Automated culture device and cell culture method using the same

Abstract

To provide an automated culture device that maintains the CO2 gas concentration required for culture in a culture vessel with a simple configuration, and to provide a cell culture method using the same.SOLUTION: An automated culture device includes: a ventilation adapter capable of supplying gas containing CO2 to a culture vessel in a state where a culture vessel for culturing cells is placed; and a gas supply unit for supplying gas containing CO2 to the ventilation adapter. The automated culture device also includes a gas retention space forming unit in which, when the culture vessel is placed in a predetermined position on the ventilation adapter, an upper part of the ventilation adapter directly or indirectly supports at least a part of a gas exchange membrane located below the culture vessel, and a lower end of the ventilation adapter contacts at least a part of an upper surface of the ventilation adapter, allowing the gas containing CO2 supplied from the gas supply unit to be airtightly retained.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to an automatic culture device for culturing cells or tissues, and a cell culture method using the same. [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 expectations are high 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 their high demand from the perspective of improving patients' quality of life (QOL).

[0004] The most common cell type is immune cells, accounting for more than 40% of the total. Cancer is the main target disease, and specific cell types include T cells, NK cells, and NKT cells. In particular, CAR-T (chimeric antigen receptor-T) cell therapy, in which T cells, which are immune cells, are extracted from the patient, modified by gene transfer so that they can attack cancer cells, and then injected back into the patient, is being put into practical use.

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

[0006] This process is carried out at a cell processing center (CPC) in accordance with standard operating procedures (SOPs) that comply with Good Manufacturing Practices (GMP), the standards for manufacturing and quality control of pharmaceuticals, etc. Therefore, operating a CPC requires significant costs and personnel with specialized culture techniques.

[0007] In addition, because 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 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 reductions, 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 always 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 within it 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.

[0010] As an example of a culture vessel, as shown in Patent Document 2, there is a technology in which a closed vessel having a ventilation membrane in part of the culture vessel is used, and the area around the ventilation surface is maintained at a desired gas concentration for cell culture. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-312668 [Patent Document 2] U.S. Patent No. 4,839,292 Summary of the Invention [Problem to be solved by the invention]

[0012] The technology described in Patent Document 1 allows for culturing in a closed culture vessel, but the culture vessel is held in a CO2 incubator, the interior of which is maintained at a constant CO2 gas concentration. Gas exchange occurs when gas in the space above the culture medium in the culture vessel dissolves in the culture medium, or when gas in the culture medium is released into the space.

[0013] In addition, the water stored inside a CO2 incubator for humidification purposes comes into contact with the air, posing a risk of microbial growth, making it difficult to use in facilities that manufacture cells for transplantation, which require high levels of cleanliness.

[0014] In Patent Document 2, a ventilation chamber is provided to maintain a desired gas concentration around the ventilation surface of the culture vessel without using a CO2 incubator. Because the inside of the ventilation chamber becomes positive pressure due to pressure loss in the piping, if a highly airtight ventilation chamber cannot be provided, the humidified gas will leak from the periphery of the culture vessel and into the incubator, causing high humidity inside the chamber and potentially causing mold and other problems, but Patent Document 2 makes no mention of this point.

[0015] An object of the present invention is to provide an automatic culture device that has a simple configuration and that maintains the CO2 gas concentration in the culture vessel required for culture, and a cell culture method using the same. [Means for solving the problem]

[0016] The present invention is configured as follows to achieve the above object. This automatic culture device is equipped with a ventilation adapter capable of supplying a gas containing CO2 to a culture vessel in which cells are cultured when the culture vessel is placed on the ventilation adapter, and a gas supply unit that supplies a gas containing CO2 to the ventilation adapter.When the culture vessel is placed in a predetermined position on the ventilation adapter, the upper part of the ventilation adapter directly or indirectly supports at least a part of the gas exchange membrane provided below the culture vessel, and the lower end of the ventilation adapter contacts at least a part of the upper surface of the ventilation adapter, so that the gas containing CO2 supplied from the gas supply unit can be airtightly held. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide an automatic culture device that has a simple configuration and that maintains the CO2 gas concentration in the culture vessel required for culture, and a cell culture method using the same. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram showing one configuration of an automatic culture device. [Figure 2] FIG. 10 is a diagram illustrating an example of a ventilation adapter. [Figure 3] FIG. 10 shows the results of gas exchange in a culture vessel using a ventilation adapter. [Figure 4] 10A and 10B are diagrams showing an example of a pressing jig for a ventilation adapter. [Figure 5] 10A and 10B are diagrams showing an example of a pressing jig for a ventilation adapter. [Figure 6] 10A and 10B are diagrams showing an example of a pressing jig for a ventilation adapter. [Figure 7] 10A and 10B are diagrams illustrating an example of a gas holding space forming portion in a ventilation adapter. [Figure 8] 10A and 10B are diagrams illustrating an example of a gas holding space forming portion in a ventilation adapter. [Figure 9] FIG. 1 is a diagram showing a flow of operation of the automatic culture device. [Figure 10A] FIG. 1 is a diagram showing an example of a flow path circuit including a closed culture vessel. [Figure 10B] FIG. 1 is a diagram showing an example of a procedure for cell seeding. [Figure 10C] FIG. 10 is a diagram showing an example of a gas exchange procedure. [Figure 10D] FIG. 1 is a diagram showing an example of a procedure for adding a medium. [Figure 10E] FIG. 1 is a diagram showing an example of a procedure for changing a culture medium. [Figure 10F] FIG. 1 shows an example of a procedure for sampling a supernatant. [Figure 10G] FIG. 1 is a diagram showing an example of a procedure for collecting cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the present invention will be described with reference to the drawings, etc. The following description shows specific examples of the contents of the present invention, and the present invention is not limited to these descriptions. Various changes and modifications can be made 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, parts having the same functions are given the same reference numerals, and repeated explanations thereof may be omitted.

[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 automated 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 components, 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-maintaining mechanism that houses the culture vessel, ventilation adapter, and rocking mechanism and controls the temperature. The culture vessel 1 is a culture vessel with a ventilation surface (also called a gas exchange membrane) 4, which has a gas-permeable membrane disposed on its bottom surface, and an open-hole bottom surface 71 in contact with its underside. The open-hole bottom surface 71 functions to horizontally support the thin membrane ventilation surface 4, has multiple openings for ventilation, and is in airtight contact with the outer periphery of the ventilation surface 4 to prevent liquid from leaking downward.

[0023] The upper end of the gas holding space forming portion 72 contacts the underside of the open bottom surface 71 so as to indirectly support the ventilation surface, and the lower end contacts the upper surface (inner surface) of the ventilation adapter 7, forming a ventilation space 8 that can airtightly maintain a desired gas concentration. "Airtight" here means that the gas supplied from the gas supply portion does not leak out of the ventilation space 8, but does not mean that the gas is sealed within the ventilation space 8 and that the sealed gas stagnates (does not flow).

[0024] Inside the culture vessel 1, cells 2 are held and cultured together with a culture medium 3. A pressure adjustment tube 5 and a vent filter 6 are connected to the culture vessel 1, allowing gas to pass in and out of the culture vessel while preventing the entry of bacteria and viruses from the outside.

[0025] The culture vessel 1 is also attached to a ventilation adapter 7, allowing the culture vessel 1 to be supplied with the gas and humidified necessary for cultivation. The gas supply unit 9, consisting of a gas cylinder 10 containing a predetermined gas concentration, a gas flow control unit (mass flow controller: MF) 11, a pressure sensor 12, and a humidification bottle 13, is connected upstream of the ventilation adapter 7. The gas supply and humidification are controlled by the gas flow control unit to a predetermined air flow rate, and the air is humidified by passing through the water inside the humidification bottle before being supplied to the ventilation adapter 7. A gas flow meter (MFM) 73, a CO2 sensor 14, and a CO2 gas vent filter 15 are connected downstream of the ventilation adapter 7, and the gas is released into the atmosphere outside the device. The gas flow meter 73 and CO2 sensor 14 allow for monitoring whether gas exchange in the ventilation adapter is appropriate based on the gas flow rate and CO2 measurements, and can also be used to predict the culture status.

[0026] The liquid or gas is fed 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. Pump 16 is configured to feed the culture medium to culture vessel 1, and is connected to a liquid feed pipe 18 of culture vessel 1 at one end and a supply pipe of culture medium bottle 20 via an electromagnetic valve 19 at the other end.

[0027] An electromagnetic valve is suitable for opening and closing the rubber tube that makes up the flow path. When electricity is applied to the closed state of the rubber tube clamped in the valve, which is actuated by spring force, the electromagnetic valve operates to control the rubber tube to open.

[0028] The pump 17 is configured to send a cell suspension to the culture vessel 1 to seed the cells, to discharge the medium 3 from the culture vessel 1, and to recover the grown cells.

[0029] The pump 17 is connected to a suction tube (also called a "drain tube") 21 of the culture vessel 1 through a tube, and the other end is connected to a cell seeding bottle 22 via an electromagnetic valve 19 and to a supernatant collection bag 23 and a supernatant analysis bag 24 via another electromagnetic valve 19.

[0030] The pump 17 is connected to a cell collection pipe 25 of the culture vessel 1 through a tube, and the other end is connected to a cell collection bottle 26 via an electromagnetic valve 19 .

[0031] 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 collection bottle 26 are measured by a weight sensor 28.

[0032] As shown in Figure 1, 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 located in the fluid control unit 29, which is the main body of the device outside the incubator 35.

[0033] 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 mechanisms 32, and a rocking stage 34 that is fixed inside the incubator.

[0034] The rocking operation of the culture vessel is performed in the left-right direction on the paper by lowering the right rocking axis downward and simultaneously raising the left rocking axis upward, and by controlling this without moving the central rocking axis, the rocking stage will move with an inclination and the culture vessel can be tilted.Next, by reversing the movement of the left and right axes, the culture vessel can be tilted in the opposite direction.

[0035] 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 medium 3 inside the culture vessel 1.

[0036] The incubator 35 is an example of a temperature maintenance mechanism, and is a so-called dry incubator consisting of a thermostatic 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 suitable temperature for cell culture. By adopting a small dry incubator instead of a large CO2 incubator and having a configuration that can supply the gases necessary for culture, the device can be made smaller, and multiple devices can be operated simultaneously in a small space.

[0037] The control unit 38 can control the operation of the gas supply unit 9, pumps 16, 17, and solenoid valve 19, as well as the operation of the rocking mechanism 30. By automatically controlling these mechanized elements at predetermined timings, it is possible to send a cell suspension from the cell seeding bottle 22 to the culture vessel 1 during cell seeding, to supply humidified gas from the humidification bottle 13 to the ventilation adapter 7 during gas exchange, to send the culture medium from the culture medium bottle 20 to the culture vessel 1 during culture medium addition, to discharge the culture medium 3 in the culture vessel 1 into the supernatant collection bag 23 during culture medium exchange and then supply the culture medium to the culture vessel 1, and to send a portion of the culture medium in the culture vessel to the supernatant analysis bag 24 during supernatant sampling.

[0038] 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 agitate the cell suspension, which can then be sent to the cell recovery bottle 26.

[0039] Figure 2 is a schematic diagram of the assembly of the ventilation adapter 7 and the culture vessel 1. The gas retention space forming part 72 is fixed at a predetermined position on the top surface of the ventilation adapter 7. In this embodiment, the gas retention space forming part 72 is formed entirely from flexible rubber. This fixing method allows for easy removal when the rubber that makes up the gas retention space forming part 72 becomes worn and needs to be replaced. Next, the culture vessel 1 is inserted from above so that it fits the external shape of the gas retention space forming part 72.

[0040] The ventilation surface 4 and the perforated bottom surface 71 of the culture vessel 1 are surrounded by a skirt portion (generally made of hard rubber, synthetic resin, etc., but may also be made of metal such as aluminum or stainless steel) that covers the bottom of the culture vessel 1. "Insert so as to fit the outer shape" means that the inner peripheral surface of this skirt portion and the gas retention space forming portion 72 are inserted so as to be in airtight contact with each other.

[0041] At this time, the height of the gas retention space forming portion 72 is at its natural length. Next, one end of the holding jig 74 is placed in contact with the outer surface of the culture vessel 1 and temporarily placed there. A fastener receiver 75 is located on the stage 31, and the fastener receiver 75 engages with a fastener 76 at a position that fits the holding jig 74. The holding jig 74 is composed of two parts and is installed symmetrically with respect to the vessel. When the fastener is rotated to engage, the holding jig 74 is pulled downward, pushing the culture vessel 1 downward. At this time, the gas retention space forming portion 72 is similarly pressed down by the perforated bottom surface 71 of the culture vessel 1, forming a tight seal due to the elastic deformation of the rubber and also coming into close contact with the ventilation adapter 7. The ventilation surface 4 then receives force from the gas retention space forming portion 72 via the perforated bottom surface 71, i.e., indirectly. The deformation of the ventilation surface 4 reduces the gap between the ventilation surface 4 and other components, forming an airtight ventilation space 8. CO2 gas is supplied from an air supply pipe 42 provided on the bottom surface of the ventilation adapter 7, passes through the ventilation space 8, contacts the gas permeable membrane 4, and is then exhausted from a port 43.

[0042] The method of installing the holding jig 74 is to install the culture vessel 1 in the ventilation adapter 7, and then install one holding jig 74 on each side of the culture vessel 1. A feature of this holding jig is that it can be installed without interfering with the four tubes connected to the culture vessel 1. Furthermore, if two plates are manufactured with the same shape, they can be installed on either the left or right side.

[0043] With the above configuration, the ventilation adapter 7 and the gas holding space forming part 72 can be connected via the open bottom surface 71 to form a highly airtight ventilation surface 4, so that humidified CO2 gas is held in the ventilation space 8 and does not leak into the incubator, allowing the incubator to be maintained at a low humidity.

[0044] The upper part of the gas holding space forming part 72 may be in direct contact with, i.e., directly support, the ventilation surface 4. In this case, the ventilation surface 4 receives a force directly from the gas holding space forming part 72, and the ventilation surface 4 is deformed, thereby reducing the gap between the ventilation surface 4 and other components, and an airtight ventilation space 8 is formed.

[0045] Furthermore, the humidified CO2 gas is continuously supplied to the internal space of the ventilation adapter 7, and due to the pressure loss caused by passing through the CO2 vent filter 15 shown in Figure 1, the internal space (ventilation space 8) 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 has 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.

[0046] Furthermore, the volume of the internal space of the ventilation adapter 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, because if the volume of the internal space is large, it takes a long time to fill the internal space with humidified CO2 gas.

[0047] On the other hand, if the internal space is too small, there is a greater concern that humidified CO2 gas may not be supplied uniformly. Therefore, it is preferable that the internal space be as thin as possible vertically and have a horizontal area 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 rectangular if the vessel is square.

[0048] In addition, the gas holding space forming portion 72 only needs to be made of a flexible material such as rubber at its upper and lower ends to maintain airtightness, and the middle portion may be made of metal or hard plastic.

[0049] Figure 3 shows the results of a comparison of the time course of CO2 gas concentration in the gas phase inside the culture vessel in Example 1 using two measurement methods: a ventilation method using a ventilation adapter 7 and a ventilation method using a 5% CO2 incubator (technology described in Patent Document 1).

[0050] To measure the ventilation method using the ventilation adapter 7, a CO2 sensor (VAISSLA 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 in which gas could only pass through the ventilation surface (gas permeable membrane) 4. The volume of the ventilation space 8 was set to 300 cc, and a 5% CO2-air mixed gas was continuously supplied through the air supply pipe 42 at a flow rate of 50 cc / min.

[0051] To measure the ventilation method using a CO2 incubator, a CO2 sensor was installed inside the incubator, which was constantly maintained at 5% CO2-air, and at the same time, a separate CO2 sensor was installed inside the closed-system culture vessel 1, creating a vessel with only the ventilation surface (gas-permeable membrane) 4 through which gas could pass.Each vessel was kept at 37°C and measurements were taken for 15 hours.

[0052] Since the CO2 gas concentration inside the incubator is maintained at 5%, the CO2 sensor indicates a CO2 gas concentration of 5% as soon as measurement begins (the dashed line bent at almost a right angle labeled "Concentration inside the incubator" in Figure 3). It can be seen that the 5% CO2 concentration was reached in 800 minutes in both the ventilation method using the CO2 incubator (the solid line labeled "Natural ventilation inside the incubator" in Figure 3) and the ventilation method using the ventilation adapter (the dashed line labeled "Ventilation adapter" in Figure 3, drawn slightly below the solid line).

[0053] Ventilation using a ventilation adapter can achieve ventilation equivalent to that achieved by a CO2 incubator by adjusting the amount of gas sent, taking into account that it takes time for the gas concentration in the ventilation space 8 to increase.

[0054] Figure 4 shows another embodiment of the holding jig 74. A feature of this holding jig 74 is that an opening 77 is provided in the center where the culture vessel 1 passes through. The installation method is to place the culture vessel 1 in the ventilation adapter 7, and then pass the four tubes through the opening 77 above the culture vessel 1, and then install the holding jig 74 on the rocking stage. A feature of this holding jig 74 is that it can be manufactured from a single plate, which is cost-effective, and it can press the vessel horizontally with a constant force. It can also be installed with the depth direction reversed.

[0055] Figure 5 shows another embodiment of the holding jig 74. This holding jig 74 is characterized by having an opening 79 that can be opened and closed around a rotation axis 78. The opening 79 has a concave-convex shape where the left and right ends fit together, and when installed and closed, the left and right sides become one, increasing horizontal strength.

[0056] The installation method is as follows: after placing the culture vessel 1 in the ventilation adapter 7, it is opened at the side of the waist of the culture vessel 1 and closed after the vessel has passed. Next, the holding jig 74 is installed on the swinging stage. This holding jig 74 has the advantage that it can be manufactured with a single set of parts, and when closed, it is able to contact the circumference of the vessel evenly and press horizontally with a constant force. It can also be installed with the depth direction reversed.

[0057] 6 shows another embodiment of the holding jig 74. This holding jig 74 is characterized by a large opening in the center through which the culture vessel 1 passes, an opening 80 through which a tube passes during installation, and a basically circular shape with a male thread 81 facing the swing stage. On the other hand, the inner side of the ventilation adapter 7 has a female thread 82 that mates with the male thread 81.

[0058] The installation method involves placing the holding jig 74 on the ventilation adapter 7 after the four tubes have passed through the opening 80 above the culture vessel 1, and then using the recessed portion 83 of the holding jig 74 to rotate the male thread 81 to connect the threads. The connection of the threads is completed when a load is applied to the gas holding space forming portion 72, bringing the bottom of the opening and the top surface of the ventilation adapter 7 into close contact. The holding jig 74 has the advantage that it can be manufactured from a single set of component plates, and when closed, it is able to press horizontally with a constant force, evenly contacting the circumference of the vessel. This pressing force presses more firmly against the gas holding space forming portion 72, allowing the ventilation adapter 7 to be installed with a stronger closing force.

[0059] FIG. 7 shows another embodiment of the gas holding space forming portion 72. In this embodiment, a groove is formed from the side to the top of a packing holder 84 made of a material with little flexibility (hard material), such as metal or hard plastic, and an L-shaped packing 85 that contacts the bottom surface 71 of the opening fits into this groove to form an integrated unit (this L-shaped packing 85 is also referred to as the second airtight sealing portion). A downward groove is also formed in the bottom of the packing holder 84, and an O-ring 86 that contacts the inner upper surface of the ventilation adapter 7 fits into this groove to form an integrated unit (this O-ring 86 is also referred to as the first airtight sealing portion). The packing holder 84 can be fixed to the inside of the ventilation adapter 7 with multiple screws (not shown). The packing holder 84 is basically pressed against the ventilation adapter 7 by the screw force, and the O-ring 86 elastically deforms to maintain the airtightness of the ventilation space 8. On the other hand, the L-shaped packing 85 is pressed downward by the pressing jig 74, and comes into close contact with the bottom surface 71 of the opening, thereby elastically deforming, and the ventilation space 8 is kept airtight.

[0060] The feature of this method is that elastic members are used only in areas where airtightness is required, and if the elastic members wear out and the airtightness decreases as the container is closed multiple times, airtightness can be restored by simply replacing the elastic members.This means that the manufacturing costs are lower than if the entire container were made of flexible rubber, etc., and running costs are also lower.

[0061] 8 shows another embodiment of the gas holding space forming portion 72. In this embodiment, a groove is formed in the upper part of the packing holder 84, and the trapezoidal packing 87 that contacts the bottom surface 71 of the opening fits into this groove to become one unit. The difference between this method and the L-shaped packing 85 is that when pressed downward by the holding jig 74, the L-shaped packing comes into close contact with the bottom surface 71 of the opening and elastically deforms in the direction from above to the side, whereas the trapezoidal packing acts downward. This results in stronger airtightness because the elastic members repel each other in only one direction, which has the effect of maintaining a stronger airtight seal in the ventilation space 8.

[0062] Fig. 9 is a flowchart showing the overall operation of cell culture in the cell culture device 100 shown in Fig. 10A. Hereinafter, the flow of cell culture using the cell culture device 100 will be described with reference to Figs. 10A to 10G.

[0063] Fig. 10A 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 example shown in Fig. 1 are given the same numbers, and for the solenoid valve 19, the unique solenoid valves -1 to -9 are given numbers 46 to 54, respectively.

[0064] 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. 59 is a connecting part that allows the connection and disconnection of pipes by means of a male-female joint. 60 is a branching part, which is the branching point of the tubes connected using a T-shaped connecting part.

[0065] The flow path circuit 45 shown here can be attached and detached as a unit from the device main 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.

[0066] In Figure 9, after "START", a flow path is installed in the cell culture device 100 (S01), and then a cell seeding bottle 22 holding a separately prepared cell suspension, a culture medium bottle 20 holding a culture medium, and a cell collection bottle 26 are connected to the flow path (S02).

[0067] Figure 10B 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 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 cell seeding bottle 22 and placed on weight sensor 28, and culture vessel 1, empty inside, is placed horizontally on ventilation adapter 7. At the start of cell seeding, solenoid valves 47, 48, 54, and 51 are opened, and the drainage pipe 21 of culture vessel 1 and the pipeline to CO2 gas vent filter 15 are opened.

[0068] Next, the pump 17 is operated to supply gas from one side of the conduit of the cell seeding bottle 22 to pressurize the cell suspension inside, and the cell suspension passes through the conduit and is sent to the culture vessel 1 through the drain pipe 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 (the same applies below).

[0069] Next, when a predetermined amount of 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 the closure of solenoid valve 47, and the gas that had been pressurizing cell seeding bottle 22 is discharged to the vent filter that is open to the outside from branching part 61, and the movement of the liquid stops.

[0070] Next, solenoid valves 48 and 49 are closed, solenoid valve 46 is opened, and pump 17 is operated, so that the cell suspension in the conduit 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, so that the cell suspension in the conduit returns to cell seeding bottle 22 due to the difference in head, and the liquid in the conduit disappears, all solenoid valves are closed, and the cell seeding process is completed (S03).

[0071] 10C shows the process of gas exchange into the culture vessel 1 in Example 1. In the initial state, the solenoid valve closes the rubber tube and is in a closed state. Water is held in the humidifying bottle 13, and the opening of a long tube is provided at the bottom of the vessel. One end of this tube is connected to the gas flow control unit 11 and the gas cylinder 10.

[0072] The opening of the short tube in the humidifying bottle 13 is provided at the top inside the vessel, and the ventilation adapter is connected to one end of this. 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 ventilation space 8 of the ventilation adapter 7 increases and is maintained at a predetermined gas concentration, so that gas exchange to the culture vessel 1 continues (S04).

[0073] 10D 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 closed by closing the rubber tube. Culture medium is held in culture medium bottle 20, which is attached to weight sensor 27. First, solenoid valve 53 is opened, opening the liquid supply pipe 18 of culture vessel 1 and the pipeline to culture medium bottle 20. Next, when pump 16 is operated, the culture medium passes through the pipeline and is sent to culture vessel 1 via liquid supply pipe 18.

[0074] 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 due to the stopping of 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 that is open to the outside at the branching point 63.

[0075] Next, by closing the solenoid valve 53 and operating the pump 16, the culture medium in the conduit 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 conduit can be removed, all the solenoid valves are closed, and the culture medium addition process is completed (S05). After that, if the culture medium needs to be replaced, the process proceeds to (S06) described below, and if the culture medium does not need to be replaced, the process proceeds to (S08) described below.

[0076] 10E 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 closed by closing the rubber tube. The 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, opening the drainage pipe 21 of the culture vessel 1 and the pipeline leading to the supernatant collection bag 23. Next, when pump 17 is operated, the culture medium is sent from the culture vessel 1 through the drainage pipe 21 and reaches the supernatant collection bag 23.

[0077] Next, when solenoid valve 49 is opened, the culture medium in the conduit closer to culture vessel 1 than branch 64 (same as branch 61 in Figure 10B) is sent to culture vessel 1 by head drop. After that, solenoid valve 46 is closed and pump 17 is operated, outside air is introduced through the vent filter, and the culture medium in the conduit reaches supernatant collection bag 23. All solenoid valves are closed, and the culture medium discharge process is completed (S06). Next, culture medium replacement can be performed by similarly performing the process of adding culture medium to culture vessel 1 described using Figure 10D (S07). After the culture medium replacement, rocking culture vessel 1 with rocking mechanism 30 to mix new culture medium with the cells will further promote cell culture.

[0078] FIG. 10F shows the process of sampling supernatant from culture vessel 1 in Example 1. In the initial state, the pump is stopped and the solenoid valve is closed by closing the rubber tube. Culture medium is held in culture vessel 1, and supernatant analysis bag 24 is installed empty. First, solenoid valve 46, solenoid valve 52, and manual valve 56 are opened, opening the drain pipe 21 of culture vessel 1 and the pipeline leading to supernatant analysis bag 24. Next, when pump 17 is operated, the culture medium is sent from culture vessel 1 through drain pipe 21 and reaches supernatant collection bag 23.

[0079] Next, when solenoid valve 49 is opened, the culture medium in the conduit closer to culture vessel 1 than branch 64 is sent to culture vessel 1 by head drop. After that, solenoid valve 46 is closed and pump 17 is operated, outside air is introduced through the vent filter, and the culture medium in the conduit reaches supernatant analysis bag 24. All solenoid valves are closed, and the supernatant sampling process ends (S08). If it is determined as a result of sampling the culture supernatant that cell culture has ended, the process proceeds to (S09) described below, and if it is determined that cell culture needs to be further continued, the process returns to (S04) described above.

[0080] The cell recovery process from the culture vessel 1 in Example 1 will be explained with reference to Figures 10E and 10G. After the cells have sufficiently proliferated, they settle to the bottom of the culture vessel and float. As in the supernatant recovery process shown in Figure 10E, the supernatant is recovered from the culture vessel 1 using the same flow path circuit, and supernatant recovery is continued until the culture medium 3 is below the opening height of the drainage pipe 21. This increases the ratio of culture medium 3 to cells in the culture vessel 1. Next, the culture vessel is rocked by the rocking mechanism 30, which agitates the culture medium and cells to form a cell suspension.

[0081] Next, the cell recovery process from the culture vessel 1 will be explained using Figures 10G and 10A. In the initial state, the pump is stopped and the solenoid valve is closed by closing the rubber tube. The culture vessel 1 holds a cell suspension 65, and the cell recovery bottle 26 is installed empty.

[0082] Next, solenoid valves 50 and 51 are opened, opening the collection pipe 25 of the culture vessel 1, the pipeline from the cell collection bottle 26, and the pipeline to the CO2 gas vent filter 15, which is open to the outside air. Next, when pump 17 is operated, cell suspension 65 is sent from the culture vessel 1 through collection pipe 25 and reaches the cell collection bottle 26.

[0083] Next, when the entire volume 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 liquid transfer stops. All solenoid valves are closed and the cell collection process ends (S09).

[0084] After the cells are collected, the waste 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.

[0085] As explained above, the automated culture device according to this embodiment can perform culture while miniaturizing the device. Furthermore, it is possible to reduce the risk of rust on mechanical components, such as the culture vessel rocking mechanism and the cell camera observation mechanism, used in the culture operation, and implement these components. Additionally, the partial supply of humidified gas to the culture vessel using the ventilation adapter can also reduce the risk of microbial growth throughout the incubator. Note that this embodiment describes the invention using humidified gas. However, if the evaporation of the culture medium 3 through the ventilation surface 4 has little impact on the culture results, dry gas, rather than humidified gas, may be supplied to the ventilation surface 4. In this embodiment, dry gas can be supplied by directly connecting the flow control unit 11 to the ventilation adapter 7 without using the humidifying bottle 13 shown in Figures 1 and 10. This further reduces the impact on mechanical components and the risk of microbial growth, and also reduces the labor required for maintenance, such as refilling the humidifying bottle with water. [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 ventilation space, 9 gas supply unit, 10 gas cylinder, 11 flow rate control unit, 12 pressure sensor, 13 humidification bottle, 14 CO2 sensor, 15 CO2 gas vent filter, 16, 17 pump, 18 liquid supply tube, 19 solenoid valve, 20 culture medium bottle, 21 suction tube (drainage 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, 71 perforated bottom surface, 72 gas retention space forming unit, 73 gas flow meter, 74 holding jig.

Claims

1. With a culture vessel for culturing cells placed on the vessel, CO ２ a ventilation adapter capable of supplying a gas containing The ventilation adapter ２ a gas supply unit that supplies a gas containing An automatic culture device comprising: When the culture vessel is placed at a predetermined position on the ventilation adapter, the upper portion thereof directly or indirectly supports at least a part of the gas exchange membrane provided below the culture vessel, and the lower end portion thereof contacts at least a part of the upper surface of the ventilation adapter, thereby allowing the CO 2 supplied from the gas supply unit to pass through the gas exchange membrane. ２ a gas holding space forming portion capable of airtightly holding a gas containing the An automatic culture device comprising:

2. The automatic culture device according to claim 1, An automatic culture device characterized in that the gas retention space forming portion has an approximately cylindrical shape and is structured to be in airtight contact with the inner surface of a skirt portion that surrounds the gas exchange membrane provided below the culture vessel.

3. The automatic culture device according to claim 1, An automatic culture device comprising a pressing jig for pressing the culture vessel against the gas holding space forming portion.

4. The automatic culture device according to claim 1, The gas holding space forming portion is characterized in that at least one of the portion that directly or indirectly supports at least a portion of the gas exchange membrane and the portion that contacts at least a portion of the upper surface of the ventilation adapter is made of a flexible material.

5. The automatic culture device according to claim 1, the gas holding space forming portion is made of a hard material having a substantially cylindrical shape, An automatic culture device characterized in that at least one of the portion that directly or indirectly supports at least a portion of the gas exchange membrane and the portion that contacts at least a portion of the upper surface of the ventilation adapter is made of a flexible material.

6. The automatic culture device according to claim 1, the gas holding space forming portion is made of a hard material having a substantially cylindrical shape, An automatic culture device characterized in that at least a portion of the portion of the gas holding space forming portion that contacts the upper surface of the ventilation adapter has a first airtight sealing portion that is interposed between the gas holding space forming portion and the upper surface of the ventilation adapter and that improves the airtightness between the gas holding space forming portion and the ventilation adapter.

7. The automatic culture device according to claim 1, An automatic culture device characterized in that it has a second airtight sealing portion interposed between the portion that directly or indirectly supports at least a portion of the gas exchange membrane of the gas retention space forming portion and the portion that directly or indirectly supports at least a portion of the gas exchange membrane, in order to improve the airtightness between the portion that directly or indirectly supports at least a portion of the gas exchange membrane and the gas retention space forming portion.

8. The automatic culture device according to claim 6, The automatic culture device is characterized in that the first airtight sealing portion is an O-ring that fits into a groove provided in a part of the lower surface of the gas holding space forming portion.

9. The automatic culture device according to claim 7, The automatic culture device is characterized in that the second airtight sealing portion is made of a ring-shaped member having an engaging portion that engages with a part of the upper end of the gas holding space forming portion.

10. The automatic culture device according to claim 9, The ring-shaped member has a cross section that is approximately L-shaped, and the shorter side of the L shape is configured to engage with a groove portion provided in the gas retention space forming portion.

11. The automatic culture device according to claim 9, The automatic culture device is characterized in that the ring-shaped member has a cross section that is approximately I-shaped, and the lower end of the I-shape has a structure that engages with a groove portion provided in the gas retention space forming portion.

12. The automatic culture device according to claim 3, The automatic culture device is characterized in that the holding jig has a structure that can be opened and closed around a rotation axis that extends vertically.

13. The automatic culture device according to claim 3, The automatic culture device is characterized in that the holding jig has a hole for fixing the ventilation adapter and the holding jig via a fastener that is inserted into a hole provided in a support member that supports the ventilation adapter.

14. The automatic culture device according to any one of claims 1 to 13, When the culture vessel is placed at a predetermined position on the ventilation adapter, the CO ２ The automatic culture device is characterized in that the pressure in the space that airtightly holds the gas containing the above-mentioned is higher than the atmospheric pressure at the location where the culture vessel is placed.

15. With a culture vessel for culturing cells placed on the vessel, CO ２ a ventilation adapter capable of supplying a gas containing The ventilation adapter ２ a gas supply unit that supplies a gas containing When the culture vessel is placed at a predetermined position on the ventilation adaptor, the upper part of the ventilation adaptor directly or indirectly supports at least a part of the gas exchange membrane provided below the culture vessel, and the lower end of the ventilation adaptor is in contact with at least a part of the upper surface of the ventilation adaptor, so that the CO 2 supplied from the gas supply unit is absorbed. ２ a gas holding space forming portion capable of airtightly holding a gas containing the A cell culture method using an automatic culture device comprising: placing the culture vessel in a predetermined position on the ventilation adapter; The CO ２ and supplying a gas containing CO ２ and retaining a gas containing the A cell culture method comprising:

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