Culture device and cell separation method

Abstract

Provided are a culture device and a cell separation method, which are capable of reducing damage to cells in a step for separating cells dispersed in a culture solution (and for extracting supernatant).　A culture device 1 comprises: a culture tank 11 in which cells in a culture solution are cultured and in which at least oxygen is supplied to the culture solution; a sedimentation chamber 12 that has an opening 121 open to the culture solution; a cooling aggregation device 13 that cools the culture solution in the sedimentation chamber 12 and aggregates the cells contained in the culture solution in the sedimentation chamber 12; and a discharge mechanism 15 that discharges the supernatant of the culture solution in the sedimentation chamber 12.

Description

Culture device and cell separation method

[0001] The present invention relates to a culture device for culturing cells in a culture medium and a cell separation method for separating cells from a culture medium in which the cells are dispersed.

[0002] In fields such as drug discovery and food, cell culture techniques are often used to produce target products, and increasing the cell density in the culture medium can improve the production efficiency of the target product. During the cell culture process, cells produce various metabolites, which can inhibit cell growth and substance production, reducing the efficiency of cell culture. One technique that overcomes this issue and achieves high-density cell culture is the perfusion culture method, which involves removing the culture medium from the culture tank, separating the cells dispersed in the medium, returning the removed cells to the culture tank (removing the supernatant from the culture medium from which the cells were separated), and adding fresh culture medium.

[0003] Japanese Patent Application Publication No. 2021-103953 Japanese Patent Application Publication No. 2005-501553 International Publication No. 2020 / 189417

[0004] Patent Documents 1 to 3 relate to perfusion culture devices for culturing cells by perfusion culture, and all of these devices are provided with a separation tank (sedimentation tank) for separating cells dispersed in a culture solution. In these culture devices, the separation tank is provided separately from the culture tank, and the culture solution is introduced from the culture tank into the separation tank, and the cells separated in the separation tank are returned to the culture tank. In such a configuration, there is a problem in that during the time between separation in the separation tank and return to the culture tank (from when the cells are removed from the culture tank until they return to the culture tank), it becomes difficult to supply sufficient oxygen to the cells (the culture conditions controlled by the culture tank are no longer met), which can result in damage to the cells.

[0005] In view of the above, an object of the present invention is to provide a culture device and a cell separation method that can reduce damage to cells during the process of separating cells dispersed in a culture medium (and extracting the supernatant).

[0006] (Configuration 1) A culture device comprising: a culture tank for culturing cells in a culture solution, in which at least oxygen is supplied to the culture solution; a settling chamber having an opening in the culture solution; a cooling and flocculating device that cools the culture solution in the settling chamber to flocculate cells contained in the culture solution in the settling chamber; and a discharge mechanism that discharges the supernatant of the culture solution in the settling chamber.

[0007] (Configuration 2) The culture apparatus according to Configuration 1, wherein the settling chamber is a tubular member arranged at an incline with respect to the vertical direction, thereby forming an inflow path for the culture medium into the settling chamber on a side surface that is on the upper side due to the incline, and a return path for the culture medium to the culture tank on a side surface that is on the lower side due to the incline.

[0008] (Configuration 3) The culture apparatus according to Configuration 2, wherein an end opening of the discharge mechanism located in the settling chamber is disposed in a position on the inflow path.

[0009] (Configuration 4) The culture device according to Configuration 2 or 3, wherein the openings include an inlet opening provided on the inlet path and a return opening provided on the return path.

[0010] (Configuration 5) The culture device according to any one of Configurations 1 to 4, wherein an end opening of the discharge mechanism located in the settling chamber is disposed at a position near the liquid surface of the culture medium in the settling chamber.

[0011] (Configuration 6) The culture apparatus according to any one of Configurations 1 to 5, wherein the opening is installed deeper than a bubble presence region where bubbles exist near the surface of the culture solution in the culture tank during culture.

[0012] (Configuration 7) The culture device according to any one of Configurations 1 to 6, wherein the liquid level of the culture medium in the culture tank is different from the liquid level of the culture medium in the settling chamber.

[0013] (Configuration 8) A culture vessel used in the culture apparatus according to any one of configurations 1 to 7, comprising a settling chamber having an opening in the culture medium and attached to the culture vessel.

[0014] (Configuration 9) A cell separation method for separating cells from a culture solution in which the cells are dispersed, comprising providing a sedimentation chamber having an opening in the culture solution, cooling the culture solution in the sedimentation chamber to aggregate the cells contained in the culture solution in the sedimentation chamber, and discharging the supernatant of the culture solution in the sedimentation chamber.

[0015] The culture device, culture tank, and cell separation method of the present invention can reduce damage to cells during the process of separating cells dispersed in a culture medium (and extracting the supernatant) with a relatively simple configuration.

[0016] 1. A block diagram showing the outline of the configuration of a perfusion culture system (perfusion culture apparatus) according to an embodiment of the present invention. 2. A conceptual diagram showing the concept of operation within the sedimentation chamber of the perfusion culture system. 3. A conceptual diagram showing another example of the sedimentation chamber (sedimentation tube). 4. A conceptual diagram showing another example of the sedimentation chamber (sedimentation tube).

[0017] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the following embodiments are merely examples of how the present invention can be realized, and are not intended to limit the scope of the present invention.

[0018] FIG. 1 is a diagram showing the schematic configuration of a perfusion culture system (perfusion culture apparatus) according to an embodiment of the present invention. The perfusion culture system 1 of this embodiment is configured as a culture apparatus for culturing various cells (e.g., animal, insect, or plant cells, microorganisms, bacteria, etc.) in a culture solution. The perfusion culture system 1 includes a culture tank 11 for holding a culture solution, a settling chamber 12 and a cooling device 13 for separating the culture solution into a supernatant and a sediment, a discharge mechanism 15 for discharging the supernatant of the culture solution in the settling chamber, a metering tank 142 for measuring the discharged solution, a storage tank 18 for storing the discharged solution, a supply mechanism 16 for supplying new culture solution to the culture tank 11, a metering tank 141 for measuring the culture solution to be supplied, and a feed solution tank 17 containing the culture solution. Although not specifically shown, the perfusion culture system 1 also includes a control unit connected to each pump, each on-off valve, each sensor, and each cooling device, and the operations described below are controlled by the control unit.

[0019] The culture tank 11 is a component that holds a culture solution in which cells to be cultured are dispersed. Although not shown, it is equipped with various mechanisms for maintaining the culture solution in a desired state, such as a mechanism for supplying oxygen to the culture solution. Adjusting the state of the culture solution in the culture tank 11 allows cells to be properly cultured in the culture solution, enabling the production of the target product. Furthermore, although not shown, the perfusion culture system 1 (culture tank 11) can be configured with various mechanisms (ancillary equipment) for properly controlling the culture process. For example, it can be configured with equipment (ancillary equipment) such as a measuring device for measuring the state of the culture solution (dissolved oxygen, pH, temperature, etc.), a measuring device for measuring the progress of the culture (a measuring device for glucose concentration, etc.), an adjustment mechanism for adjusting the state of the culture solution, a supply mechanism for supplying culture solution (fresh liquid medium) and nutrients, etc., and a sampling mechanism for extracting the culture solution. The culture tank 11 and various ancillary equipment maintain the culture solution held in the culture tank 11 in an appropriate state, enabling cells to be cultured in the culture solution, and therefore these can be referred to as a culture device.

[0020] The settling chamber 12 is a member for separating cells from the culture solution in the culture tank 11 in order to perform perfusion culture, and serves to separate the culture solution into a supernatant and a precipitate by sedimentation. The settling chamber 12 is provided in the culture tank 11, and has an opening 121 in the culture solution, so that the culture solution is contained therein and the culture solution can flow in and out of the culture tank 11. The settling chamber 12 in this embodiment is a tubular member that is arranged at an incline with respect to the vertical direction, and, as will be explained later, in combination with the function of the cooling device 13, "the inflow path P of the culture solution into the settling chamber at the side that is on the upper side due to the incline" is formed. ｉｎ (See FIG. 2)” and “the return path P of the culture fluid to the culture tank on the side surface that becomes the lower side due to the inclination ｏｕｔ". In this embodiment, the settling chamber 12 is configured so that a portion of its tubular member (here, the upper side) protrudes outside the culture tank 11 and is attached to the culture tank 11. In this attachment structure, the position of the settling chamber 12 in the height direction relative to the culture tank 11 is adjustable. This makes it possible to respond to changes in the liquid level of the culture solution due to differences in culture conditions, etc., and also makes it possible to adjust the position of the lower end (opening 121) of the settling chamber relative to the liquid level of the culture solution in the culture tank in order to adjust the separation function described below. Note that, although an example in which the attachment height of the settling chamber 12 is adjustable is shown here, the attachment height of the settling chamber 12 may of course be fixed based on a predetermined design. A discharge pipe 151 is provided inside the settling chamber 12, and the supernatant of the culture solution separated in the settling chamber 12 is discharged through the discharge pipe 151.

[0021] The cooling device (cooling and flocculating device) 13 is installed at the portion of the sedimentation chamber 12 that protrudes outside the culture tank 11, and has the function of cooling the culture solution in the sedimentation chamber 12 and flocculating the cells contained in the culture solution (the function of increasing the sedimentation rate by flocculating the cells). In addition, by cooling the culture solution, convection is generated, and in combination with the inclined sedimentation chamber 12, the aforementioned "inflow path P ｉｎ " and "Return route P ｏｕｔ The cooling device 13 of this embodiment is configured by using a Peltier element and wrapping it around the outer periphery of the settling chamber 12. However, the present invention is not limited to this, and any device capable of cooling the settling chamber 12 can be used. For example, the cooling device may be configured by providing a circulation path that circulates a cooling medium from a chiller near or inside the settling chamber.

[0022] The discharge mechanism 15 forms a flow path for discharging the supernatant of the culture medium in the settling chamber 12 to the storage tank 18, and includes: a discharge pipe 151 installed in the settling chamber 12; a pump P2 provided on the flow path for sending the discharged liquid; a metering tank 142 for metering the discharged liquid; flow path components such as pipes and tubes that interconnect these components and the settling chamber 12 and the storage tank 18; and on-off valves V2 and V3 provided on the flow path for switching and opening and closing the flow path, thereby connecting the settling chamber 12 and the storage tank 18. The discharge pipe 151 is formed of a hollow pipe, and as shown in FIG. 2 , the tip is curved in a J-shape, with an end opening 1511 at the tip facing upward. In this embodiment, the end opening 1511 of the discharge pipe 151 is located near the upper side surface 12u of the inclined settling chamber 12. This results in "the end opening of the discharge mechanism opening in the settling chamber being positioned on the inflow path." Note that the metering tank 142, pump P2, and on-off valves V2 and V3 (and the flow path members and filter F connecting them) are conceptually similar to the supply / discharge unit 16 shown in Figure 2 of Patent Document 1, and therefore a description thereof will be omitted here. By using a unit similar to the supply / discharge unit 16 shown in Patent Document 1, the amount of culture medium (supernatant) extracted can be accurately controlled.

[0023] The supply mechanism 16 constitutes a flow path that supplies new culture solution to the culture tank 11 in an amount corresponding to the culture solution discharged by the discharge mechanism 15. The supply mechanism 16 includes: a pump P1 provided on the flow path for supplying the culture solution; a metering tank 141 for metering the culture solution to be supplied; flow path components such as pipes and tubes that interconnect these components and the culture tank 11 and the feed liquid tank 17; and an on-off valve V1 provided on the flow path for switching and opening and closing the flow path, thereby connecting the culture tank 11 and the feed liquid tank 17. The supply mechanism 16 is conceptually similar to the supply unit 18 shown in FIG. 3 of Patent Document 1, and therefore will not be described here. By using a unit similar to the supply unit 18 shown in Patent Document 1, the amount of culture solution supplied can be accurately controlled. The storage tank 18 and feed liquid tank 17 are each provided with an aeration unit that connects the internal space of the tank with the external space via a sterile filter F. Although not specifically shown, the culture tank 11 may also be configured with an aeration unit.

[0024] The perfusion culture system 1 of this embodiment having the above configuration efficiently separates the culture solution into a supernatant and a precipitate using the settling chamber 12 and the cooling device 13, discharges the supernatant into the storage tank 18 using the discharge mechanism 15, and supplies new culture solution in an amount corresponding to the discharged supernatant from the feed liquid tank 17 to the culture tank 11 using the supply mechanism 16, thereby performing perfusion culture.

[0025] In the above processing operation, the culture solution is separated in the settling chamber 12 arranged in the culture tank 11. In this embodiment, the separation of the culture solution is based on two functions provided by the settling chamber 12 and the cooling device 13. First, the culture solution in the settling chamber 12 is cooled by the cooling device, causing the cells to aggregate and settle, thereby improving the efficiency of separation of the cells from the culture solution. Second, the culture solution in the settling chamber 12 is cooled by the cooling device, causing convection within the settling chamber, and the settling chamber 12 is tilted, thereby improving the "inflow path P of the culture solution into the settling chamber on the side that becomes the upper side due to the tilt." ｉｎ " and "the return path P of the culture fluid to the culture tank on the side that becomes the lower side due to the inclination ｏｕｔ" and efficiently return the settling cells to the culture tank 11. FIG. 2 is a conceptual diagram showing the operation concept within the settling chamber 12. The settling chamber 12 has a lower opening 121 that opens into the culture fluid CF, and the upper end is sealed. When suction is performed through the discharge pipe 151 (discharge mechanism 15 described above) disposed within the settling chamber 12, negative pressure is generated within the settling chamber 12, causing the liquid level within the settling chamber 12 to rise. When the liquid level reaches the end opening 1511, the culture fluid (supernatant) is drawn out by the discharge pipe 151 (discharge mechanism 15). With this configuration, "the end opening of the discharge mechanism is positioned near the liquid level of the culture fluid within the settling chamber." The cells C within the settling chamber 12 aggregate due to cooling by the cooling device 13, increasing their settling rate. As a result, the supernatant is discharged from the end opening 1511, which is positioned near the liquid level within the settling chamber 12 (first function). During this extraction operation, the culture fluid CF in the culture tank 11 flows into the settling chamber 12, and convection occurs in the settling chamber 12 because the culture fluid in the settling chamber 12 is cooled. As shown in FIG. 2, this convection occurs along the inflow path P of the culture fluid into the settling chamber 12 on the side of the upper side surface 12u of the settling chamber 12. ｉｎ and a return path P of the culture medium to the culture tank 11 on the side of the lower side surface 12d. ｏｕｔ (This tendency is created by the inclination of the settling chamber 12.) Due to this convection, as can be seen from FIG. 2, the settling cells C have a return path P ｏｕｔ The end opening 1511 is connected to the inflow path P ｉｎ Because it is located near the upper liquid surface (the return path P, which is the path along which the aggregated cells settle, ｏｕｔ"), allowing the supernatant containing fewer cells to be discharged. As described above, the settling chamber 12 of this embodiment allows cell separation while circulating the culture solution between the culture tank 11 and the settling chamber 12, thereby reducing damage to the cells during the process of separating the cells dispersed in the culture solution (and removing the supernatant) (by providing a constant and appropriate supply of oxygen, etc.). Note that this embodiment is preferable because it has both the first function of cell aggregation by cooling and the second function of forming an inflow path and a return path by convection and tilting, but the present invention is not limited to having both of these functions, and it is also possible to use only one of these functions.

[0026] As described above, the perfusion culture system (culture apparatus), culture tank, and cell separation method of this embodiment can reduce damage to cells during the process of separating cells dispersed in a culture medium (and removing the supernatant), and can achieve this with a relatively simple configuration. The simple configuration also makes it suitable for single-use applications. For example, it becomes possible to provide a culture tank portion (a culture tank with a sedimentation chamber attached at a position with an opening in the culture medium) as a replacement part (single-use item) at a relatively low cost.

[0027] In order to improve the separation efficiency, the inflow path P ｉｎ It is preferable to control the pump P2 so that the increasing component of the linear velocity of the inflowing culture solution in the inflow path P is slower than the settling velocity of the aggregated cells C. On the other hand, when the cell density in the culture tank 11 becomes too high and it is desired to extract the cells (adjust the amount of cells in the culture tank 11), the suction is increased (conversely to the above, the inflow path P ｉｎThis can be easily achieved by simply setting the linear velocity of the inflowing culture medium in the culture tank 11 to be faster than the settling velocity of the aggregated cells C. When extracting the cells, the cooling by the cooling device 13 may be stopped. These controls (control of the pump, etc.) may be performed according to a set value (the control unit controls the operation of each unit based on the set value), or may be performed by measuring the cell density in the culture tank 11 using a sensor or the like (for example, using the measuring unit 22 described in Patent Document 1), and operating by feedback control such as PID so that the cell density falls within a predetermined range based on the measured value (the control unit controls the operation of each unit based on the sensor value).

[0028] In this embodiment, the opening 121 of the settling chamber 12 is formed at one end (lower end) of the tubular member, but the present invention is not limited thereto. Any opening can be used as long as it allows the culture medium to pass between the culture tank and the opening (at least allows the culture medium to flow in from the culture tank). Here, the position (depth) of the opening relative to the liquid surface is preferably deeper than the bubble presence region where bubbles exist near the liquid surface of the culture medium in the culture tank during culture. FIG. 3 is a schematic diagram illustrating this point. During culture, bubbles generated in the culture medium in the culture tank may float near the liquid surface WS1. Cells tend to adhere to these bubbles. If the opening 121 is located in the bubble presence region BZ (the shaded area in FIG. 3 ) where these bubbles exist, the cells adhering to the bubbles may be easily sucked in, potentially hindering cell separation efficiency. In contrast, as shown in FIG. 3 , by setting the depth D1 of the opening 121 deeper than the bubble presence region BZ, cell separation efficiency can be improved.

[0029] In addition, in this embodiment, an example in which there is one opening is shown, but the present invention is not limited to this, and there may be a plurality of openings. For example, an inlet opening provided on the "inlet path" and a return opening provided on the "return path" may be formed as openings. An example of such an opening is shown in Figure 5. In the settling chamber 12-2 of Figure 5, the inlet path P ｉｎ and a return path Pｏｕｔ The return opening 1212 for the inlet and the outlet are formed as separate openings. By providing dedicated openings on each path, it is expected that the inlet and the return paths will be formed more easily. ｉｎ Therefore, the inflow opening 1211 may be formed larger than the return opening 1212.

[0030] In this embodiment, the settling chamber is a tubular member, but the present invention is not limited thereto. The settling chamber may be a member of any shape having an opening in the culture medium in the culture tank (i.e., the culture medium is retained therein (enclosing the culture medium in a predetermined compartment) while allowing the culture medium to pass in and out of the culture tank). Furthermore, in this embodiment, the upper side of the settling chamber 12 is configured to protrude outside the culture tank, but a portion of the settling chamber 12 may protrude from the side of the culture tank. Furthermore, in this embodiment, a portion of the settling chamber 12 is configured to protrude outside the culture tank, and the protruding portion is cooled by a cooling device. However, the present invention is not limited thereto. For example, the entire settling chamber may be disposed inside the culture tank. For example, in this embodiment, a cooling device is configured to cool the settling chamber from outside the settling chamber, but the entire settling chamber may be disposed inside the culture tank, while a cooling device is provided inside the settling chamber. In this case, by constructing the sedimentation chamber from a material with low thermal conductivity, the impact on the temperature control of the culture solution in the culture tank can be reduced, while the culture solution in the sedimentation chamber can be cooled by a cooling device installed in the sedimentation chamber to aggregate the cells.

[0031] In this embodiment, the end of the discharge pipe 151 is curved in a J-shape with the end opening 1511 facing upward, but the present invention is not limited to this, and the end opening of the discharge pipe 151 may face to the side, or may face downward, as in the case of end opening 1511-1 of discharge pipe 151-1 in Fig. 3. By configuring the end opening of the discharge mechanism to be positioned near the liquid surface of the culture medium in the sedimentation chamber, it is possible to extract (discharge) supernatant that does not contain many cells (improving separation efficiency).

[0032] In this embodiment, the end opening is positioned higher than the liquid level of the culture medium in the culture tank, so that the liquid level WS2 of the culture medium in the settling chamber 12-1 is higher than the liquid level WS1 of the culture medium in the culture tank, as shown in Figure 3. This is preferable because it is thought that cells are less likely to rise to a higher position, thereby achieving the effect of increasing cell separation efficiency, but the present invention is not limited to this. Figure 4 is a schematic diagram of an example in which the liquid level of the culture medium in the culture tank and the liquid level of the culture medium in the settling chamber are different, but the liquid level WS2 of the culture medium in the settling chamber 12-1 is lower than the liquid level WS1 of the culture medium in the culture tank. As described above, the upper side of the settling chamber is sealed (the discharge pipe is inserted at the top of the settling chamber, but the air inside the settling chamber cannot escape once the end opening of the discharge pipe reaches the same height as the liquid level WS2). Therefore, by setting the position of end opening 1511-1 of discharge pipe 151-1 lower than the liquid level WS1 of the culture medium in the culture tank, the liquid level WS2 becomes lower than the liquid level WS1. With this configuration, water pressure is applied, which reduces the load on the extraction (discharge) pump (pump P2 in FIG. 1). Here, an example is shown in which the liquid level of the culture medium in the culture tank and the liquid level of the culture medium in the settling chamber are different, but the liquid level of the culture medium in the culture tank and the liquid level of the culture medium in the settling chamber may also be the same. In addition, if there is no problem with the capacity of the pump for withdrawal (discharge), from the viewpoints of the cooling effect of the settling chamber, reducing the impact of that cooling on the culture solution in the culture tank, and also efficient formation of convection, a configuration in which the end opening is set higher than the liquid level of the culture solution in the culture tank, as in this embodiment, so that the liquid level in the settling chamber is higher than the liquid level of the culture solution in the culture tank during withdrawal, is more preferable. In this embodiment, an example is shown in which the attachment height of the settling chamber 12 relative to the culture tank 11 is adjustable, but instead (or in addition), the height of the discharge pipe 151 (end opening 1511) relative to the settling chamber 12 may be adjustable.

[0033] In this embodiment, due to its configuration, the end opening of the discharge pipe is always located near the liquid level in the settling chamber when the supernatant is discharged, but the present invention is not limited to this. For example, the end opening of the discharge pipe may be positioned lower than the liquid level of the culture medium in the settling chamber by creating a negative pressure in the settling chamber 12 using a negative pressure means separate from the discharge pipe. Even when the end opening is located lower than the liquid level of the culture medium in the settling chamber, the end opening 1511 is located at the inlet path P ｉｎ It is preferable to provide it on the top.

[0034] 1. Perfusion culture system (culture device) 11. Culture tank 12. Settling chamber 121. Opening 13. Cooling device (cooling and flocculation device) 15. Discharge mechanism 1511. End opening 16. Supply mechanism

Claims

1. A culture apparatus comprising: a culture tank for culturing cells in a culture solution, the culture tank being configured to supply at least oxygen to the culture solution; a settling chamber having an opening in the culture solution; a cooling and flocculating device for cooling the culture solution in the settling chamber to flocculate cells contained in the culture solution in the settling chamber; and a discharge mechanism for discharging the supernatant of the culture solution in the settling chamber.

2. The culture apparatus according to claim 1, wherein the settling chamber is a tubular member arranged at an incline with respect to the vertical direction, thereby forming an inflow path for the culture medium into the settling chamber on the side that is the upper side due to the incline, and a return path for the culture medium to the culture tank on the side that is the lower side due to the incline.

3. The culture apparatus according to claim 2, wherein an end opening of the discharge mechanism located within the settling chamber is disposed in a position on the inflow path.

4. The culture device according to claim 2 or 3, wherein the openings include an inlet opening provided on the inlet path and a return opening provided on the return path.

5. The culture apparatus according to claim 1, wherein an end opening of the discharge mechanism located within the settling chamber is disposed at a position near the liquid surface of the culture medium within the settling chamber.

6. The culture apparatus according to claim 1, wherein the opening is installed to a depth deeper than a bubble presence region in which bubbles exist near the surface of the culture medium in the culture tank during culture.

7. The culture device according to claim 1, which is configured so that the liquid level of the culture medium in the culture tank is different from the liquid level of the culture medium in the settling chamber.

8. A cell separation method for separating cells from a culture solution in which the cells are dispersed, comprising providing a sedimentation chamber having an opening in the culture solution, and discharging the supernatant of the culture solution in the sedimentation chamber while cooling the culture solution in the sedimentation chamber to aggregate the cells contained in the culture solution in the sedimentation chamber.

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