Automated cultivation device

Abstract

To provide a culture device that can use multiple types of culture vessels, that is, a device capable of culturing multiple types of cells. [Solution] An automated culture apparatus equipped with a rocking mechanism for rocking placed culture vessels, wherein the rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights inside, and the rocking mechanism is capable of being fitted with a stand that adjusts the height of the culture vessels so that the distance from the rocking center of the rocking mechanism to the culture surface is approximately equal. Furthermore, the automated culture apparatus is equipped with a rocking mechanism for rocking the placed culture vessels, the rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights, and the rocking mechanism can be fitted with a stand that adjusts the height of the culture vessels so that the stress on the cultured cells near the culture surface is approximately equal.

Description

Technical Field

[0001] The present invention relates to an automatic culture device. [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​[Problems that the invention aims to solve]

[0008] While the variety of cells that can be cultured is increasing, a culture vessel suitable for each type of cell is selected. Culture equipment is designed and manufactured specifically for each culture vessel, meaning users must purchase the appropriate culture vessel and culture equipment for each type of cell they wish to culture. In other words, if a user wants to culture multiple types of cells, they need to prepare expensive culture equipment for each type, which is costly.

[0009] The objective of the present invention is to provide an automated culture device that can use multiple types of culture vessels, that is, a device capable of culturing multiple types of cells. [Means for solving the problem]

[0010] The configuration of the present invention for achieving the above objective is as follows. An automated culture apparatus equipped with a rocking mechanism for rocking placed culture vessels, wherein the rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights, and the rocking mechanism is capable of being fitted with a stand that makes the distance from the culture surface of the two or more culture vessels to the rocking center of the rocking mechanism approximately equal.

[0011] Furthermore, the automated culture apparatus is equipped with a rocking mechanism for rocking the placed culture vessels, the rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights, and the rocking mechanism can be fitted with a stand that ensures the stress on cultured cells near the culture surface of the two or more culture vessels is approximately equal.

[0012] Furthermore, the automatic culture apparatus is equipped with a rocking mechanism for rocking the placed culture vessels, the rocking mechanism is capable of rocking two or more culture vessels with different heights of culture surfaces inside the vessels, and the rocking mechanism is equipped with culture vessel mounting sections on both the vertical and horizontal sides on which culture vessels can be placed, and the distance from the rocking center of the rocking mechanism to the culture surfaces of the two or more culture vessels placed on the culture vessel mounting section is approximately equal.

[0013] Furthermore, the automatic culture apparatus is equipped with a rocking mechanism for rocking the placed culture vessels, the rocking mechanism is capable of rocking two or more culture vessels with different heights of culture surfaces inside the vessels, and the rocking mechanism is equipped with culture vessel mounting sections on both the vertical and horizontal sides on which culture vessels can be placed, and the stress on the culture surfaces of the two or more culture vessels placed in the culture vessel mounting section is approximately equal. [Effects of the Invention]

[0014] According to the present invention, it is possible to provide an automated culture device that can use multiple types of culture vessels, that is, a device capable of culturing multiple types of cells. [Brief explanation of the drawing]

[0015] [Figure 1] This figure shows one configuration of the automated culture apparatus according to Example 1. [Figure 2] This diagram shows a side view of one configuration of the automated culture apparatus according to Example 1. [Figure 3A] This figure shows an example of a flow channel circuit including a closed-system culture vessel according to Example 1. [Figure 3B] This figure shows an example of the cell seeding procedure according to Example 1. [Figure 3C] This figure shows an example of the gas exchange procedure according to Example 1. [Figure 3D] This figure shows an example of the procedure for adding culture medium according to Example 1. [Figure 3E] This figure shows an example of the procedure for changing the culture medium according to Example 1. [Figure 3F] This figure shows an example of the procedure for sampling the supernatant according to Example 1. [Figure 3G] It is a diagram showing an example of the procedure for cell collection according to Example 1. [Figure 4] It is a diagram showing the flow during the operation of the automatic culture device according to Example 1. [Figure 5] It is a diagram showing the state of the inclination of the culture vessel during cell collection according to Example 1. [Figure 6] It is a schematic diagram of a flask culture vessel applicable to Example 1. [Figure 7] It is a schematic diagram of a peel-off culture vessel applicable to Example 1. [Figure 8] It is a schematic diagram of the pedestal according to Example 1. [Figure 9] It is a perspective view of the automatic culture device to which the pedestal according to Example 1 is applied. [Figure 10] It is a diagram for explaining the rocking moment applied to the culture surface. [Figure 11] It is a schematic diagram in which a container installation part is provided above and below the rocking mechanism according to the present example. [Figure 12] It is a schematic diagram in which a plurality of culture vessels are placed on the rocking stage according to the present example. [Figure 13] It is a diagram for explaining the holder according to Example 2. [Figure 14] It is a diagram for explaining the holder according to Example 2.

Embodiments for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The following description shows specific examples of the content of the present invention, and the present invention is not limited to these descriptions, and various changes and modifications can be made by those skilled in the art within the scope of the technical idea disclosed in this specification.

[0017] Also, in all the drawings for explaining the present invention, those having the same function are denoted by the same reference numerals, and the repeated description thereof may be omitted.

Examples

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Figure 3A shows an example of a flow channel circuit 45 including a closed 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.

[0037] 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.

[0038] 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.

[0039] Figure 4 is a flowchart showing the overall operation of cell culture in the automated culture device 100 shown in Figure 3A. After "START", the flow path is installed in the automated culture device 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 path (S02).

[0040] Figure 3B 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.

[0041] 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).

[0042] 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.

[0043] Next, when solenoid valves 48 and 49 are closed, solenoid valve 46 is opened, and pump 17 is activated, the cell suspension in the conduit 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 conduit returns to the cell seeding bottle 22 due to gravity, and the liquid in the conduit is removed, all solenoid valves are closed, and the cell seeding process is completed (S03).

[0044] Figure 3C 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.

[0045] 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).

[0046] Figure 3D 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.

[0047] 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.

[0048] 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).

[0049] Figure 3E 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 activated, the culture medium is pumped from culture vessel 1 through the suction tube 21 and reaches the supernatant collection bag 23.

[0050] 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.

[0051] 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.

[0052] Figure 3F 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.

[0053] 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.

[0054] 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).

[0055] Figures 3E and 3G illustrate the cell recovery process from 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 3E, the supernatant is recovered from 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 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.

[0056] Next, the cell retrieval process from culture vessel 1 will be explained with reference to Figures 3G and 3A. 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.

[0057] Figure 5 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 must be able to tilt in a certain 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 positioned so that it can tilt toward the direction where the opening of the retrieval tube 25 is located. At that time, a mark indicating the orientation of the culture vessel 1 may be placed on the ventilation adapter 7 fixed to the rocking part 70.

[0058] Next, solenoid valves 50 and 51 are opened, opening the pipeline from the culture vessel 1's recovery pipe 25 to the cell recovery bottle 26, and the pipeline to the CO2 gas vent filter 15 which is open to the outside air.

[0059] Next, when the pump 17 is activated, the cell suspension 65 is delivered from the culture vessel 1 through the recovery tube 25 and reaches the cell recovery bottle 26.

[0060] 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).

[0061] 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.

[0062] Incidentally, in addition to the bottle-shaped container shown in Figure 1, there are also other types of culture vessels, such as the flask culture vessel shown in Figure 6 and the peel-off culture vessel shown in Figure 7. Flask-type culture vessels are mainly used for cell culture where cells adhere to the bottom surface of the container. Typically, two screw holes 101 are provided on the top surface of the main body, and each is closed with a ventilation cap 103 that has a gas-permeable membrane 102 at its opening.

[0063] When a flask culture vessel is used as a culture vessel in the automated culture apparatus shown in Figure 1, the ventilation cap 103 on the left screw opening 101 in Figure 6 is used as is for gas exchange by the ventilation adapter 7. On the right screw opening 101 in Figure 6, the gas-permeable membrane 102 and ventilation cap 103 are removed, and a ported cap 104 with a liquid delivery pipe is installed to perform automatic liquid delivery by the automated culture apparatus. The culture vessel 1 is placed on the 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. The ventilation adapter 7 is closely installed on the left ventilation cap 103 of the culture vessel 1. At this time, a predetermined gas space 8 is formed inside the ventilation cap 103 and the ventilation adapter 7.

[0064] The ported cap 104 is equipped with a liquid delivery tube 18, a suction tube 21, and a recovery tube 25 (also referred to as a "cell recovery tube"). These three ports are connected in the same way as the automated culture apparatus and flow path circuit in Figure 1 (Figure 3A), and the cell seeding process, gas exchange process, culture medium addition process, culture medium exchange process, supernatant sampling process, and cell recovery process described above can be carried out.

[0065] Furthermore, the peel-off culture vessel shown in Figure 7 has a culture area about the same size as an A4 sheet of copy paper, making it suitable for culturing large quantities of adherent cells. It is shallow, only a few centimeters deep, and the top is made of a film structure. After cell culture is complete, the film 701 can be peeled off, and cells can be collected from every corner of the vessel using a scraper. Similar to the flask culture vessel, the main body usually has two screw holes on the top surface, each closed with a ventilation cap 702 that has a gas-permeable membrane at the opening. It can be set in the automated culture device 100 and cell culture can be performed in the same way as described for the flask culture vessel.

[0066] When culture vessels 1 of different sizes and shapes are placed on the rocking stage 31 of the rocking mechanism 30 of the automated culture apparatus shown in Figure 1 and rocked, the height of the culture surface differs for each culture vessel, and the force (stress) applied to the cells by the rocking differs. In some cases, excessive stress may be applied, which could prevent cell culture from progressing properly.

[0067] Specifically, in the bottle-shaped culture vessel shown in Figure 1, the culture surface is directly above the ventilation surface 4. However, since the culture vessel 1 is placed on the rocking stage 31 via the ventilation adapter 7, the culture surface is raised by the height of the ventilation adapter 7. In the flask culture vessel shown in Figure 6, the bottom of the vessel is the culture surface. However, since the bottom of the vessel is placed in contact with the rocking stage 31, the height of the rocking stage 31 is approximately equal to the height of the culture surface. In the peel-off culture vessel shown in Figure 7, the bottom of the vessel is the culture surface, similar to the flask culture vessel, so the height of the rocking stage 31 is approximately equal to the height of the culture surface.

[0068] Furthermore, the force applied to cells by shaking can vary depending on the area of ​​the bottom surface of the culture vessel and the amount of culture medium liquid. Thus, although the automated culture apparatus shown in Figure 1 can accommodate culture vessels 1 of different shapes and sizes, the inventors have found that simply shaking the culture vessel 1 while performing cell culture can sometimes reduce the efficiency of the culture. To solve this problem, the inventors have invented a stand that is placed on the shaking stage 31 and adjusts the height of the culture surface for each type of culture vessel 1, thereby adjusting the stress on the culture surface.

[0069] Figure 8 shows an example of the structure of the stand 200. Four support columns 203, 204, 205, and 206 are provided between two opposing aluminum plates 201 and 202. The culture vessel 1 is placed on the upper aluminum plate 201. The height of the culture surface of the culture vessel 1 can be adjusted by changing the height (length) of the support columns 203, 204, 205, and 206. The stand 200 can be fixed on the rocking stage 31 using screws or a locking mechanism that can be fastened / released with a single touch (in Figure 8, 207, 208, 209, and 210 are locking mechanisms, and the knobs on top can be rotated clockwise to lock and counterclockwise to unlock) to prevent the stand 200 from shifting position even when the rocking stage 31 rocks.

[0070] Furthermore, it is desirable to provide a stopper 211 (only the left stopper is shown in Figure 8, but there is a similar stopper on the right side as well) on the upper surface of the aluminum plate 201 to hold down the outside of the installed culture container 1. The height (length) of the support columns 203, 204, 205, and 206 should be set so that the height of each culture surface is the same regardless of whether the culture container is a bottle-type culture container, a flask culture container, or a peel-off culture container. However, in practice, it is desirable to experimentally determine the optimal height according to the area of ​​the bottom surface of the culture container, the amount of culture medium used for cultivation, etc. Note that the amount of stress on the culture surface of each culture container due to the oscillation of the oscillation stage 31 will have an effect as an oscillation moment, as will be described later, but the culture efficiency will not change dramatically due to a difference in oscillation moment of, for example, a few percent. Considering the constraints such as the height, width, and depth inside the incubator 35 in Figure 1, the optimal support column height can be determined after experimentally confirming that it does not affect the culture efficiency.

[0071] Furthermore, the three holes 212, 213, and 214 provided in the upper aluminum plate 201 in Figure 8 are weight-reducing holes for the aluminum plate 201, and it is preferable to provide openings that do not affect the strength of the aluminum plate 201.

[0072] Figure 9(a) shows a peel-off culture container 301 placed on the rocking stage 31 via the stand 200, and Figure 9(b) shows an overhead view of a bottle-type culture container 302 placed directly on the rocking stage 31. As shown by the dashed lines, the culture surfaces are at approximately the same height. Although the rocking stage 31 is not shown in Figure 9(b), the same rocking stage 31 as in Figure 9(a) is also present in Figure 9(b).

[0073] The technical concept of aligning the height of the culture surface will be further explained using Figure 10. Figures 10(a) and 10(b) show the concept of the culture surface height when using a peel-off culture vessel and a bottle-type culture vessel, respectively, with the rocking stage in a horizontal state (the rocking stage is not operating). If the respective culture vessels are placed directly on the rocking stage 31, the culture surface heights will differ between the peel-off culture vessel 301 and the bottle-type culture vessel 302. Therefore, when using the peel-off culture vessel 301, a stand 200 is placed on the rocking stage, and the peel-off culture vessel 301 is placed on the stand 200 to ensure that the culture surface heights are the same.

[0074] Figures 10(c) and 10(d) show the state in which the oscillating stage 31 is tilted when it is operated. The oscillating stage 31 oscillates left and right and front and back (forward and depth directions in Figure 10) around the oscillation center indicated by the black circle. When this occurs, an oscillation moment is applied to the culture surface, and the magnitude of the oscillation moment changes as the distance between the oscillation center and the oscillation surface changes. Since this oscillation moment represents the magnitude of the stress on the cells, the technical concept of this invention is to keep the distance from the oscillation center to the culture surface the same so that the oscillation moment remains the same even when the culture vessel changes.

[0075] If the distance from the center of oscillation to the culture surface is the same, the stress on the culture surface will be the same. Therefore, as shown in Figure 11(a), by creating a two-tiered structure for the installation surface of the culture vessels and making the distance between each culture vessel installation surface and the center of oscillation the same, the number of culture vessels that can be cultured can be doubled while keeping the bottom area of ​​the incubator 35 (which can also be called the footprint of the automated culture device) the same. The culture vessels to be installed can be the same peel-off culture vessel, or one can be a bottle-type culture vessel and the other a peel-off culture vessel.

[0076] In the latter case, as shown in Figure 11(b), it is preferable to excavate the lower installation surface of the two-story installation surface, install the bottle-type culture container below the container installation surface (upper surface of the rocking stage), and lower the culture surface of the bottle-type culture container.

[0077] To further increase the number of culture vessels that can be installed, four culture vessels may be installed on the rocking stage at equidistant from the center of oscillation, as shown in Figure 12(a). Furthermore, by creating a two-story structure as shown in Figure 11, it is possible to perform cultivation using 4 × 2 = 8 culture vessels, as shown in Figure 12(b). [Examples]

[0078] In the automated culture apparatus shown in Figure 1, the amount of culture medium in the culture medium bottle 20 is measured by a weight sensor 27. However, when the apparatus operator swaps the culture medium bottle 20 with the supernatant collection bag 23, the weight sensor 27 also measures the weight of the supernatant collection bag 23. In this case, since the culture medium bottle 20 is made of rigid resin, it is possible to measure its weight by placing it on the weight sensor 27. However, since the supernatant collection bag 23 is made of flexible resin, if it is placed directly on the weight sensor 27, the bag may bend or fold in the middle, making it difficult to accurately measure its weight.

[0079] Therefore, a holder 80 was created that can measure the weight of the supernatant collection bag 23 using a weight sensor 27, without interfering with the weight measurement of the culture medium bottle 20.

[0080] Figure 13(a) shows a perspective view of the gas supply unit 9, which corresponds to the right half of the automated culture apparatus shown in Figure 1, and Figure 13(b) shows a perspective view of the gas supply unit 9 viewed from the right. Figure 13 shows the culture medium bottle 20 placed on the weight sensor 27. A wedge-shaped jig 81 is placed on top of the weight sensor 27, and a holder 80 is placed on top of the wedge-shaped jig 81, so that all of the culture medium contained in the culture medium bottle 20 can be sucked up through a tube inserted from the opening at the top of the culture medium bottle 20.

[0081] The holder 80 has a large opening at the front so that the culture medium bottle 20 does not interfere with the holder 80 when placed on the wedge-shaped jig 81. Furthermore, if the culture medium bottle is rectangular when viewed from above, the holder has a groove 83 (a ridge when viewed from the back) in the center of the back surface so that the tip of the tube can be positioned at the corner of the culture medium bottle (towards the innermost part in Figure 13(a)) and the culture medium can be drawn up to the end.

[0082] Figure 14 shows the supernatant collection bag 23 placed on the weight sensor 27. The procedure for swapping the culture medium bottle 20 and the supernatant collection bag 23 is as follows. First, the device operator lifts the culture medium bottle 20, which is on the weight sensor 27, upwards and places it on the culture medium bottle holder 84 on the left. Next, the supernatant collection bag 23, which was placed behind the culture medium bottle 20, is pulled out. At this time, since a tube is connected to the bottom of the supernatant collection bag 23, the tube is pulled out to the front while avoiding the holder 80, and together with the supernatant collection bag 23, it is set into the holder 80 through the notch 82 at the front of the holder 80. At this time, it is desirable to provide a groove 85 for the tube to pass through on the upper surface of the wedge-shaped jig 81 and the holder 80 so that the tube at the bottom of the supernatant collection bag 23 is not crushed by the supernatant collection bag 23.

[0083] As the supernatant is collected, the supernatant collection bag 23 may tilt forward due to the weight of the upper part of the bag. To prevent the supernatant collection bag 23 from detaching from the holder 80 even if it tilts, it is preferable that the notch 82 provided on the front surface of the holder 80 has a narrowed portion 86 that is narrower at the top.

[0084] From the above, the holder 80 of Example 2 is characterized in that it can hold both a hard container (culture medium bottle 20) and a soft container (supernatant collection bag 23), and has a groove on one side that fits the corner of the hard container. Alternatively, the soft container is placed in the holder 80 so that the tube of the soft container is facing downwards, and the holder 80 has a groove for passing the tube through. Alternatively, the notch provided on the front of the holder 80 has a narrowed portion 86 that is narrower at the top. Furthermore, a wedge-shaped jig 81 is provided below the holder 80, and a weight sensor 27 is provided below the jig. One or more of these features are incorporated into the automatic culture apparatus and holder 80 of the present invention. [Explanation of symbols]

[0085] 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 Liquid delivery tube, 19 Solenoid valve, 20 Culture medium bottle, 21 Suction tube, 22 Cell seeding bottle, 23 Supernatant collection bag, 24 Supernatant analysis bag, 25 Collection tube, 26 Cell collection bottle, 27 Weight sensor, 28 Weight sensor, 29 Fluid control unit, 30 Oscillating mechanism

Claims

1. An automated culture apparatus equipped with a rocking mechanism for rocking the culture vessel on which it is placed, The rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights within the culture vessel, The automatic culture apparatus is characterized in that the rocking mechanism can be fitted with a stand that causes the distance from the culture surface of two or more of the culture vessels to the rocking center of the rocking mechanism to be approximately equal.

2. An automated culture apparatus equipped with a rocking mechanism for rocking the culture vessel on which it is placed, The rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights within the culture vessel, The automatic culture apparatus is characterized in that the rocking mechanism can be fitted with a stand that causes the stress on cultured cells near the culture surface of two or more types of culture vessels to be approximately equal.

3. An automated culture apparatus equipped with a rocking mechanism for rocking the culture vessel on which it is placed, The rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights within the culture vessel, An automated culture apparatus characterized in that it is provided with culture vessel mounting sections on each of the vertical directions of the rocking mechanism on which the culture vessels can be placed, and the distance from the rocking center of the rocking mechanism to the culture surface of two or more types of culture vessels installed in the culture vessel mounting section is approximately equal.

4. An automated culture apparatus equipped with a rocking mechanism for rocking the culture vessel on which it is placed, The rocking mechanism is capable of rocking two or more culture vessels with different culture surface heights within the culture vessel, An automated culture apparatus comprising a culture vessel mounting section on each of the vertical directions of the rocking mechanism on which the culture vessels can be placed, wherein the culture vessel mounting section is characterized in that the stress on the culture surfaces of two or more types of culture vessels installed in the culture vessel mounting section is substantially equal.

5. In the automated culture apparatus according to claim 1, An automated culture apparatus characterized in that multiple culture vessels can be placed on the stand, and the distance from the culture surface in the multiple culture vessels to the center of oscillation is approximately equal.

6. In the automated culture apparatus according to claim 2, An automated culture apparatus characterized in that multiple culture vessels can be placed on the frame, and the stress on the culture surface in the multiple culture vessels is approximately equal.

7. In the automated culture apparatus according to claim 1 or 2, The automatic culture apparatus is characterized in that the frame has a height adjustment section that allows the height to be adjusted.

8. In the automated culture apparatus according to any one of claims 1 to 6, An automated culture apparatus characterized by having a gas supply unit capable of directly supplying the gas necessary for cell culture to the culture vessel.

9. In the automated culture apparatus according to claim 4, An automated culture apparatus characterized in that a plurality of culture vessels can be installed in the culture vessel installation section.

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