Automated culture apparatus, 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 cell or tissue culture device, and particularly to an automatic cell culture device that can be miniaturized and an automatic cell culture system using a plurality of such devices.

Background Art

[0002] Medical treatment using regenerated human cells and cells with modified functions by gene transfer has raised expectations for its spread because it can overcome diseases that have been difficult to cure so far.

[0003] Cancer is the most common treatment target, followed by various organs. Autologous transplantation using the patient's own cells has a low possibility of rejection reaction, and it is considered that this is because there is a high need from the perspective of improving the patient's QOL (quality of life).

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

[0005] In the manufacturing process of cells for transplantation, biological samples collected from the patient himself or others are separated and purified, and processed such as amplification and gene transfer.

[0006] This process is carried out in a cell processing facility (CPC: Cell Processing Center) according to a standard operating procedure (SOP: Standard Operating Procedure) that meets the appropriate manufacturing standards (GMP: Good Manufacturing Practice), which are the manufacturing management and quality control standards for pharmaceuticals and the like. Therefore, the operation of CPC requires a great deal of cost and personnel with specialized culture techniques.

[0007] Furthermore, since the manufacturing process is primarily manual, there are limits to increasing production volume. Low productivity and high manufacturing costs are hindering the widespread adoption of regenerative medicine, and automation of the culture process, which requires particular effort and cost, is needed. Automating the culture process would enable labor savings, cost reduction, and mass production.

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

[0009] A closed-loop system allows for the movement of liquids and gases held within it through the operation of valves, pumps, etc., installed on its exterior. This enables automated culture devices to automatically perform cell seeding, culture medium changes, microscopic observation, etc., while maintaining the closed nature of the culture space. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2007-312668 [Overview of the project] [Problems that the invention aims to solve]

[0011] In the technology described in Patent Document 1, cultivation is possible in a closed culture vessel, but the culture vessel is held inside a CO2 incubator that maintains a constant CO2 gas concentration inside in order to perform the cultivation. Gas exchange occurs when gas in the space above the culture medium inside the culture vessel dissolves into the medium, or when gas in the medium is released into the space.

[0012] However, CO2 incubators are large due to the need to ensure airtightness to keep the internal space filled with gas, making it difficult to miniaturize automated culture systems using this configuration.

[0013] Furthermore, because CO2 incubators humidify the interior, metal parts are prone to rusting, making it difficult to implement mechanical elements such as the rocking mechanism of the culture vessel used in culture operations and the camera mechanism for observing cells.

[0014] In addition, CO2 incubators pose a risk of microbial growth due to humidification, posing operational hurdles to their use in facilities that manufacture cells for transplantation, where high levels of cleanliness are required.

[0015] The object of the present invention is to provide an automated culture apparatus that can be miniaturized and operate multiple apparatuses simultaneously in a space-saving manner, and an automated culture system using multiple such apparatuses. [Means for solving the problem]

[0016] The configuration of the present invention for achieving the above objective is as follows. An automated culture apparatus capable of housing a culture vessel for a subject to be cultured, comprising: a temperature holding mechanism for maintaining the culture vessel at a predetermined temperature; a gas supply unit for supplying CO2 gas necessary for culture; and a ventilation adapter attached to the culture vessel for supplying and exchanging gas necessary for culture to the culture vessel, wherein the culture vessel is On the bottom surface of the screw opening provided on the ceiling The ventilation adapter has a gas permeable section made of a gas-permeable material, and is connected to an air supply pipe that connects to the gas supply section and an exhaust pipe that releases gas from the culture vessel to the atmosphere, and is mounted opposite the gas permeable section of the culture vessel, and is characterized by exchanging the humidified CO2 gas inside the culture vessel. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide an automated culture apparatus that can be miniaturized and operate multiple apparatuses simultaneously in a space-saving manner, as well as an automated culture system using multiple such apparatuses. [Brief explanation of the drawing]

[0018] [Figure 1]It is a diagram showing one configuration of an automatic culture device according to Example 1. [Figure 2] It is a diagram showing one configuration of an automatic culture device according to Example 1 from the side. [Figure 3A] It is a diagram showing an example of a ventilation adapter according to Example 1. [Figure 3B] It is a diagram showing an example of a ventilation adapter according to Example 1. [Figure 3C] It is a diagram showing an example of a ventilation adapter according to Example 1. [Figure 4] It is a top view of the ventilation adapter in Example 1. [Figure 5] It is a diagram showing the gas exchange result in the culture vessel by the ventilation adapter in Example 1. [Figure 6A] It is a diagram showing an example of a flow path circuit including a closed culture vessel according to Example 1. [Figure 6B] It is a diagram showing an example of a cell seeding procedure according to Example 1. [Figure 6C] It is a diagram showing an example of a gas exchange procedure according to Example 1. [Figure 6D] It is a diagram showing an example of a medium addition procedure according to Example 1. [Figure 6E] It is a diagram showing an example of a medium exchange procedure according to Example 1. [Figure 6F] It is a diagram showing an example of a supernatant sampling procedure according to Example 1. [Figure 6G] It is a diagram showing an example of a cell recovery procedure according to Example 1. [Figure 7] It is a diagram showing the state of inclination of the culture vessel at the time of cell recovery according to Example 1. [Figure 8] It is a diagram showing the flow during operation of the automatic culture device according to Example 1. [Figure 9] It is a diagram showing an example of a ventilation adapter for a flask culture vessel according to Example 2. [Figure 10] It 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. [Modes for carrying out the invention]

[0019] Embodiments of the present invention will be described below with reference to the drawings and other illustrations. The following description provides specific examples of the content 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 herein.

[0020] Furthermore, in all the figures used to illustrate the present invention, components having the same function are denoted by the same reference numerals, and repeated explanations may be omitted. [Examples]

[0021] Using Figure 1, the components of the automated culture apparatus used in this embodiment, which performs culture using a closed-system culture vessel, will be explained.

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

[0023] Furthermore, the culture vessel 1 is attached to the ventilation adapter 7, allowing for the supply and exchange of gas necessary for culture in the culture vessel 1. The gas supply unit 9 consists of a gas cylinder 10 maintaining a predetermined gas concentration, a gas flow control unit (mass flow controller: MF) 11, a pressure sensor 12, and a humidifying bottle 13, and is connected upstream of the ventilation adapter 7. Downstream of the ventilation adapter 7, a CO2 sensor 14 and a CO2 gas vent filter 15 are connected, and the gas is released into the atmosphere outside the device. The CO2 sensor 14 can monitor whether gas exchange in the ventilation adapter is being performed appropriately and can also be used to predict the culture state.

[0024] Such liquid or gas delivery is performed by pumps 16 and 17, and a preferred pump is a tube pump that generates pressure by squeezing a rubber tube with a rotating roller. Pump 16 is configured to deliver the culture medium to the culture vessel 1 and is connected by a tube to the liquid delivery pipe 18 of the culture vessel 1, and the other end is connected to the supply pipe of the culture medium bottle 20 via a solenoid valve 19.

[0025] Solenoid valves are suitable for opening and closing the rubber tubes that make up the flow path. When the rubber tubes, which are clamped in a valve actuated by spring force, are energized, the solenoid valve operates and can control the rubber tubes to open.

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

[0027] Pump 17 is connected via a tube to the suction tube 21 of the culture vessel 1, and the other end is connected via a solenoid valve 19 to the cell seeding bottle 22 and via another solenoid valve 19 to the supernatant collection bag 23 and supernatant analysis bag 24.

[0028] Furthermore, the pump 17 is connected via a tube to the cell recovery tube 25 of the culture vessel 1, and the other end is connected to the cell recovery bottle 26 via a solenoid valve 19.

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

[0030] As shown in Figures 1 and 2, the culture medium bottle, cell seeding bottle, cell harvesting bottle, supernatant harvesting bag, supernatant analysis bag, the channels connecting each component, the pump, solenoid valve, and weight sensor are located in the fluid control unit 29, which is the main body of the device outside the incubator 35.

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

[0032] The rocking operation of the culture vessel can be controlled by lowering the right rocking axis downwards and simultaneously raising the left rocking axis upwards, without moving the central rocking axis. This will cause the rocking stage to move with an inclination, tilting the culture vessel. Then, by reversing the movement of the left and right axes, 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, thereby agitating the cells 2 and culture medium 3 inside the culture vessel 1.

[0034] The incubator 35 is an example of a temperature maintenance mechanism and is a so-called dry incubator consisting of a constant temperature section 36 and an opening / closing door 37. The incubator 35 can house the culture vessel 1, the rocking mechanism 30, and the humidifying bottle 13, and can maintain the temperature inside the incubator at a temperature suitable for cell culture. By adopting a small dry incubator instead of a large CO2 incubator and having a configuration that can supply the gas necessary for culture, the device can be miniaturized, making it possible to operate multiple devices simultaneously in a small space. The control unit 38 can control the operation of the gas supply unit 9, pumps 16 and 17, and solenoid valve 19, as well as the operation of the rocking mechanism 30. By automatically controlling these mechanized elements at predetermined timings, during cell seeding, the cell suspension is delivered from the cell seeding bottle 22 to the culture vessel 1; during gas exchange, humidified gas from the humidifying bottle 13 is supplied to the ventilation adapter 7; when adding culture medium, the culture medium is delivered from the culture medium bottle 20 to the culture vessel 1; during culture medium exchange, the culture medium 3 in the culture vessel 1 is discharged into the supernatant recovery bag 23 and then the culture medium is supplied to the culture vessel 1; and during supernatant sampling, a portion of the culture medium in the culture vessel can be delivered to the supernatant analysis bag 24.

[0035] During cell retrieval, the culture medium in the culture vessel 1 is discharged into the supernatant retrieval bag 23, and then the cell suspension is agitated by the agitation mechanism 30 before being transferred to the cell retrieval bottle 26.

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

[0037] The side cover 40 acts as a windbreak to shield the movement of gas in the entire space where the culture medium bottle 20, cell seeding bottle 22, and cell harvesting bottle 26 are placed on top of the weight sensors 27 and 28 for weighing, thereby ensuring stable weighing.

[0038] Furthermore, the bottle unit door 41 is open when in a horizontal position, and serves as a temporary stand for placing bottles and tubes when installing the flow path. After the flow path tubes are connected to the pump and valves, it can be closed vertically and used as a windbreak. In addition, because the opening and closing directions of the bottle unit door 41 and the opening and closing door 37 of the incubator 35 are different, the two doors can be opened and closed without interfering with each other, making it easy to access the equipment during work.

[0039] The components of the ventilation adapter 7 according to Embodiment 1 will be explained using Figures 3A, 3B, and 3C. In Figure 3A, the ventilation adapter 7 is installed on the rocking stage 31 and is connected to the air supply pipe 42 connected to the humidifying bottle 13 and the exhaust pipe 43 connected to the CO2 sensor. The culture container 1 is placed on a horizontal platform (not shown) and is closely attached to the ventilation adapter 7 mainly by being pressed against the rubber packing 44 by the weight of the culture container 1 and the elasticity of the rubber packing 44. At this time, a predetermined gas space 8 is formed below the culture container 1 and inside the ventilation adapter 7.

[0040] Figure 3A shows an example of a ventilation adapter in which CO2 gas is supplied from the outer circumference of the ventilation adapter 7 and exhausted from the outer circumference. This type of ventilation adapter has the advantage of being able to reduce its height.

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

[0042] The continuous supply of humidified CO2 gas to the internal space of the ventilation adapter 7 maintains a pressure slightly higher than atmospheric pressure. In other words, the internal space of the ventilation adapter 7 is under positive pressure relative to atmospheric pressure. This reduces the risk of airborne bacteria entering the internal space of the ventilation adapter 7.

[0043] Furthermore, regarding the volume of the internal space of the ventilation adapter 7, it is preferable that it be as small as possible, as long as it is a volume sufficient to uniformly supply humidified CO2 gas to the gas permeable membrane of the culture vessel. This is because a larger internal volume would increase the time required 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 humidifying CO2 gas will not be supplied uniformly. Therefore, it is preferable that the internal space be as thin vertically as possible and have a horizontal area equal to or slightly larger than the area of ​​the gas permeable membrane of the culture vessel. The shape of the space is circular if the container is cylindrical, and rectangular if the container is square.

[0045] Figure 3B shows a ventilation adapter that supplies and exhausts CO2 gas from the bottom. Figure 3B shows the culture container 1, rubber gasket 44, and ventilation adapter 7 before installation, while Figure 3C shows the state during use. This type of ventilation adapter has the advantage of allowing the internal space of the ventilation adapter 7 to be smaller compared to the type shown in Figure 3A. In addition, in Figure 3B, the cross-sectional shape of the rubber gasket 44 follows the outer circumference of the culture container in order to improve the airtightness between the rubber gasket 44 and the culture container. Even if the inner circumference of this shape is smaller than the outer edge of the culture container, the rubber gasket 44 is tightly fitted to the ventilation adapter 7 by utilizing the elasticity of the rubber gasket. The form of the ventilation adapter can be appropriately selected according to the required specifications of the culture device (size of the culture container, culture rate, etc.). In addition to the forms shown in Figures 3A and 3B, it is also possible to supply CO2 gas from the outside of the circumference and provide an exhaust pipe at the bottom.

[0046] Figure 4 is a top view of the ventilation adapter 7 in the shape of Figure 3A, viewed from above. As mentioned above, the internal space of the ventilation adapter 7, when viewed from above, is circular in shape, slightly larger than the circular gas permeable membrane provided in the culture vessel. In this figure, there is one humidifying CO2 gas supply pipe 42, but the supply pipe may be divided into multiple pipes to ensure that the humidifying CO2 gas is supplied to the internal space as uniformly as possible, or the opening may be a single large opening. Furthermore, the supply pipe 42 is not limited to connection from the side of the ventilation adapter 7, but may also be connected from the bottom.

[0047] Figure 5 shows the time-dependent change in the CO2 gas concentration in the gas phase of the culture vessel in Example 1, compared using two measurement methods: ventilation using the ventilation adapter 7 and ventilation 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 container through which gas could only pass through the ventilation surface (gas permeable membrane) 4. The volume of the gas space 8 was set to 300cc, and a 5% CO2-air mixed gas was continuously supplied from the supply pipe 42 at a flow rate of 50cc / min.

[0049] To measure the ventilation method using a CO2 incubator, a CO2 sensor was installed inside the chamber of an incubator that was constantly maintained with 5% CO2-air. Simultaneously, a separate CO2 sensor was installed inside the chamber of a closed-system culture vessel 1. This created a container where gas could only pass through the ventilation surface (gas permeable membrane) 4. The measurements were taken over 15 hours while each container was maintained at 37°C.

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

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

[0052] Figure 6A shows an example of a flow channel circuit 45 including a closed-system culture vessel according to Example 1. The same elements as in the example shown in Figure 1 are numbered the same way, and for the solenoid valves 19, the specific solenoid valves -1 to -9 are numbered 46 to 54, respectively.

[0053] Furthermore, the rubber tube connected to the supernatant collection bag 23 is equipped with a manual valve 55 that allows the tube to be opened and closed manually, and the rubber tube connected to the supernatant analysis bag 24 is equipped with a manual valve 56. 59 is a connecting part, which is a component that allows the pipes to be joined and cut by a male-to-female connector. 60 indicates a branching point, which is the branching point of the tubes joined using a T-shaped connector.

[0054] The flow path circuit 45 shown here, with the exception of the mechanical elements such as the solenoid valve 19 in the pumps 16 and 17, the weight sensor (electronic balance) 28, the gas flow control unit 11, and the pressure sensor 12, can be attached and detached as an integral part of the apparatus body shown in Example 1, and can be applied to cell culture by sterilizing only the flow path circuit.

[0055] Figure 8 is a flowchart showing the overall operation of cell culture in the cell culture apparatus 100 shown in Figure 6A. Following "START", the flow channel is installed in the cell culture apparatus 100 (S01), and then the cell seeding bottle 22 holding the separately prepared cell suspension, the culture medium bottle 20 holding the culture medium, and the cell harvesting bottle 26 are connected to the flow channel (S02).

[0056] Figure 6B shows the cell seeding process according to Example 1. Initially, the pump is stopped and the roller is stopped by clamping the rubber tube, so pumps 16 and 17 are closed as valves. The solenoid valve is also closed by clamping 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 container 1 is empty and placed horizontally on the ventilation adapter 7. At the start of cell seeding, solenoid valves 47, 48, 54, and 51 are opened, and the piping from the suction tube 21 of the culture container 1 to the CO2 gas vent filter 15 is opened.

[0057] Next, the pump 17 is activated, and gas is supplied from one end of the tubing of the cell seeding bottle 22 to pressurize the cell suspension inside. The cell suspension then passes through the tubing and is delivered to the culture vessel 1 via the suction tube 21 (S03). The liquid flow state at this time is shown by a solid line, and the gas flow state is shown by a dashed line (the same applies below).

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

[0059] Next, when solenoid valves 48 and 49 are closed, solenoid valve 46 is opened, and pump 17 is activated, the cell suspension in the pipeline closer to the culture vessel than the branching section 62 is delivered to the culture vessel 1. After that, when pump 17 is stopped and solenoid valves 47, 48, and 49 are opened, the cell suspension in the pipeline returns to the cell seeding bottle 22 due to gravity, and the liquid in the pipeline is removed, all solenoid valves are closed, and the cell seeding process is completed (S03).

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

[0061] Furthermore, the opening of a short tube in the humidifying bottle 13 is located at the top of the container, and the ventilation adapter is connected to one end of this opening. When the gas flow control unit 11 is activated, 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. Subsequently, 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] Figure 6D shows the process of adding culture medium to culture vessel 1 in Example 1. Initially, the pump is stopped and the solenoid valve is closed by closing the rubber tube. Culture medium is held in culture medium bottle 20 and is installed on weight sensor 27. First, the solenoid valve 53 is opened, opening the pipeline from the liquid delivery pipe 18 of culture vessel 1 to the culture medium bottle 20. Next, when the pump 16 is activated, the culture medium passes through the pipeline and is delivered to culture vessel 1 from the liquid delivery pipe 18.

[0063] Next, when a predetermined amount of culture medium has moved, the solenoid valve 51 is opened and the pump 16 is stopped at the same time. At this time, the flow of culture medium is temporarily paused in the pipeline when the pump 16 stops, and the culture medium in the pipeline closest to the culture medium bottle 20 is returned to the inside of the culture medium bottle 20 by gravity as outside air enters through the vent filter which is open to the outside from the branching section 63.

[0064] Next, when the solenoid valve 53 is closed and the pump 16 is activated, the culture medium in the pipeline closer to the culture vessel from the branching section 63 is delivered to the culture vessel 1. After that, when the pump 16 is stopped, the liquid in the pipeline is removed, all the solenoid valves are closed, and the culture medium addition process is completed (S05).

[0065] Figure 6E shows the process of discharging the culture medium during the exchange of culture medium to culture vessel 1 in Example 1. Initially, the pump is stopped and the solenoid valve is closed by closing the rubber tube. Culture medium is held in culture vessel 1, and the supernatant collection bag 23 is installed empty. First, solenoid valves 46 and 52 and manual valve 55 are opened, opening the piping from the suction tube 21 of culture vessel 1 to the supernatant collection bag 23. Next, when pump 17 is started, the culture medium is pumped from 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 moved, the solenoid valve 51 is opened and the pump 16 is stopped at the same time. At this time, the flow of culture medium is temporarily paused in the pipeline when the pump 16 stops, and the culture medium in the pipeline closest to the culture medium bottle 20 is returned to the inside of the culture medium bottle 20 by gravity as outside air enters through the vent filter which is open to the outside from the branching section 62.

[0067] Next, when the solenoid valve 49 is opened, the culture medium in the conduit closer to the culture vessel 1 from the branching section 64 is transferred to the culture vessel 1 by gravity. After that, when the solenoid valve 46 is closed and the pump 17 is activated, outside air is introduced from the vent filter and the culture medium in the conduit reaches the supernatant collection bag 23. All solenoid valves are then closed and the culture medium discharge process is completed (S06). Next, the culture medium can be replaced by performing the process of adding culture medium to the culture vessel 1 as described using Figure 6D (S07). After the culture medium replacement, the culture vessel 1 is shaken by the shaking mechanism 30 to mix the new culture medium and cells, which further promotes cell culture.

[0068] Figure 6F shows the process of sampling the supernatant from culture vessel 1 in Example 1. Initially, the pump is stopped and the solenoid valve is closed by closing the rubber tube. Culture vessel 1 contains culture medium, and the supernatant analysis bag 24 is empty. First, solenoid valves 46, 52, and manual valve 56 are opened, opening the tubing from the suction tube 21 of culture vessel 1 to the supernatant collection bag 23. Next, when the pump 17 is activated, the culture medium is delivered from 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 moved, the solenoid valve 51 is opened and the pump 16 is stopped at the same time. At this time, the flow of culture medium is temporarily paused in the pipeline when the pump 16 stops, and the culture medium in the pipeline closest to the culture medium bottle 20 is returned to the inside of the culture medium bottle 20 by gravity as outside air enters through the vent filter which is open to the outside from the branching section 62.

[0070] Next, when the solenoid valve 49 is opened, the culture medium in the pipeline closer to the culture vessel 1 from the branching section 64 is transferred to the culture vessel 1 by gravity. After that, when the solenoid valve 46 is closed and the pump 17 is activated, outside air is introduced from the vent filter and the culture medium in the pipeline reaches the supernatant analysis bag 24. All solenoid valves are then closed and the supernatant sampling process is completed (S08).

[0071] Figures 6E and 6G illustrate the cell recovery process from the culture vessel 1 in Example 1. After the cells have sufficiently proliferated, they settle and float at the bottom of the culture vessel. Similar to the supernatant recovery process shown in Figure 6E, the supernatant is recovered from the culture vessel 1 using the same flow channel 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 increases the ratio of culture medium 3 to cells in the culture vessel 1. Next, the culture vessel is shaken by the aforementioned shaking mechanism 30 to agitate the culture medium and cells, creating a cell suspension.

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

[0073] Figure 7 illustrates the tilting of the culture vessel during cell retrieval. First, the rocking part 70 of the rocking mechanism 30 is tilted toward the direction where the retrieval tube 25 is located, causing the cell suspension to collect on the wall side of the culture vessel 1 where the retrieval tube 25 is located. The tilt angle of the rocking part 70 should be greater than 0 degrees and less than 90 degrees, as long as it can collect the cell suspension on the wall side of the culture vessel 1. The rocking part 70 needs to be able to tilt in one direction, but it may also be able to tilt in multiple directions. When placing the culture vessel 1 on the rocking mechanism, the culture vessel 1 should be placed in a direction that allows it to tilt toward the direction where the opening of the retrieval tube 25 is located. At that time, a mark indicating the placement direction of the culture vessel 1 may be written on the ventilation adapter 7 fixed to the rocking part 70.

[0074] Next, solenoid valves 50 and 51 are opened, opening the pipeline from the culture vessel 1 to the recovery tube 25 and the cell recovery bottle 26, as well as the pipeline to the CO2 gas vent filter 15 which is open to the outside air. Then, when the pump 17 is activated, the cell suspension 65 is pumped from the culture vessel 1 through the recovery tube 25 and reaches the cell recovery bottle 26.

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

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

[0077] As described above, the automated culture apparatus according to this embodiment allows for culture while miniaturizing the apparatus. Furthermore, it is possible to implement and suppress the risk of rust formation on mechanical elements such as the rocking mechanism of the culture vessel and the camera observation mechanism for cells used in the culture operation. In addition, the risk of microbial growth throughout the incubator chamber can be suppressed by supplying humidifying gas to the culture vessel partially using a ventilation adapter. [Examples]

[0078] Figure 9 shows an example of a ventilation adapter for a flask culture vessel according to Example 2.

[0079] Using Figure 9, the components of the ventilation adapter 7 according to Example 2 will be explained. The culture vessel 1 is a flask-type culture vessel, mainly used for cell culture where cells adhere to the bottom surface of the vessel. Normally, two screw holes 101 are provided on the top surface of the main body, and each is closed by a ventilation cap 103 with a gas-permeable membrane 102 at its opening.

[0080] In this embodiment, one screw-ended ventilation cap is left as is, and a method of gas exchange using a ventilation adapter 7 is shown. The other screw-ended cap is fitted with a ported cap 104 equipped with a liquid supply pipe, and automatic liquid supply is performed by an automated culture device. More specifically, the culture container 1 is placed on a rocking stage 31 and connected to the air supply pipe 42 connected to the humidifying bottle 13 and the exhaust pipe 43 connected to the CO2 sensor. One ventilation cap 103 in the culture container 1 is fitted closely with the 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. These three ports are connected in the same way as the automated culture apparatus (Figure 1) and flow path circuit (Figure 6A) described in Example 1, and the cell seeding process, gas exchange process, culture medium addition process, culture medium exchange process, supernatant sampling process, and cell recovery process can be carried out. [Examples]

[0082] Figure 10 shows an example of an automated culture system in which multiple automated culture devices according to Example 1 are installed, connected to a network, and controlled by a central management PC.

[0083] An automated culture system, consisting of an incubator 35 and a fluid control unit 29, is installed in four sets on a stand 105. In this embodiment, a stand is shown with four sets of devices installed, two sets on the upper and two sets on the lower level. However, the number of devices per level in this embodiment is arbitrary, making it easy to increase the number of devices that can be accumulated in a limited CPC facility space. From the perspective of precise process control and aseptic operation, it is also possible to fully automate all processes of installing and removing flow paths using robots, and the number of devices that can be accumulated can be made beyond the reach of human hands.

[0084] Each control unit 38 is mounted on the front of the door 37 of the incubator 35, and each is connected to the central control PC 106 via a network. The central control PC 106 has functions for monitoring the status of each automated culture device, managing updates and modifications to the operating program, and transmitting information when an abnormality occurs in the device or when the device has finished operating.

[0085] By adopting the configuration described in this embodiment, it is possible to provide an automated culture apparatus that can operate multiple devices simultaneously in a space-saving manner, and an automated culture system using multiple such apparatuses. [Explanation of Symbols]

[0086] 1 Culture vessel, 2 Cells, 3 Culture medium, 4 Ventilation surface, 5 Pressure regulating 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 Fluid 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 Oscillating mechanism

Claims

1. An automated culture apparatus capable of accommodating culture vessels for containing the subject to be cultured, A temperature-maintaining mechanism for maintaining the culture vessel at a predetermined temperature, CO required for culture 2 The gas supply unit where gas is supplied, The culture vessel is fitted with a ventilation adapter that supplies and exchanges the gas necessary for cultivation to the culture vessel, The culture vessel has a screw-hole opening on the top surface or a gas permeable section on the bottom surface made of a gas-permeable material. The ventilation adapter is connected to an air supply pipe that is connected to the gas supply unit and an exhaust pipe that releases gas from the culture vessel into the atmosphere, and is mounted opposite the gas permeable portion of the culture vessel, and humidifies CO2 inside the culture vessel. 2 An automated culture device characterized by gas exchange.

2. In the automated culture apparatus according to claim 1, An automated culture apparatus characterized in that the ventilation adapter is constructed separately from the culture vessel and is detachable from the culture vessel.

3. In the automated culture apparatus according to claim 1, The automatic culture apparatus is characterized in that the ventilation adapter is structured such that the culture vessel is placed above the ventilation adapter and attached to the ventilation adapter by pressing down on the culture vessel.

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

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

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

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

8. In the automated culture apparatus according to claim 1, The automatic culture apparatus is characterized in that the temperature holding mechanism comprises at least the ventilation adapter and a housing that houses the culture vessel, and includes a temperature control mechanism that maintains the temperature inside the housing at the predetermined temperature.

9. In the automated culture apparatus according to claim 8, An automated culture apparatus characterized in that the gas released from the exhaust pipe of the ventilation adapter is discharged to the outside of the housing.

10. In the automated culture apparatus according to claim 9, An automated culture apparatus characterized by having a gas sensor in the flow path of the exhaust pipe for measuring the amount of gas discharged from the ventilation adapter.

11. In the automated culture apparatus according to claim 8, An automated culture apparatus characterized by having a rocking mechanism for rocking the culture vessel located inside the housing.

12. In the automated culture apparatus according to claim 8, An automated culture apparatus characterized in that the inside of the enclosure is equipped with a humidifier as part of the gas supply unit for humidifying the supplied gas.

13. In the automated culture apparatus according to claim 8, An automated culture apparatus characterized by comprising, outside the housing of the temperature-holding mechanism, a fluid control unit having a first container capable of containing a supply culture medium, a second container capable of containing drainage liquid, and a pump that enables fluid operation.

14. A plurality of automated culture devices according to any one of claims 1 to 13, An automated culture system characterized by comprising a centralized control computer that controls the aforementioned multiple automated culture devices.

15. An automated culture system according to claim 14, An automated culture system characterized by arranging the aforementioned multiple automated culture devices in parallel, either vertically or horizontally.

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