JP7292706B2 - Perfusion culture system and centrifuge - Google Patents

Description

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

In the fields of drug discovery, food, and the like, cell culture techniques are sometimes used to produce target products, and if the density of cells in a culture medium can be increased, the production efficiency of target products can be increased.
By the way, in the process of culturing cells, cells produce various metabolites, and these metabolites may inhibit cell growth and substance production, thereby lowering the efficiency of cell culturing.
As a technology to solve this problem and realize high-density culture of cells, the culture solution is removed from the culture tank, the metabolites are separated using a filtration membrane and centrifugal force, and the removed cells are returned to the culture tank. , a perfusion culture technique in which fresh culture medium is added.

JP-A-52-114085 JP-A-63-252558

Patent Document 2 discloses a technique related to a cell separation device and a cell culture method using centrifugation. Patent document 2 is a technique using a disk-shaped separation layer (FIGS. 1 to 6), but it is possible to form (manufacture) a separation layer as shown in FIGS. 1 to 6 of patent document 2. However, it cannot be said that it is easy, and there is a problem that the cost is high.
In addition, because of its high cost, it is not suitable for disposable use, and it is assumed that it will be used repeatedly. There is also the problem that it is difficult to prevent

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

(Configuration 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 and a discharge mechanism for discharging the culture solution from the provided opening.

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

(Composition 3)
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.

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

(Composition 5)
5. The perfusion culture system of configuration 4, wherein the pump comprises a sealing member that aseptically seals the interior of the cylinder.

(Composition 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.

(Composition 7)
The introduction mechanism and the discharge mechanism include 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, and the and a fourth end connected to a reservoir for storing the centrifugal supernatant of the culture solution, the channel member for feeding the culture solution, wherein the channel member is arranged so that the pump moves the culture switching among a first state in which the pump is connected only to the tank, 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 reservoir 7. A perfusion culture system according to configuration 6, which is configured to be capable of.

(Composition 8)
The flow path member includes a fixed tube extending along the rotation axis of the rotating mechanism, a rotary tube fitted in the fixed tube in a liquid-tight manner and rotatable relative to the fixed tube, The perfusion culture system according to configuration 7, further comprising a transport path connecting the end of the rotary tube and the opening.

(Composition 9)
A centrifuge tube containing a treatment liquid and a rotation mechanism for centrifuging the treatment liquid in the centrifuge tube by rotating the centrifuge tube, wherein the treatment liquid is placed on the centrifugal side of the centrifuge tube. A centrifuge with an outlet for discharging.

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

1 is a diagram showing an outline of the configuration of a perfusion culture system according to an embodiment of the present invention; FIG. A perspective view showing a centrifuge included in the perfusion culture system of the embodiment. Cross-section of a centrifuge Enlarged view of part of the centrifuge 4 is a flow chart showing an 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. It should be noted that the following embodiment is one mode for embodying the present invention, and does not limit the scope of the present invention.

FIG. 1 is a diagram showing a schematic 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 (eg, cells of animals, insects, plants, etc., microorganisms, bacteria, etc.) in a culture solution.
The perfusion culture system 1 roughly comprises a culture tank 14, a centrifuge 11, a pump 12, a feed liquid tank 16, a storage tank 15, and flow path members interconnecting these components. , a control unit 13, and the like.

The culture vessel 14 is a member that holds a culture solution in which cells to be cultured are dispersed, and includes various mechanisms (not shown) 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.
In addition, although not shown, the apparatus may be configured to include various mechanisms (incidental facilities) for appropriately controlling the culture process. For example, a measuring device that measures the state of the culture solution (dissolved oxygen, pH, temperature, etc.), a measuring device that measures the progress of the culture (a measuring device that measures cell density, glucose concentration, etc.), and adjusts the state of the culture solution. It can be configured to include equipment (ancillary equipment) such as an adjustment mechanism for this purpose, a supply mechanism for supplying a culture solution (fresh liquid medium) and nutrients, and a sampling mechanism for extracting the culture solution.

The centrifuge 11 is a device equipped with a rotation mechanism for centrifuging the culture solution in the centrifuge tube 111 by rotating the centrifuge tube 111, and the culture solution sent from the culture tank 14 is centrifuged. , serves to separate into centrifugation supernatant and precipitate.
The centrifuge tube 111 is provided with an opening OP on the centrifugal side, and the sediment (cells) and centrifugation supernatant are discharged from the opening OP on the centrifugal side. A cap is provided on the side of the center of rotation of the centrifugation tube 111, and a ventilating unit is provided that communicates the internal space and the external space of the centrifugation tube 111 via the sterile filter F1.
A more specific configuration of the centrifuge 11 will be described later.

The pump 12 has the function of introducing the culture solution in the culture tank 14 into the centrifuge tube 111 of the centrifuge 11, the function of returning the sediment (cells) in the centrifuge tube 111 to the culture tank 14, and the function of centrifuging in the centrifuge tube 111. It has a function of discharging the clear water to the storage tank 15 .
The pump 12 in this embodiment is a syringe pump (a pump that draws fluid into the cylinder and discharges the fluid from the cylinder) including a cylinder 121 and a plunger 122 . Although not shown in the drawings, the pump 12 includes a driving device that drives the plunger 122 in and out of the cylinder 121 by controlling its speed and amount.
Although not shown, the pump 12 includes a sealing member that aseptically seals the inside of the cylinder. The sealing member is intended to prevent bacteria from entering the cylinder 121, that is, to prevent bacteria from entering the culture solution. Alternatively, the plunger portion inserted into the cylinder 121 is sealed with a sealing structure (for example, a bellows structure that sealingly 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 something that is done.

The storage tank 15 is a tank in which the centrifugation supernatant obtained by centrifugation by the centrifuge 11 is discharged and stored. The storage tank 15 is provided with a ventilation unit that communicates the internal space and the external space of the storage tank 15 via the sterile 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 channel member for feeding the culture solution.
The culture tank 14 and the centrifuge tube 111 are connected by a channel 171, a channel 172, a channel 173 and a channel 175 with the pump 12 interposed therebetween. In addition, the centrifuge tube 111 and the storage tank 15 are connected by a flow path 175, a flow path 173, a flow path 172 and a flow path 174 with the pump 12 interposed therebetween.
Channels 171, 172 and 174 are interconnected by a branch connector 179A, and channels 172, 173 and pump 12 are interconnected by a branch connector 179B.
On-off valves 178A and 178B are provided in the flow paths 171 to 174 so that the flow paths can be opened and closed individually.
The channels 171, 172, 173, 175, the pump 12, and the on-off valves 178A and 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. In addition, the flow paths 172, 173, 174, and 175, the pump 12, and the on-off valves 178A and 178B constitute a discharge mechanism for discharging the culture fluid to the storage tank 15 from the opening OP provided on the centrifugal side of the centrifuge tube 111. be.
A channel member configured by the introduction mechanism and the discharge mechanism includes a first end e1 connected to the culture tank 14, a second end e2 connected to the opening OP of the centrifuge tube 111, and a pump 12. and a fourth end e4 connected to the reservoir 15 .
In addition, each flow path is appropriately formed of various pipe materials, tube materials, and the like. Further, each flow path may be constructed so as to be divisible by appropriately providing a connection connector in the middle for the purpose of improving handling.

The feed liquid tank 16 is a tank in which a feed liquid (culture liquid) to replenish the culture tank 14 is stored. ing. In addition, the feed liquid tank 16 is provided with a ventilation unit that communicates 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 component (the centrifuge 11, the pump 12, the on-off valves 178A and 178B, and the liquid feed pump 19), and is composed of a microcomputer or other electronic devices.

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

The housing 112 is composed of a frame body 1121 which is a frame structure that supports the basic structure of the device, and panels 1122 provided on the side surfaces of the frame body 1121 .
Any structure can be employed as long as it functions as a housing.

The rotating mechanism 114 is a mechanism for holding and rotating the centrifuge tube 111, and includes a rotary table 1143 that holds the centrifuge tube, a centrifuge tube holding member 1142 provided on the rotary table 1143, and a structure integrated with the rotary table 1143. A rotary jig 1141 for chucking the rotary tube 177 and a motor unit 1144 for rotating the rotary table 1143 and the rotary jig 1141 are provided.
The motor section 1144 is supported by the frame body 1121 , and a rotary table 1143 is attached to the rotating shaft of the motor section 1144 . By holding the centrifuge tube 111 by the centrifuge tube holding member 1142 provided on the rotary table 1143, the centrifuge tube 111 is held and rotated.
Further, the rotary jig 1141 is connected to the rotary table 1143 to form an integral structure, and the rotary tube 177 is chucked on the rotary shaft by the rotary jig 1141 . As a result, the rotary tube 177 also rotates with 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 a rotary tube 177 . That is, the flow path 175 is a transport path that connects the end of the rotary tube 177 and the opening OP. The channel 175 and the rotary tube 177 (internal tube 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 port for introducing/discharging the culture medium into/from the centrifuge tube 111 in the centrifuge 11. As shown in FIG.
As described above, the centrifuge tube 111 is a member that rotates for centrifugal separation, and a structure that absorbs rotation is required in order to take out the flow path connected to this to the outside of the centrifuge tube 111 .
In the centrifuge 11, a fixed tube (non-rotating tube) 176 is a port for introducing/discharging the culture solution into the centrifuge tube 111, and the rotating tube 177 is fitted to the fixed tube 176 in a liquid-tight manner. , and is configured to be rotatable relative to the fixed tube 176, thereby absorbing the rotation.

As shown in FIG. 4, the rotating tube 177 and the fixed tube 176 inserted into the rotating tube 177 in a liquid-tight manner are held coaxially along the rotation axis by the tube holding portion 113 supported by the frame body 1121. is held to
The fixed tube 176 is non-rotatably fixed by a fixing jig 116 fixed to the tube holding portion 113 . On the other hand, the rotating tube 177 is rotatably held by being attached to the tube holding portion 113 via two ball bearings BB.
With each of the above configurations, the rotating tube 177 is rotated by the rotating jig 1141, while the stationary tube 176 inserted into the rotating rotating tube 177 in a liquid-tight manner does not rotate. Rotation is absorbed in the flow path from the inside to the outside.
In this embodiment, the rotating pipe 177 has a double structure of an inner pipe 1771 and an auxiliary pipe 1772 as an example. The internal pipe 1771 and the fixed pipe 176 are pipes having the same diameter, and are configured such that the internal pipe 1771 and the fixed pipe 176 collide with each other with a slight gap in the auxiliary pipe 1772 . With this configuration, the continuity as a flow path from the internal tube 1771 to the fixed tube 176 is enhanced, and the strength is enhanced by adopting a double-pipe structure. Alternatively, the fixed tube may be inserted into the rotating tube (or the rotating tube may be inserted into the fixed tube).
In addition, in this embodiment, the fixed pipe 176 is provided from above so as to leave a slight gap with respect to the internal pipe 1771 extending upward from the bottom within the auxiliary pipe 1772, and is positioned below the upper end of the rotating pipe 177 by a predetermined distance. WHEREIN: It is set as the structure which provides the silicon washer 115. As shown in FIG. The silicon washer 115 is provided between the inner wall of the tube holding portion 113 and the outer periphery of the rotating tube 177, and is a member that seals between these members in a liquid-tight manner. Although the rotating tube 177 and the fixed tube 176 are fitted in a liquid-tight manner, they rotate relative to each other, and liquid may leak from the gap between them. On the other hand, by forming a sealed space between the inner wall of the tube holding part 113 and the outer periphery of the rotary tube 177 with the silicon washer 115, even if the liquid leaks slightly, the liquid is retained in the sealed space. is.

Next, the outline of the processing operation of the perfusion culture system 1 configured as described above will be described with reference to the flowchart of FIG. In addition, although the subject of control processing is omitted below, the following processing is performed by the control unit 13 by controlling the operations of the centrifuge 11, the pump 12, the on-off valves 178A and 178B, and the liquid feed pump 19. It is.

First, the culture solution is sucked from the culture tank 14 by the pump 12 . That is, by raising the plunger 122, the culture solution is sucked into the cylinder 121 (step 601). In this process, the on-off valves 178A and 178B are controlled so that only the flow paths 171 and 172 are opened, and the first state in which the pump 12 is connected only to the culture tank 14 is established.
Next, the on-off valve 178B is controlled so that only the flow path 173 is opened, and the second state in which the pump 12 is connected only to the centrifuge tube 111 is established (step 602). The culture medium is injected into the centrifuge tube 111 (step 603).

After the culture solution is poured into the centrifuge tube 111, the centrifuge 11 is operated to perform centrifugal separation (step 604).
After the centrifugation process, the centrifuged cells are sucked from the centrifuge tube 111 by the pump 12 (the channel is in the "second state"). That is, the cells are aspirated from the centrifuge tube 111 by raising the plunger 122 by an amount necessary to extract the cells (step 605).
In the field of cell culture, cells are generally classified into components with high specific gravity, and thus cells are collected at the tip of centrifuge tube 111 by centrifugation. By raising the plunger 122 as much as necessary to extract the collected cells from the opening OP formed in the tip portion, the centrifuged cells are aspirated. Most of the centrifugal supernatant remains in the centrifuge tube 111 . By adopting a configuration in which the flow path is connected to the opening OP formed at the tip of the centrifuge tube 111, cells can be efficiently extracted.
Note that the centrifugal separation process may cause cells to clump together at the tip of the centrifuge tube 111, and with an impeller-type pump, the cells may not be successfully sucked from the opening OP. By using a syringe pump as in the embodiment, cells can be sucked from the opening OP relatively smoothly.
From the same point of view, centrifugation and cell extraction may be finely divided and repeated multiple times. If the centrifugation tube 111 is rotated for a long time until the centrifugation is completed, the cells tend to clump together at the tip of the centrifugation tube 111 . Therefore, the above problem is reduced by repeating the process of extracting the cells by shortening the rotation time.

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

Next, the channel is set to the second state (step 608), and the remaining centrifugation supernatant is aspirated from the centrifuge tube 111 by the pump 12 (step 609).
After that, the on-off valves 178A and 178B are controlled so that only the flow paths 172 and 174 are opened, and a third state in which the pump 12 is connected only to the storage tank 15 is established (step 610), and the centrifugation supernatant is discharged. 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 processes of steps 601 to 611 (step 620). This is a process for appropriately replenishing the discharged centrifugation supernatant.
By repeating the above series of processes, the culture solution is taken out from the culture tank to separate metabolites and the like, and the cells are returned to the culture tank and perfusion culture is performed by adding fresh culture solution.
The operation of the centrifuge 11 may be performed only during step 604 (or repeated processing of steps 604 and 605), or the rotating state can be maintained through the processing of steps 601 to 611. .

The perfusion culture system 1 of this embodiment can be manufactured and operated at relatively low cost.
That is, the syringe, the centrifuge tube, and the flow path portion (excluding the on-off valves 178A and 178B) used for the pump 12 can be formed using conventional products such as plastic syringes and plastic tubes, and are 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 to 175, 18, the cylinder 121 and the plunger 122, the branch connectors 179A, B, the fixed tube 176, and the rotating tube 177 are integrated as a single-use product. can be handled, etc.
Disposable use also solves the problem of difficulty in cleaning (prevention of contamination) during repeated use.

In addition, according to the perfusion culture system 1 (centrifuge 11) of the present embodiment, the opening OP is formed at the tip of the centrifuge tube 111, and the cells are discharged from the opening OP, whereby the cells can be efficiently extracted. can be done.
In the present embodiment, the introduction of the culture solution into the centrifuge tube is also performed from the opening OP, but the introduction of the culture solution into the centrifuge tube may be performed from another location (for example, the such as providing an inlet on the center side of the sink tube).

Although a syringe pump is used as an example of the pump in this embodiment, any type of pump can be used. However, it is preferably a positive displacement pump, particularly preferably a syringe pump. As described above, by using the syringe pump, the cells accumulated at the tip of the centrifuge tube 111 can be relatively smoothly pulled out from the opening OP by the centrifugation process.
Further, in the present embodiment, one pump 12 is arranged between the flow paths 172 and 173 as an example, but the present invention is not limited to this. For example, as described above, when the 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 centrifugation tube itself may be used as a cylinder, and the plunger may enter and exit from the center side of the centrifugation tube.

1. . . Perfusion culture system 11 . . . centrifuge 111 . . . Centrifuge tube OP. . . opening 12 . . . pump 121 . . . Cylinder 122 . . . plunger 14 . . . culture tank 15 . . . Reservoir 176 . . . Fixed tube 177 . . . Rotating tube F1-3. . . Sterile filter e1-4. . . First to fourth ends

Claims (6)

an introduction mechanism for introducing the culture solution held in the culture tank into the centrifuge tube;
a rotating mechanism for centrifuging the culture medium in the centrifuge tube by rotating the centrifuge tube;
A discharge mechanism for discharging the culture solution from an opening provided on the centrifugal side of the centrifuge tube;
with
The perfusion culture system , wherein the introducing mechanism introduces the culture solution from the opening into the centrifuge tube . an introduction mechanism for introducing the culture solution held in the culture tank into the centrifuge tube;
a rotating mechanism for centrifuging the culture medium in the centrifuge tube by rotating the centrifuge tube;
A discharge mechanism for discharging the culture solution from an opening provided on the centrifugal side of the centrifuge tube;
with
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 . Perfusion culture system. 3. The perfusion culture system of claim 2 , wherein the pump comprises a sealing member that aseptically seals the interior of the cylinder. 4. The perfusion culture system according to claim 2 , wherein said introducing mechanism uses said pump to introduce said culture solution into said centrifuge tube through said opening. The introduction mechanism and the discharge mechanism include 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, and the A channel member for feeding the culture solution, having a fourth end connected to a reservoir for storing the centrifugal 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 a state in which the pump is connected only to the storage tank. 5. The perfusion culture system according to claim 4 , which is configured to be switchable between the third state and the third state. The flow path member is
a fixed tube extending along the rotation axis of the rotating mechanism;
a rotary tube that is fluid-tightly fitted to the fixed tube and configured to be rotatable relative to the fixed tube;
6. The perfusion culture system according to claim 5 , further comprising a transport path connecting the end of the rotary tube and the opening.

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