Automatic culture device, automatic culture system, and cell culture method

Abstract

The following configuration was used to provide an automatic culture device, an automatic culture system, and a culture method with which it is possible to accurately feed a liquid regardless of the type of cell culture vessel, cell suspension vessel, or cell collection vessel used and with which it is possible to supply and collect cells with reduced physical damage to the cells by feeding a cell suspension between any containers without passing through the drive part of a pump mechanism.　An automatic culture device according to the present invention includes: a cell suspension storage vessel capable of storing a cell suspension; a cell processing vessel for processing a cell suspension fed from the cell suspension storage vessel; a flow path for connecting at least the cell suspension storage vessel and the cell processing vessel; a first buffer space connected to the flow path and having a predetermined pressure resistance; and a pressure source for drawing the cell suspension from the cell suspension storage vessel into the first buffer space by creating negative pressure within the first buffer space.

Description

Automatic culture device, automatic culture system, and cell culture method

[0001] The present invention relates to an automatic culture device for culturing cells or tissues, an automatic culture system, and a cell culture method.

[0002] In recent years, cell therapy, which involves transplanting cultured cells and tissues to repair and restore damaged or dysfunctional tissues, has attracted attention. Until now, cell products have been manufactured manually, but issues such as high manufacturing costs and quality variations due to the level of operator skill have led to the promotion of automation. However, the scale of cell product manufacturing varies depending on whether it is autologous therapy using the patient's own cells or allogeneic therapy using cells from healthy donors. Furthermore, the manufacturing process spans a wide range of steps, including cell selection, washing, gene transfer, proliferation, differentiation induction, and organization. Therefore, cell manufacturing may involve the use of multiple devices specialized for each process, with each device requiring the supply and recovery of cells.

[0003] When cells are supplied to each device, it is common to provide a liquid delivery mechanism (drive unit) such as a peristaltic pump between a container containing the cell suspension and a container to which the liquid is to be delivered, and to connect them with a tube. For example, Patent Document 1 describes such a technique.

[0004] International Publication No. 2015 / 025425

[0005] When a culture medium is delivered using a fluid delivery mechanism with a drive unit such as a peristaltic pump, there is a concern that the cells may be physically damaged as they pass through the drive unit. Patent Document 1 (Patent Document 1) (Figure 11) discloses a method for delivering cells without passing through a drive unit by supplying gas to a container containing a cell suspension to pressurize it. However, since users use various containers (e.g., flexible synthetic resin containers called bags) as cell culture containers, containers containing cell suspensions, and cell collection containers, there is a concern that if the containers pressurized to deliver the cell suspension have low pressure resistance, problems with fluid delivery may occur.

[0006] The object of the present invention is to provide an automatic culture device, an automatic culture system, and a cell culture method that enable accurate liquid transfer regardless of the type of cell culture vessel, container containing cell suspension, or cell collection vessel used, and that enable the supply and collection of cells with reduced physical damage to the cells by transferring the cell suspension between any containers without passing through the drive part of the pump mechanism.

[0007] The present invention achieves the above-mentioned object by providing an automatic culture device comprising: a cell suspension storage container capable of storing a cell suspension; a cell processing container for processing the cell suspension delivered from the cell suspension storage container; a flow path connecting at least the cell suspension storage container and the cell processing container; a first buffer space having a predetermined pressure resistance connected to the flow path; and a pressure source for drawing the cell suspension from the cell suspension storage container into the first buffer space by creating a negative pressure within the first buffer space. Also provided is an automatic culture system comprising the automatic culture device and a control device for controlling each mechanism of the automatic culture device, including the pressure source. Also, there is provided a cell culture method for an automatic culture device comprising a cell suspension storage container capable of storing a cell suspension, a cell processing container for processing the cell suspension delivered from the cell suspension storage container, a flow path connecting at least the cell suspension storage container and the cell processing container, a first buffer space having a predetermined pressure resistance connected to the flow path, and a pressure source capable of creating a negative pressure within the first buffer space, the cell culture method comprising the steps of: setting a liquid containing cells to be cultured in the cell suspension storage container; operating the pressure source to create a negative pressure within the first buffer space and transferring the liquid containing cells set in the cell suspension storage container into the first buffer space; and operating the pressure source to create a positive pressure within the first buffer space and transferring the liquid containing cells transferred to the first buffer space to the cell processing container.

[0008] According to the present invention, it is possible to provide an automatic culture device, an automatic culture system, and a cell culture method that can accurately transfer cells regardless of the type of cell culture vessel, cell suspension container, or cell collection container used, and that can supply and collect cells with reduced physical damage by transferring the cell suspension between any containers without passing through the drive unit of a pump mechanism. Problems, configurations, and effects other than those described above will be made clear by the description of the following embodiments.

[0009] FIG. 1 is a diagram showing a configuration of a cell suspension supply and recovery mechanism according to Example 1. FIG. 2 is a diagram showing an example of a procedure for supplying and recovering a cell suspension according to Example 1. FIG. 3 is a diagram showing a configuration of a flow path circuit of a culture device according to Example 1. FIG. 4 is a diagram showing a configuration of a cell suspension supply and recovery mechanism according to Example 2. FIG. 5 is a diagram showing a configuration of a cell suspension supply and recovery mechanism according to Example 3. FIG. 6 is a diagram showing an example of a procedure for supplying and recovering a cell suspension according to Example 3. FIG. 7 is a diagram showing a configuration of a cell suspension supply and recovery mechanism according to Example 4. FIG. 8 is a diagram showing an example of a procedure for supplying and recovering a cell suspension according to Example 4. FIG. 9 is a diagram showing a decrease in cell proliferation rate due to passage through a peristaltic pump.

[0010] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The following description shows specific examples of the contents of the present invention, but 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.

[0011] 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.

[0012] In this example, an example of supplying and recovering cells that do not pass through a drive unit using a pressure-resistant container will be described with reference to Figures 1 to 4. Figure 1 shows an example of the configuration of a closed flow path according to Example 1 of the present invention, and Figure 2 shows an example of the procedure for the flow path in Figure 1.

[0013] First, a cell processing device (also referred to as a "cell culture vessel" or "cell processing vessel") 1 that supplies cells is connected to a flow path (shown by a solid line without a symbol) using a connector 3 (S01 in FIG. 2 ). A container (also referred to as a "cell suspension container"; in this embodiment, the aforementioned "bag" is used) 7 containing a cell suspension (also referred to as a "suspension") is then connected to the flow path (S02 in FIG. 2 ). Next, solenoid valves 8 and 16 are opened (S03 in FIG. 2 ), and a peristaltic pump (also referred to as a "pressure source" or simply a "pump") 17 is rotated clockwise (S04 in FIG. 2 ) to create a negative pressure in a buffer space 13 (also referred to as a "first buffer space"), thereby drawing the suspension from the container 7 into the buffer space 13. Because the buffer space 13 is a container with sufficient pressure resistance, the container is not deformed by the negative pressure, and the suspension can be drawn into the buffer space 13.

[0014] Examples of pressure-resistant containers include those made of high-strength synthetic resins and glass containers. While transparent materials are preferred so that the state of the drawn-in suspension can be seen from the outside, containers can also be manufactured using metal materials such as stainless steel or aluminum, with a transparent window in one portion of the container providing visibility into the interior of the container. The required pressure-resistant capacity varies depending on the elevation difference and the diameter and length of the fluid delivery channel. For example, when using a channel with an inner and outer diameter of several millimeters, the required pressure-resistant capacity is approximately several hundred kPa. Qualitatively, "predetermined pressure resistance" refers to a level of pressure resistance that does not collapse even when negative pressure is applied to the buffer space 13. Therefore, if negative pressure is applied to a flexible synthetic resin container called a "bag," the bag will simply collapse and become flat, preventing the suspension from being drawn into the bag. Therefore, a "bag" does not qualify as a container with a predetermined pressure-resistant capacity.

[0015] The weight of the suspension drawn in is measured by measuring the weight of the buffer space with the weighing scale 14 (S05 in FIG. 2), and when the desired weight is reached, the pump 17 is stopped (S06 in FIG. 2) and the solenoid valves 8 and 16 are closed (S07 in FIG. 2).

[0016] Next, the solenoid valves 6 and 16 are opened (S08 in FIG. 2), and the pump 17 is rotated counterclockwise (S09 in FIG. 2), creating a positive pressure in the buffer space 13 and sending the suspension to the cell processing device 1. The weight of the buffer space is measured with the weighing scale 14 (S10 in FIG. 2), and when the desired weight is reached, the pump 17 is stopped (S11 in FIG. 2), and the solenoid valve is closed (S12 in FIG. 2).

[0017] After cell processing (e.g., cell culture, etc.) (S13 in FIG. 2), solenoid valves 5 and 15 are opened (S14 in FIG. 2) and pump 17 is rotated clockwise (S15 in FIG. 2) to create a negative pressure in buffer space 11 (also referred to as the "second buffer space"), and the processed suspension is drawn into buffer space 11 from cell processing device 1. The weight of the buffer space is measured with weighing scale 12 (S16 in FIG. 2), and when the desired weight is reached, pump 17 is stopped (S17 in FIG. 2), solenoid valves 5 are closed, and 9 are opened (S18 in FIG. 2).

[0018] Next, the pump 17 is rotated counterclockwise (S19 in FIG. 2) to create a positive pressure in the buffer space 11, and the suspension is sent to the cell collection container (also called a "collection container"; in this embodiment, a bag is used) 10. The weight of the buffer space is measured with the weighing scale 12 (S20 in FIG. 2), and when the desired weight is reached, the pump 17 is stopped (S21 in FIG. 2) and the solenoid valve is closed (S22 in FIG. 2).

[0019] The collection container 10 is then disconnected from the flow path and collected (S23 in FIG. 2). The reason for using two buffer spaces, the first and second, is that there is a concern that if the pre-processing cell suspension and the post-processing cell suspension mix, the pre-processing (seeded) cells will remain and die. Depending on the type of cells being processed and the processing content, mixing may not have any effect. In such cases, the first buffer space alone allows for the transfer of the cell suspension from the container 7 containing the cell suspension to the cell processing device 1 and the collection of the processed cell suspension into the cell collection container 10. This configuration allows for the miniaturization and cost reduction of the automatic culture device.

[0020] 1, the buffer space 13 is provided in the flow path between the connection part of the container 7 containing the cell suspension and the peristaltic pump 17, but it can also be provided in the flow path between the cell processing device 1 and the connection part of the container 7 containing the cell suspension. In that case, the flow path length can be shortened compared to the configuration in Fig. 1, which may enable the device to be made smaller and less expensive. Fig. 3 shows an example of the cell supply and recovery mechanism shown in Fig. 1 incorporated into a flow path capable of automatic culture (illustration of an example of the procedure in the flow path in Fig. 3 is omitted).

[0021] First, by opening solenoid valves 8, 16, and 26 and rotating pump 17 clockwise, negative pressure is created within buffer space 13 and cell suspension is drawn from container 7 into buffer space 13. After the desired amount has been drawn in, the pump is stopped and all valves are closed. Next, by opening solenoid valves 6, 16, and 26 and rotating pump 17 counterclockwise, positive pressure is created within buffer space 13 and the cell suspension is sent from buffer space 13 to cell processing container 1, after which the pump is stopped and all solenoid valves are closed.

[0022] During the culture, gas such as 5% CO is supplied from a gas cylinder 30 via a regulator 29 and a mass flow controller (also called a "mass flow meter") 28. ２A desired gas, such as a gas, is supplied to the cell treatment vessel 1 at a desired flow rate. In this embodiment, the supply gas is humidified using a humidifying bottle 32 to prevent evaporation of the culture medium in the vessel. When replacing the culture medium, first, solenoid valves 21 and 22 are opened and pump 17 is rotated clockwise to send the culture supernatant to a drainage container (also referred to as a "drainage collection container") 23. After the culture supernatant is completely discharged, the pump is stopped and all solenoid valves are closed. Next, solenoid valve 24 is opened and pump 20 is rotated counterclockwise to supply the culture medium from a culture medium bottle (also referred to as a "culture medium or other reagent supply bottle") 25 to the cell treatment vessel 1. After the desired amount of culture medium has been supplied, the pump is stopped and all solenoid valves are closed. When recovering cells at the end of the culture, solenoid valves 5, 15, and 26 are opened and pump 17 is rotated clockwise to create a negative pressure in the buffer space 11 and draw the cell suspension from the cell treatment vessel 1 into the buffer space 11. After all the suspension has been recovered, the pump is stopped and all solenoid valves are closed. Next, the solenoid valves 9, 15, and 26 are opened and the pump 17 is rotated counterclockwise to create a positive pressure in the buffer space 11, and the cell suspension is sent from the buffer space 11 to the cell collection container 10. After the sending is complete, the cell collection container 10 is aseptically separated from the flow path and moved to another operation.

[0023] In Example 1, a container was used as the buffer space, but the buffer space can also be a tube with a certain level of pressure resistance, as shown in Figure 4. By using a tube, a vent filter connected to a bottle or the like is no longer necessary, and the flow path configuration can be simplified.

[0024] Such a tube can be, for example, a coiled rubber tube made of silicone or thermoplastic elastomer with sufficient strength. The number of turns of the coil should be adjusted depending on the amount of suspension to be delivered. It is desirable to use a system in which the flow path diameter can be controlled by the pump or solenoid valve used and which has a certain level of pressure resistance. It is also possible to use a metal pipe. Although it is more expensive to introduce than a rubber tube, if the tube length is long it can be reused by sterilization, and costs can be reduced by using it multiple times.

[0025] In this example, an example in which a single buffer space is used for cell supply and cell recovery will be described with reference to Figures 5 and 7. Because a closed flow path prevents contamination by external contaminants, the buffer space used for cell supply can also be used for cell recovery. However, as mentioned above, depending on the type of cells being processed and the processing content, there is a concern that if the pre-processing cell suspension and the post-processing cell suspension are mixed, the pre-processing (seeded) cells may remain and die.

[0026] For this reason, in this example, a mechanism was provided to wash the flow path through which the cell suspension before processing was delivered and the used buffer space with a washing liquid such as physiological saline (PBS). This example is shown in Figure 5, which shows an example of the configuration of a closed flow path, and Figure 6, which shows an example of the procedure.

[0027] As in Example 1, the flow path is installed (S101 in FIG. 6 ), and the cell insertion container 7 is connected to the flow path (S102 in FIG. 6 ). Then, the solenoid valves 8, 16, and 24 are opened (S103 in FIG. 6 ), and the peristaltic pump 17 is rotated clockwise (S104 in FIG. 6 ) to create a negative pressure in the buffer space 13, drawing the suspension from the container 7 into the buffer space 13. The weight of the buffer space is measured with the weighing scale 14 (S105 in FIG. 6 ), and when the desired weight is reached, the pump 17 is stopped (S106 in FIG. 6 ), and the solenoid valves 8, 16, and 24 are closed (S107 in FIG. 6 ). Next, the solenoid valves 6, 16, and 24 are opened (S108 in FIG. 6 ), and the pump 17 is rotated counterclockwise (S109 in FIG. 6 ) to create a positive pressure in the buffer space 13, and the suspension is delivered to the cell processing device 1. The weight of the buffer space is measured by the weighing scale 14 (S110 in FIG. 6), and when the desired weight is reached, the pump 17 is stopped (S111 in FIG. 6) and the solenoid valves 6, 16, and 24 are closed (S112 in FIG. 6).

[0028] Next, solenoid valves 15, 22, and 26 are opened (S113 in FIG. 6 ), and pump 17 is rotated counterclockwise (S114 in FIG. 6 ), thereby supplying cleaning liquid from cleaning liquid bottle 31 to buffer space 13. The weight of the buffer space is measured with weighing scale 14 (S115 in FIG. 6 ), and when the desired weight is reached, solenoid valve 22 is closed and solenoid valve 24 is opened (S116 in FIG. 6 ). By maintaining counterclockwise rotation of pump 17 for a predetermined time, all of the cleaning liquid in the flow path is pushed into buffer space 13, and then pump 17 is stopped (S117 in FIG. 6 ), and solenoid valves 15, 22, and 26 are closed (S118 in FIG. 6 ).

[0029] Next, the solenoid valves 16, 21, and 24 are opened (S119 in FIG. 6 ), and the pump 17 is rotated counterclockwise (S120 in FIG. 6 ) to create a positive pressure in the buffer space 13, thereby sending the cleaning solution to the waste liquid collection container 23. The weight of the buffer space 13 is measured with the weighing scale 14 (S120 in FIG. 6 ), and when the desired weight is reached, the pump 17 is stopped (S121 in FIG. 6 ), and the solenoid valves 16, 21, and 24 are closed (S122 in FIG. 6 ). After cell processing in the culture processing device 1 (S123 in FIG. 6 ), the solenoid valves 5, 16, and 24 are opened (S124 in FIG. 6 ), and the pump 17 is rotated clockwise (S125 in FIG. 6 ) to create a negative pressure in the buffer space 13, and the suspension is drawn into the buffer space 13 from the cell processing device 1. The weight of the buffer space is measured by the weighing scale 14 (S126 in FIG. 6), and when the desired weight is reached, the pump 17 is stopped (S127 in FIG. 6) and the solenoid valves 5, 16, and 24 are closed (S128 in FIG. 6).

[0030] Next, the solenoid valves 9, 16, and 24 are opened (S129 in FIG. 6 ), and the pump 17 is rotated counterclockwise (S130 in FIG. 6 ) to create a positive pressure in the buffer space 13, and the suspension is sent to the cell collection container 10. The weight of the buffer space 13 is measured with the weighing scale 14 (S131 in FIG. 6 ), and when the desired weight is reached, the pump 17 is stopped (S132 in FIG. 6 ), and the solenoid valves 9, 16, and 24 are closed (S133 in FIG. 6 ). Thereafter, the collection container 10 is disconnected from the flow path and collected (S134 in FIG. 6 ).

[0031] In this example, an example of measuring the weight of a cell collection container when collecting cells will be described with reference to Figures 7 and 8. Figure 7 shows an example of the configuration of a closed flow path according to Example 4 of the present invention, and Figure 8 shows an example of the procedure for the flow path of Figure 7. By measuring the weight of the collection container 10 when collecting cells, it is possible to confirm the exact amount of collected cells. Furthermore, although collection is performed in a single collection container in Figure 7, it is also possible to distribute equal amounts by connecting multiple collection containers.

[0032] When a bag is used as the collection container 10, space can be saved by using a hanging balance, but the shape of the bag changes when the suspension is supplied, which changes the center of gravity and makes it impossible to measure the weight accurately.

[0033] 8, steps S01 to S18 are exactly the same as those in Fig. 2, and therefore will not be described further. The difference from the steps in Example 1 is that when the suspension is sent from the buffer space 11 to the collection container 10, the pump 20 is rotated left (S219 in Fig. 8) to simultaneously supply gas to the collection container 10, thereby keeping the shape of the collection container 10 constant and enabling accurate weight measurement. The pressure inside the collection container 10 may be measured, and feedback may be applied to the operation of the pump 20 to maintain the pressure constant.

[0034] The weights of the buffer space 11 and the collection container 10 were measured (S220 in FIG. 8 ). When the weight scale 12 for the buffer space 11 was below a specified value and the weight scale 33 for the collection container 10 was above a specified value, the pumps 17 and 20 were stopped (S21 and S221 in FIG. 8 ), the solenoid valves 9 and 15 were closed (S22 in FIG. 8 ), and the cell collection container 10 was collected from the flow path (S23 in FIG. 8 ). <Note> FIG. 9 shows an evaluation of physical damage to cells caused by a peristaltic pump using iPS cells (strain 201B7). Cells were detached and left to stand, passed through the peristaltic pump 10 times, and pumped 10 times using the non-pumping method described in Patent Document 1. The proliferation rates were evaluated after 7 days of expansion culture. The proliferation rate was significantly lower when the cell suspension was passed through the peristaltic pump. Thus, by configuring the cell suspension to not pass through the drive unit when pumping the cell suspension as described in this example, it is possible to supply and collect cells with reduced physical damage to the cells.

[0035] 1 Cell processing device, 2 Vent filter, 3 Connector between cell processing device and flow path, 4 Branch connector, 5 / 6 / 8 / 9 / 15 / 16 / 21 / 22 / 24 / 26 Solenoid valve, 7 Container for cell suspension, 10 Cell collection container, 11 / 13 Buffer space, 12 / 14 Weight scale, 17 / 20 Driving unit such as peristaltic pump, 23 Drainage collection container, 25 Culture medium or other reagent supply bottle, 27 Pressure sensor, 28 Mass flow meter, 29 Regulator, 30 Gas cylinder, 31 Washing solution bottle, 32 Humidification bottle, 33 Hanging balance.

Claims

1. An automatic culture device comprising: a cell suspension storage container capable of storing a cell suspension; a cell processing container for processing the cell suspension delivered from the cell suspension storage container; a flow path connecting at least the cell suspension storage container and the cell processing container; a first buffer space having a predetermined pressure resistance connected to the flow path; and a pressure source for drawing the cell suspension from the cell suspension storage container into the first buffer space by creating a negative pressure within the first buffer space.

2. An automatic culture device according to claim 1, characterized in that the pressure source has the function of creating a positive pressure within the first buffer space, thereby sending the cell suspension within the first buffer space to the cell processing container.

3. An automatic culture device according to claim 1, characterized in that a collection container for collecting treated cells is connected to the flow path, and the automatic culture device is equipped with a cleaning mechanism for washing away untreated cells remaining in the flow path and / or the first buffer space before transferring treated cells in the cell processing container to the collection container.

4. An automatic culture device according to claim 1, wherein the flow path is connected to a collection container for collecting treated cells and a second buffer space having a predetermined pressure resistance, and the pressure source has the function of transferring the cell suspension containing treated cells in the cell processing container into the second buffer space by creating a negative pressure within the second buffer space.

5. An automatic culture device according to claim 4, characterized in that the pressure source has the function of creating a positive pressure in the second buffer space, thereby sending the cell suspension containing the processed cells in the second buffer space to a collection container.

6. An automatic culture device according to claim 5, characterized in that at least one of the cell suspension storage container, the cell processing container and the recovery container is a bag.

7. The automatic culture device according to claim 1, wherein the buffer space is made of a coiled tube.

8. An automatic culture device according to claim 5, wherein the collection container is a bag, and the automatic culture device has a gas supply mechanism that supplies gas to the bag to prevent the bag from shrinking when the cell suspension containing the treated cells in the second buffer space is transferred to the bag.

9. An automatic culture system comprising the automatic culture device according to any one of claims 1 to 8 and a control device that controls each mechanism of the automatic culture device, including the pressure source.

10. A cell culture method for an automatic culture device comprising: a cell suspension storage container capable of storing a cell suspension; a cell processing container for processing the cell suspension delivered from the cell suspension storage container; a flow path connecting at least the cell suspension storage container and the cell processing container; a first buffer space having a predetermined pressure resistance connected to the flow path; and a pressure source capable of creating a negative pressure within the first buffer space, the cell culture method comprising the steps of: setting a liquid containing cells to be cultured in the cell suspension storage container; operating the pressure source to create a negative pressure within the first buffer space and transferring the liquid containing cells set in the cell suspension storage container into the first buffer space; and operating the pressure source to create a positive pressure within the first buffer space and transferring the liquid containing cells transferred to the first buffer space to the cell processing container.

11. A cell culture method according to claim 10, wherein a bag for collecting treated cells and a second buffer space having a predetermined pressure resistance are connected to the flow path, and the cell culture method comprises the steps of: operating the pressure source to create negative pressure in the second buffer space and transferring the liquid containing the treated cells in the cell processing container into the second buffer space; and operating the pressure source to create positive pressure in the second buffer space and transferring the liquid containing the treated cells transferred to the second buffer space to a collection container for collecting the treated cells.

12. A cell culture method according to claim 10, wherein a collection container for collecting treated cells and a second buffer space are connected to the flow path, and the method includes a step of generating negative pressure in the second buffer space by operating the pressure source, and transferring the cell suspension containing treated cells in the cell treatment container into the second buffer space.

13. A cell culture method according to claim 12, characterized in that it includes a step of generating a positive pressure in the second buffer space by operating the pressure source, and transferring the cell suspension containing the treated cells in the second buffer space to a collection container.

14. A cell culture method according to claim 13, wherein the collection container is a bag, and the method includes a step of supplying gas to the bag to prevent the bag from collapsing when the cell suspension containing the treated cells in the second buffer space is transferred to the bag.

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