising a centralized management computer for controlling the plurality of automatic culture devices.

15. The automated culture system according to claim 14, An automatic culture system characterized in that a plurality of the automatic culture devices are arranged in parallel vertically or horizontally.

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