WO2020179166A1

WO2020179166A1 - Perfusion culture system and centrifuge

Info

Publication number: WO2020179166A1
Application number: PCT/JP2019/048249
Authority: WO
Inventors: 周太郎石川
Original assignee: エイブル株式会社
Priority date: 2019-03-05
Filing date: 2019-12-10
Publication date: 2020-09-10
Also published as: JP7292706B2; JP2020141579A

Abstract

This perfusion culture system 1 can be manufactured at relatively low costs by comprising: an introducing mechanism that introduces a culture medium held in a culturing tank 14 into a centrifuge tube 111; a rotary mechanism that centrifuges the culture medium inside the centrifuge tube 111 by rotating the centrifuge tube 111; and a discharging mechanism that discharges the culture medium from an opening OP provided on the centrifuge side of the centrifuge tube 111.

Description

Perfusion culture system and centrifuge

The present invention relates to a perfusion culture system, particularly a perfusion culture system using a centrifuge, and a centrifuge.

BACKGROUND ART In fields such as drug discovery and food products, cell culturing technology may be used for production of a target product, and if the density of cells in a culture solution can be increased, the production efficiency of the target product can be increased.
By the way, in the process of culturing cells, cells produce various metabolites, which may cause inhibition of cell growth and inhibition of substance production, which may reduce the efficiency of culturing cells.
As a technology to solve this problem and realize high-density culture of cells, the culture solution is taken out from the culture tank, metabolites are separated using a filtration membrane or centrifugal force, and the extracted cells are returned to the culture tank. A perfusion culture method in which a fresh culture solution is added is known.

Japanese Patent Laid-Open No. 52-114085 JP 63-252558A

Patent Document 2 discloses a technique relating to a cell separation device and a cell culture method using centrifugation. Patent Document 2 is a technique using a disc-shaped separation layer (FIGS. 1 to 6), but forming (manufacturing) a separation layer as shown in FIGS. 1 to 6 of Patent Document 2. Is not easy and has a problem of high cost.
In addition, because of its high cost, it is not suitable for disposable use, and it is assumed that it will be used repeatedly. However, cleaning (contamination) during repeated use, such as cleaning the inside of a disc-shaped separation layer. It is difficult to prevent).

In view of the above points, an object of the present invention is to provide a perfusion culture system and a centrifuge that are perfusion culture systems using a centrifuge and have a configuration that can be manufactured at a relatively low cost.

(Structure 1)
An introduction mechanism for introducing the culture solution held in the culture tank into the centrifuge tube, a rotation mechanism for centrifuging the culture solution in the centrifuge tube by rotating the centrifuge tube, and a centrifugal side of the centrifuge tube. A perfusion culture system comprising a discharge mechanism for discharging the culture solution from an provided opening.

(Configuration 2)
The perfusion culture system according to configuration 1, further comprising an aeration unit that is provided on the rotation center side of the centrifuge tube and that communicates the internal space and the external space of the centrifuge tube via a sterile filter.

(Structure 3)
The perfusion culture system according to configuration 1 or 2, wherein the introduction mechanism introduces the culture solution from the opening into the centrifuge tube.

(Structure 4)
The discharge mechanism includes a pump that sucks fluid into the cylinder and discharges the fluid from the cylinder, and sucks the culture solution with the pump to discharge the culture solution from the centrifuge tube. The perfusion culture system according to any one of 1 to 3.

(Structure 5)
The perfusion culture system according to Configuration 4, wherein the pump includes a sealing member that aseptically seals the inside of the cylinder.

(Structure 6)
The perfusion culture system according to configuration 4 or 5, wherein the introduction mechanism uses the pump to introduce the culture solution into the centrifuge tube through the opening.

(Structure 7)
The introduction mechanism and the discharge mechanism, a first end connected to the culture tank, a second end connected to the opening of the centrifuge tube, a third end connected to the pump, A flow path member for feeding the culture solution, the flow path member having a fourth end connected to a storage tank for storing a centrifugation supernatant of the culture solution; Switching between a first state in which only the tank is connected, a second state in which the pump is connected only to the centrifuge tube, and a third state in which the pump is connected only to the storage tank 7. The perfusion culture system according to configuration 6, which is configured to be possible.

(Structure 8)
The flow path member includes a fixed pipe extending along the rotation axis of the rotating mechanism, a rotating pipe that is liquid-tightly fitted to the fixed pipe and is configured to be rotatable relative to the fixed pipe. 8. The perfusion culture system according to configuration 7, comprising a transport path connecting the end of the rotary tube and the opening.

(Configuration 9)
A centrifuge tube containing a treatment solution, and a rotating mechanism for centrifuging the treatment solution in the centrifuge tube by rotating the centrifuge tube are provided, and the treatment solution is provided on the centrifugal side of the centrifuge tube. A centrifuge with a discharge outlet.

According to the perfusion culture system and centrifuge of the present invention, a perfusion culture system using a centrifuge can be manufactured at a relatively low cost.

The figure which shows the outline of the structure of the perfusion culture system of embodiment which concerns on this invention. A perspective view showing a centrifuge included in the perfusion culture system of the embodiment. Cross section of centrifuge Enlarged view of a part of the centrifuge Flow chart showing the outline of the processing operation of the perfusion culture system of the embodiment

Hereinafter, embodiments of the present invention will be specifically described with reference to the drawings. The following embodiment is one mode for embodying the present invention and does not limit the present invention within the scope thereof.

FIG. 1 is a diagram showing an outline of the configuration of a perfusion culture system according to an embodiment of the present invention.
The perfusion culture system 1 of the present embodiment is configured as an apparatus for culturing various cells (for example, cells such as animals, insects and plants, microorganisms, bacteria, etc.) in a culture medium.
The perfusion culture system 1 has, as its rough structure, a culture tank 14, a centrifuge 11, a pump 12, a feed liquid tank 16, a storage tank 15, and a flow path member for interconnecting these components. And a control unit 13 and the like.

The culture tank 14 is a member that holds the culture solution in which the cells to be cultured are dispersed, and is not particularly shown, but includes various mechanisms for maintaining the culture solution in a desired state. By adjusting the state of the culture solution in the culture tank 14, the cells are appropriately cultured in the culture solution and the target product can be produced.
Further, although not particularly shown, it is possible to adopt a configuration provided with various mechanisms (auxiliary equipment) for appropriately controlling the culture process. For example, a measuring device for measuring the state of the culture medium (dissolved oxygen, pH, temperature, etc.), a measuring device for measuring the progress of the culture (measuring device for cell density, glucose concentration, etc.), and adjusting the state of the culture medium. It is possible to adopt a configuration including an adjusting mechanism for the above, a supply mechanism for supplying a culture solution (fresh liquid medium) and nutrients, and a sampling mechanism for extracting the culture solution (ancillary equipment).

The centrifuge 11 is a device provided with a rotation mechanism for centrifuging the culture solution in the centrifuge tube 111 by rotating the centrifuge tube 111, and centrifuges the culture solution sent from the culture tank 14. , Plays a role of separating into centrifugation supernatant and precipitate.
The centrifuge tube 111 is provided with an opening OP on the centrifugal side, and the precipitate (cells) and the centrifugation supernatant are discharged from the opening OP on the centrifugal side. In addition, a cap is provided on the rotation center side of the centrifuge tube 111, and a ventilation unit that connects the internal space and the external space of the centrifuge tube 111 via the aseptic filter F1 is provided.
A more specific configuration of the centrifuge 11 will be described later.

The pump 12 has a function of introducing the culture solution in the culture tank 14 into the centrifuge tube 111 of the centrifuge 11, a function of returning the precipitate (cells) in the centrifuge tube 111 to the culture tank 14, and a centrifugal separation in the centrifuge tube 111. It has a function of discharging the liquid to the storage tank 15.
The pump 12 in the present embodiment is a syringe pump (a pump that sucks fluid into the cylinder and discharges fluid from the cylinder) provided with a cylinder 121 and a plunger 122. Although not shown in the drawing, the pump 12 is provided with a drive device that drives the plunger 122 to and from the cylinder 121 by controlling the speed and amount thereof.
Further, although not particularly shown, the pump 12 includes a sealing member that aseptically seals the inside of the cylinder. The sealing member is for preventing the invasion of bacteria into the inside of the cylinder 121, that is, for preventing the invasion of bacteria into the culture solution, and may be configured to entirely cover the pump 12. Alternatively, it is configured by a sealed structure that seals the plunger portion inserted into the cylinder 121 (for example, a bellows structure that tightly connects the flange portion at the rear end of the cylinder 121 and the flange portion at the rear end of the plunger 122). It may be one that is performed.

The storage tank 15 is a tank in which the centrifugation supernatant obtained by performing the centrifugal separation process by the centrifuge 11 is discharged and stored. The storage tank 15 is provided with a ventilation unit that connects the internal space and the external space of the storage tank 15 via the aseptic filter F2.

The culture tank 14, the centrifuge 11 (centrifuge tube 111), the pump 12, and the storage tank 15 are connected to each other by a flow path member for sending the culture solution.
The culture tank 14 and the centrifuge tube 111 are connected to each other by a flow path 171, a flow path 172, a flow path 173, and a flow path 175 via a pump 12. Further, the centrifuge pipe 111 and the storage tank 15 are connected to each other by a flow path 175, a flow path 173, a flow path 172, and a flow path 174 via a pump 12.
The flow path 171 and the flow path 172 and the flow path 174 are connected to each other by the branch connector 179A, and the flow path 172, the flow path 173 and the pump 12 are connected to each other by the branch connector 179B.
On-off

valves

178A and 178 B are provided in the flow paths 171 to 174 so that the respective flow paths can be opened and closed individually.
The

flow paths

171, 172, 173, 175, the pump 12, and the on-off

valves

178A, 178B constitute an introduction mechanism for introducing the culture solution held in the culture tank 14 into the centrifuge tube 111 through the opening OP. Further, the

flow paths

172, 173, 174, 175, the pump 12 and the on-off valves 178A, B constitute a discharge mechanism for discharging the culture solution to the storage tank 15 from the opening OP provided on the centrifugal side of the centrifuge tube 111. To.
The flow path member composed of the introduction mechanism and the discharge mechanism is connected to the first end portion e1 connected to the culture tank 14, the second end portion e2 connected to the opening OP of the centrifuge tube 111, and the pump 12. And a fourth end e4 connected to the storage tank 15.
In addition, each flow path is appropriately formed by various pipe materials, tube materials, and the like. Further, each flow path may be configured to be divisible by appropriately providing a connector in the middle for the purpose of improving handleability.

The feed liquid tank 16 is a tank for storing the feed liquid (culture liquid) to be replenished in the culture tank 14, the culture tank 14 and the feed liquid tank 16 are connected by a flow path 18, and a liquid feed pump 19 is provided. ing. Further, the feed liquid tank 16 is provided with a ventilation unit that connects the internal space and the external space of the feed liquid tank 16 via the sterile filter F3.

The control unit 13 controls the operation of each of the above configurations (centrifuge 11, pump 12, on-off valve 178A, B, liquid feed pump 19), and is composed of a microcomputer or other electronic device.

2 to 4 are views showing the centrifuge 11, respectively, FIG. 2 is a perspective view, FIG. 3 is a cross-sectional view taken along the line AA of FIG. 2, and FIG. 4 is an enlarged portion B of FIG. FIG.
As shown in FIGS. 2 to 4, the centrifuge 11 of the present embodiment is provided with a centrifuge tube 111, a rotation mechanism 114 for rotating the centrifuge tube 111, and the like inside a substantially rectangular parallelepiped casing 112. The fixed tube 176 serves as a port of the centrifuge 11 for introducing / discharging the culture solution into the centrifuge tube 111.

The housing 112 is composed of a frame body 1121 which is a skeleton structure that supports the basic structure of the device, and a panel 1122 provided on a side surface of the frame body 1121.
Any configuration can be adopted as long as it can function as a housing.

The rotation mechanism 114 is a mechanism for holding and rotating the centrifuge tube 111, and includes a rotary table 1143 for holding the centrifuge tube, a centrifuge tube holding member 1142 provided on the rotary table 1143, and a rotary table 1143. A rotary jig 1141 for chucking the rotary tube 177, a motor unit 1144 for rotating the rotary table 1143 and the rotary jig 1141 and the like are provided.
The motor unit 1144 is supported by the frame body 1121, and the rotary table 1143 is attached to the rotation shaft of the motor unit 1144. The centrifuge tube 111 is held and rotated by the centrifuge tube holding member 1142 provided on the rotary table 1143.
Further, the rotary jig 1141 is connected to the rotary table 1143 to form an integral structure, and the rotary jig 1141 chucks the rotary tube 177 on the rotary shaft. As a result, the rotary tube 177 also rotates at the same rotation as the centrifuge tube 111.
An opening OP is formed on the centrifugal side of the centrifuge tube 111, a flow path 175 is connected to the opening OP, and the flow path 175 is connected to the rotary tube 177. That is, the flow path 175 is a transport path that connects the end of the rotary pipe 177 and the opening OP. The flow path 175 and the rotary pipe 177 (internal pipe 1771) can be connected by, for example, a flexible tube (silicon tube or Teflon (registered trademark) tube).

FIG. 4 is an enlarged view (portion B in FIG. 3) of a portion of the centrifuge 11 that serves as a port for introducing/extracting the culture solution into the centrifuge tube 111.
As described above, the centrifuge tube 111 is a member that rotates for centrifugal separation, and in order to take out the flow path connected to this to the outside of the centrifuge tube 111, a structure that absorbs the rotation is required.
In the centrifuge 11, the fixed tube (non-rotating tube) 176 is a port for introducing/exhausting the culture solution into the centrifuge tube 111, and the rotating tube 177 is fitted in the fixed tube 176 in a liquid-tight manner. In addition, the rotation is absorbed by being configured to be rotatable relative to the fixed pipe 176.

As shown in FIG. 4, the fixed tube 176 inserted in a liquid-tight manner inside the rotary tube 177 and the rotary tube 177 by the tube holding portion 113 supported by the frame body 1121 is coaxially aligned along the rotation axis. Is held in.
The fixed pipe 176 is non-rotatably fixed by a fixing jig 116 fixed to the pipe holding portion 113. On the other hand, the rotary pipe 177 is rotatably held by being attached to the pipe holding portion 113 via two ball bearings BB.
According to each of the above configurations, the rotating tube 177 is rotated by the rotating jig 1141, while the fixed tube 176 inserted in a liquid-tight manner inside the rotating rotating tube 177 does not rotate, whereby the centrifuge 11 The rotation is absorbed in the flow path from the inside to the outside.
In the present embodiment, the rotary tube 177 has a double structure of an internal tube 1771 and an auxiliary tube 1772 as an example. The inner pipe 1771 and the fixed pipe 176 have the same diameter, and the auxiliary pipe 1772 is configured such that the inner pipe 1771 and the fixed pipe 176 abut with each other with a slight gap. With this configuration, the continuity as a flow path from the internal pipe 1771 to the fixed pipe 176 is enhanced, and the strength is enhanced by adopting a double pipe structure, but the rotating pipe is not made into a double structure and is simply. The fixed tube may be inserted into the rotating tube (or the rotating tube may be inserted into the fixed tube).
Further, in the present embodiment, the fixed tube 176 is provided from above so as to leave a slight gap with respect to the inner tube 1771 extending from the bottom to the top in the auxiliary tube 1772, and the fixed tube 176 is located below the upper end of the rotary tube 177 by a predetermined distance. The silicon washer 115 is provided at the position. The silicon washer 115 is a member provided between the inner wall of the pipe holding portion 113 and the outer circumference of the rotary pipe 177, and seals between these members in a liquidtight manner. The rotating pipe 177 and the fixed pipe 176 are fitted in a liquidtight manner, but they rotate relative to each other, and there is a possibility that liquid may leak from the gap between the two. On the other hand, the silicon washer 115 forms a closed space between the inner wall of the pipe holding portion 113 and the outer circumference of the rotating pipe 177, so that even if the liquid leaks slightly, the liquid is retained in the closed space. Is.

Next, an outline of the processing operation of the perfusion culture system 1 having the above configuration will be described with reference to the flowchart of FIG. Although the main body of the control process is omitted below, the following process is performed by controlling the operations of the centrifuge 11, the pump 12, the on-off valves 178A and B, and the liquid feed pump 19 by the control unit 13. It is a thing.

First, the pump 12 sucks the culture solution from the culture tank 14. That is, the culture liquid is sucked into the cylinder 121 by raising the plunger 122 (step 601). In this process, the on-off valves 178A and 178 are controlled so that only the

flow paths

171 and 172 are opened, and the pump 12 is in the first state in which it is connected only to the culture tank 14.
Next, the on-off valve 178B is controlled so that only the flow path 173 is opened to bring the pump 12 into the second state in which the pump 12 is connected only to the centrifuge tube 111 (step 602), and the pump 12 collects the pump 12 in the cylinder 121. The culture solution is injected into the centrifuge tube 111 (step 603).

After the culture solution is injected into the centrifuge tube 111, the centrifuge 11 is operated to perform the centrifugation process (step 604).
After the centrifugation, the pump 12 sucks the cells spun down from the centrifuge tube 111 (the flow path is in the "second state"). That is, cells are aspirated from the centrifuge tube 111 by raising the plunger 122 by an amount required to extract the cells (step 605).
In the field of cell culture, cells are generally classified into components having a large specific gravity, and therefore cells are collected at the tip of the centrifuge tube 111 by centrifugation. The centrifuge cells are suctioned by raising the plunger 122 as much as necessary to remove the collected cells from the opening OP formed at the tip portion. Most of the centrifugation supernatant remains in the centrifuge tube 111. By connecting the flow path to the opening OP formed at the tip of the centrifuge tube 111, cells can be efficiently extracted.
It should be noted that the centrifugation treatment may cause the cells to solidify at the tip of the centrifuge tube 111, and if the pump is an impeller type pump, the suction of cells from the opening OP may not be successful. By using the syringe pump as in the embodiment, the cells can be sucked through the opening OP relatively smoothly.
From the same point of view, the process of centrifuge and cell extraction may be finely divided into a plurality of times and repeated. If the cells are rotated for a long time until the centrifugation is completely performed, the cells are more likely to be solidified at the tip portion of the centrifugation tube 111. Therefore, the above problem is reduced by repeatedly performing the process of extracting cells by shortening the rotation time.

When the centrifuged cells are sucked into the cylinder 121 of the pump 12, the flow path is set to the first state (step 606), and the cells are returned to the culture tank 14 (step 607).

Next, the flow path is set to the second state (step 608), and the remaining centrifuge supernatant is sucked from the centrifuge tube 111 by the pump 12 (step 609).
Thereafter, the on-off valves 178A and 178 are controlled so that only the

flow paths

172 and 174 are opened to bring the pump 12 into the third state in which it is connected only to the storage tank 15 (step 610), and the centrifugation supernatant is removed. It is discharged to the storage tank 15 (step 611).
The process of replenishing the feed liquid from the feed liquid tank 16 to the culture tank 14 by the liquid feed pump 19 is performed in parallel with the above-described processing of steps 601 to 611 (step 620). This is a process for appropriately supplementing the discharged centrifugal supernatant.
By repeating the above series of processing, the culture solution is taken out from the culture tank to separate metabolites and the like, the cells are returned to the culture tank, and the perfusion culture in which the fresh culture solution is added is performed.
The operation of the centrifuge 11 may be rotated only during step 604 (or the repetitive processing of steps 604 and 605), or the rotating state may be maintained through the processing of steps 601 to 611 described above. ..

According to the perfusion culture system 1 of the present embodiment, it can be manufactured and operated at a relatively low cost.
That is, the syringe used for the pump 12, the centrifuge tube, and the flow path portion (excluding the on-off valves 178A and B) can be formed by using a conventional product such as a plastic syringe or a plastic tube, and is practically single-use. Can be For example, the culture tank 14 is also made into a plastic bag, and the culture tank 14, the flow paths 171-175, 18, the cylinder 121 and the plunger 122, the branch connectors 179A and B, the fixed tube 176, and the rotary tube 177 are integrated as a single-use product. You can handle
By making it disposable, it is possible to solve the problem that cleaning (contamination prevention) is difficult when repeatedly used.

Further, according to the perfusion culture system 1 (centrifuge 11) of the present embodiment, an opening OP is formed at the tip of the centrifuge tube 111, and the cells are efficiently extracted by the configuration in which the cells are discharged from the opening OP. Can be done.
In the present embodiment, the introduction of the culture solution into the centrifuge tube is also performed through the opening OP as an example, but the introduction of the culture solution into the centrifuge tube may be performed from another location (for example, far away). Provide an inlet on the center side of the sinking tube).

In the present embodiment, a syringe pump is taken as an example of the pump, but any type of pump can be used. However, a positive displacement pump is preferable, and a syringe pump is particularly preferable. As described above, with the syringe pump, the cells collected in the distal end portion of the centrifuge tube 111 can be relatively smoothly drawn out from the opening OP by the centrifugal separation process.
Further, in the present embodiment, one pump 12 is arranged between the flow path 172 and the flow path 173, but the present invention is not limited to this. For example, as described above, when an introduction port is provided on the center side of the centrifuge tube, a pump for introducing the culture solution and a pump for discharging the culture solution may be provided respectively.
Further, for example, the centrifuge tube itself may function as a syringe pump. That is, the centrifuge tube itself may be used as a cylinder, and the plunger may move in and out from the center side of the centrifuge tube.

1. ．． Perfusion culture system 11. ．． Centrifuge 111. ．． Centrifuge tube OP. ．． Opening 12. ．． Pump 121. ．． Cylinder 122. ．． Plunger 14. ．． Culture tank 15. ．． Water tank 176. ．． Fixed tube 177. ．． Rotating tube F1-3. ．． Aseptic filter e1-4. ．． First to fourth ends

Claims

An introduction mechanism for introducing the culture solution held in the culture tank into the centrifuge tube,
By rotating the centrifuge tube, a rotation mechanism for centrifuging the culture solution in the centrifuge tube,
A discharge mechanism for discharging the culture solution from an opening provided on the centrifugal side of the centrifuge tube.
A perfusion culture system comprising:
The perfusion culture system according to claim 1, further comprising an aeration unit provided on the rotation center side of the centrifuge tube to communicate the internal space and the external space of the centrifuge tube via a sterile filter.
The perfusion culture system according to claim 1 or 2, wherein the introduction mechanism introduces the culture solution into the centrifuge tube from the opening.
The discharge mechanism includes a pump that sucks a fluid into a cylinder and discharges the fluid from the cylinder, and sucks the culture solution with the pump to discharge the culture solution from the centrifuge tube. Item 4. The perfusion culture system according to any one of Items 1 to 3.
The perfusion culture system according to claim 4, wherein the pump includes a sealing member that aseptically seals the inside of the cylinder.
The perfusion culture system according to claim 4 or 5, wherein the introduction mechanism uses the pump to introduce the culture solution into the centrifuge tube through the opening.
The introduction mechanism and the discharge mechanism, a first end connected to the culture tank, a second end connected to the opening of the centrifuge tube, a third end connected to the pump, A flow path member for feeding the culture solution, which has a fourth end connected to a storage tank for storing the centrifugation supernatant of the culture solution,
The flow path member has a first state in which the pump is connected only to the culture tank, a second state in which the pump is connected only to the centrifuge tube, and the pump is connected only to the storage tank. The perfusion culture system according to claim 6, which is configured to be switchable to any of the third states.
The flow path member,
A fixed tube extending along the axis of rotation of the rotating mechanism;
A rotary tube that is liquid-tightly fitted to the fixed tube and is configured to be rotatable relative to the fixed tube.
The perfusion culture system according to claim 7, further comprising a transport path connecting the end of the rotary tube and the opening.
A centrifuge tube for storing the processing liquid,
A rotation mechanism that centrifuges the treatment liquid in the centrifuge tube by rotating the centrifuge tube.
Equipped with
A centrifuge having a discharge port for discharging the treatment liquid on the centrifugal side of the centrifuge tube.