JPH022884A - Centrifugal separator for biological cell and separation of the same cell - Google Patents

Abstract

PURPOSE:To separate and recover cells from a liq. under an aseptic condition without damaging in a short time by setting a baffle so that it is freely moved to positions below and above the surface of the liq. in a rotor and by moving the baffle to a position above the surface of the liq. at the time of centrifugal separation. CONSTITUTION:A freely rotatable rotor 2 is placed in a hermetically sealed rotor chamber 1 whose wall withstands steam pressure and a baffle 5 is set so that it is freely moved to positions below and above a liq. in the rotor 2. The baffle 5 is moved to a position above the surface of the liq. at the time of centrifugal separation. Cells can be repeatedly separated and recovered from the liq. under an aseptic condition without damaging in such a short time that the physiological activity is not lowered.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to an apparatus and method for separating and recovering cells from a solution containing biological cells, and particularly to a suitable centrifugation method that does not damage biological cells and prevents the invasion of microorganisms. The present invention relates to a separation device and a separation method.

[Conventional technology]

In order to culture animal cells, so-called perfusion culture is required, in which a liquid medium containing nutrients is exchanged with a waste liquid containing waste components while the cells are captured in a culture tank. In order to maintain cell proliferation through perfusion culture, it is necessary to repeatedly perform cell separation operations for a long period of one month or more without mechanically damaging fragile cells and under physiological conditions and free of microorganisms. be done.

Conventionally, cell separation operations have generally been carried out manually at the laboratory level [Hitachi Review Vol. 69, No.
, 4 (1987) PI3-17) In other words, an operator fills a centrifuge tube with culture solution in a sterile room, taking great care to avoid microbial contamination, and then centrifuges it in an open centrifuge. The cells were separated, the supernatant was discarded, and the precipitated cells were resuspended in fresh medium and returned to the culture tank. This operation has an extremely high risk of microbial contamination and must be performed in small batches.

Moreover, since one batch requires 1 to 2 hours, the cells are forced to be exposed to non-physiological conditions such as oxygen deficiency, nutrient deficiency, and temperature drop during that time.

On the other hand, closed-system centrifugal separators have recently been developed to separate blood and serum in hospitals. However, because the cell-containing liquid contacts the solid surface at high speed and the rotating shaft penetrates the mechanical seal to the outside, there is a high risk of damaging the fragile cells and allowing microorganisms to enter through the seal.

[Problem to be solved by the invention]

The purpose of the present invention is to improve the above-mentioned drawbacks of the prior art, to prevent the culture solution from coming into contact with a solid surface at high speed, to provide a rotor with a pressure-tight seal structure, and to resuspend separated and concentrated cells in a medium. The purpose of this invention is to devise a centrifugal separator that can perform the following steps, and to provide a method for separating cells using this device.

[Means to solve the problem]

The first feature of the centrifugal separator for biological cells of the present invention is that the rotor is rotatable in a closed rotor chamber made of a steam-pressure resistant wall and is installed to be able to move up and down below and above the liquid level in the rotor. The present invention consists of a baffle, and the baffle is moved above the liquid surface at least only during centrifugation.

A second feature of the present invention is that the rotor is installed outside the wall of the rotor chamber and horizontally rotated by a magnetic force transmission mechanism.

The third feature of the present invention is: a) cell-containing liquid introduction piping, centrifugation supernatant liquid discharge piping,
It also serves as a baffle for cell suspension, piping for introducing medium, and piping for discharging cell resuspension, b) It can be expanded and contracted vertically in the rotor chamber below and above the liquid level in the rotor, and C) It is attached to the wall of the rotor chamber. It is equipped with a tubular body that is fixed and communicates with piping that runs outside the wall. This makes it possible to completely avoid parts that come into contact with the rotating liquid at high circumferential speeds. Furthermore, the centrifugation supernatant can be discharged and cells can be resuspended effectively.

The fourth feature of the present invention is that all piping leading to the outside of the rotor room is sealed with a valve outside the room. This makes it possible to handle the liquid in a state where it is completely isolated from the outside world.

Next, the contents of the present invention will be sequentially explained.

The steam-resistant rotor chamber of the present invention, the piping extending outside from the rotor chamber, and the pulp on the piping must have materials and structures that satisfy the following conditions.

1) It can be maintained at a temperature effective for steam sterilization, that is, 120°C or higher, for at least 10 minutes.

2) At least the inner surface, which comes into contact with gas and liquid, is made of a material that does not substantially elute toxic components.

An example of a material suitable for the above is SOS 316.5US
Examples include 316L and titanium. The wall thickness for pressure resistance is appropriately selected depending on the material used.

The centrifugal separator of the present invention uses a concave, for example, pillow-shaped rotor with an opening at the top, and rotates the rotor horizontally within the rotor chamber to sediment and separate cells on the side wall of the rotor chamber. . The rotational force at this time is generated by a motor whose shaft is equipped with a permanent magnet placed under the bottom wall of the rotor chamber. A permanent magnet is also placed at the bottom of the rotor to receive the rotational force of the motor, and the rotor rotates by lubricating the bottom of the rotor with the bottom of the rotor chamber. As for the lubrication method,
If it is made of steam-resistant material, a conventionally known technique is selected.

For example, ball bearings can be used. The rotational speed of the rotor is selected depending on the diameter of the rotor, but it is necessary to obtain a rotational speed as small as 2000 G, which is necessary for cell sedimentation.
0Orpm is required.

Note that the rotor chamber bottom is formed of a non-magnetic material or a low-magnetic material.

In the present invention, the vertically elastic piping which also serves as a baffle for supplying and discharging liquid into the rotor and for resuspending cells has a structure specifically as shown in FIGS. 1 to 6.

The features of the above-mentioned centrifugal separator will be explained in more detail as follows.

d) The rotor is built into a rotor chamber made of steam pressure-resistant walls.

2) A bowl-shaped rotor having an opening at the top receives the rotational force of the electric motor located outside the wall of the bottom of the rotor chamber by magnetic force through the bottom wall of the rotor chamber, and rotates in the horizontal direction.

3) The rotor chamber has a tubular body having the following characteristics.

a) Cell-containing liquid introduction piping, centrifugation supernatant liquid discharge piping,
Serves as a baffle for cell suspension, piping for introducing cell suspension medium, and piping for introducing cell-containing liquid.

b) It can expand and contract vertically within the rotor chamber below and above the liquid level in the rotor.

C) It is fixed inside the rotor chamber and communicates with the pipe leading outside the wall.

4) All piping from the rotor chamber to the outside is sealed with pulp outdoors.

Furthermore, the specific method and structure are as follows.

In the above-mentioned centrifugal separator, l) A structure in which the flow path of the fluid moving in the tubular body is communicated by a flexible tube running parallel to the tubular body.

2) A structure in which the flexible tube is a helical tube made of a rigid material.

3) A structure in which the tubular body is composed of a tube connecting an outer cylinder and an inner cylinder.

4) A structure in which either the outer cylinder or the inner cylinder of the tubular body is fixed in the vertical direction, and the other is movable in the vertical direction.

5) A structure in which the contact surface between the outer cylinder and the inner cylinder has a highly sealable surface, or a sealing material such as an elastic O-ring is arranged on the cylinder wall surface.

6) A structure in which the baffle is plate-shaped and fixed to the outer wall of the vertically moving cylinder of the tubular body, so that the baffle can move in and out of the rotor together with the core.

7) A structure in which the baffle is attached perpendicularly to the pipe wall and moves parallel to the pipe wall.

8) A structure in which a disk is placed in contact with the outer surface of the tube that moves up and down in the tubular body, and the tube moves up and down by rotating the disk.

9) In the above-mentioned tubular body, a structure in which the outer surface of the cylinder on the vertically movable side and the disc are in contact with each other and are made of a material having high resistance. For example, a structure in which the spur gear is on the outside of the bay and the gear on the disc side.

10) It consists of an outer cylinder whose upper end is connected to the upper wall of the rotor chamber and an electromagnet or solenoid is attached to the lower part of the outer wall of the tube, and an inner cylinder which has a magnetic metal or permanent magnet on the upper part and a baffle on the lower part, and has an electromagnet or solenoid. The structure allows the outer wall of the inner cylinder to slide vertically against the inner wall of the outer cylinder.

11) A structure in which the tube that moves up and down in the tubular body is connected to the winding mechanism in the rotor chamber via a flexible linear object.

12) A structure using a rotor with permanent magnets arranged on the bottom surface of the rotor.

13) A rotor structure in which the rotor side surface is vertically concave. A rotor having a structure in which the diameter of the inner surface of the rotor side wall increases along the downward direction.

14) At least 2 of the rotor chamber volume during rotor rotation
A rotor with a structure that allows it to hold more than 0% volume of liquid.

15) A structure having a lubrication mechanism on the outside of the rotor bottom surface that is in contact with the inner surface of the rotor chamber bottom wall. An example of a lubricating mechanism is a bearing.

16) In the above-mentioned rotor, a structure having a rotor holding mechanism that allows the rotor to be fitted into a rotor holder on the bottom of the rotor chamber so that the rotation axis of the rotor does not move. An example is a structure in which the neck is formed between the rotor bottom and the rotor-side magnet, and the rotor is supported by a support mechanism that is slidable on the side of the neck and fixed to the inner wall of the rotor. Additionally, either the support mechanism side or the sliding surface of the rotor neck is constructed with bearings.

17) A rotor chamber or rotor having a structure in which the rotor chamber or rotor is at least vertically separable.

18) A structure in which at least the bottom wall of the rotor chamber wall is made of a non-magnetic or low-magnetic material.

19) A structure that connects two or more steam pressure-resistant pipes from outside the rotor room to the rotor room.

20) A structure in which a sterile filter is placed in the middle of the steam and gas piping connected to the rotor chamber.

21) Rotor chamber, rotor chamber internal structure and piping are 120
A structure in which the gas-liquid contact part is made of a material that can be kept in steam at a temperature of 10°C or higher for at least 10 minutes and does not substantially elute toxic components.

22) A structure with a mechanism to adjust the temperature inside the rotor chamber to a set temperature of 40°C or less.

23) A structure in which the heat transfer surface of the temperature control mechanism is inside the rotor chamber.

24) A structure in which the temperature inside the rotor is controlled by at least a constant temperature water leakage jacket on the side of the rotor chamber.

25) A structure that adjusts the temperature inside the rotor chamber by introducing constant-temperature, sterile air into the rotor chamber.

26) The air introduced must be humidity-controlled.

27) A structure in which the increase, constant speed ratio, and decrease of the rotor rotational speed are performed by controlling the rotational speed of the motor.

28) Structure that allows temperature history to be set.

29) Centrifugal separator and culture tank or cell-containing liquid storage tank,
A cell culture system characterized by connecting a centrifugal supernatant liquid storage tank and a liquid medium storage tank with piping, and opening and closing valves on the piping for each element using a process sequencer.

30) A method for separating cells from a cell-containing solution, which comprises repeatedly repeating a cycle of centrifuging the cell-containing solution and centrifuging the cell-containing solution after steam sterilization using a centrifugal separator.

31) A cell separation method characterized by performing steam sterilization in the next step.

1) Introducing steam into the rotor chamber 2) Holding the temperature above 120°C for 10 minutes or more 3) Closing the valve on the piping exiting the rotor chamber 4) Cooling 5) Introducing sterile air into the rotor chamber to bring the pressure in the rotor chamber to equilibrium with the external pressure 6) Discharging the drain inside the rotor chamber 7) Pulling up the inner cylinder of the tubular structure above the fixed liquid level inside the rotor.

32) A cell separation method characterized by performing the following centrifugation operation.

1) Quantitative supply of cell-containing solution from the tubular structure into the rotor 2) Centrifugation by rotor rotation 3) Stopping the rotor 4) Lowering the inner cylinder to the bottom of the rotor 5) Transferring the centrifugal supernatant from the tubular structure outside the rotor room 6) Quantitative supply of cell suspension medium into the rotor 7) Resuspension of cells by low speed rotation of the rotor 8) Discharge of cell suspension from the tubular structure to the outside of the rotor chamber 9) Tubular structure A cell separation method comprising: lifting the inner cylinder of the object; 10) standby; 33) returning from step 10 to step 1 in step 32).

34) A cell separation method comprising using a cell culture solution as a cell-containing solution, using a liquid medium as a culture solution, and storing a cell suspension in a cell suspension storage tank.

35) A method for separating cells consisting of introducing sterile air into a cell suspension reservoir.

36) The method for separating cells according to claim 34, which comprises returning the cell resuspension to the culture tank.

[Effect]

When a biological cell culture solution is supplied to the biological cell centrifugation device configured as above and centrifuged, the baffle plate is raised to the top during centrifugation, so mechanical damage may occur due to contact between the cells and the baffle plate. There isn't. Since the rotor is rotated by a magnetic force transmission machine from a drive source installed outside the rotor room, centrifugal separation can be performed in a completely closed system. As a result, cells can be safely separated from the culture solution without the risk of contamination of the cells with microorganisms.

〔Example〕

Next, the present invention will be explained by examples. However, the present invention is not limited to this example.

Example 1 This will be explained with reference to FIG.

The rotor chamber 1 is isolated from the outside of the yarn by steam pressure-resistant walls, piping, and valves. The material must be heat-resistant, pressure-resistant, and water-resistant so that the interior of the rotor chamber, rotor chamber mechanism, and piping can be maintained at a temperature of at least 120"C or higher for at least 10 minutes, and material that does not substantially elute toxic components such as heavy metals. , for example SOS 316.SO5316L
, titanium, etc.

The rotor 2 is of a magnetic force transmission type, rotates horizontally, and has a pillow shape with an opening at the top. During centrifugation, the structure is such that at least 20% of the internal volume of the rotor is retained. It is installed at the bottom of the outer surface of the rotor via a bearing so that it slides smoothly on the bottom of the rotor chamber. A permanent magnet 7 is also incorporated in the bottom of the rotor. The motor 8 is located under the bottom of the rotor chamber.
A permanent magnet 7 connected to the rotor 2 is disposed, and the rotor 2 is rotated in a horizontal plane by magnetic force transmission in synchronization with the rotation of the motor. It has a piping mechanism that extends and contracts vertically from the top of the rotor chamber into the rotor opening. This mechanism is composed of an inner cylinder 4 and an outer cylinder 3. The outer cylinder 3 has a spur gear groove in the vertical direction on the outside, and is vertically expanded and contracted by the gear meshing with this groove. A baffle 5 for cell suspension is provided below the outer cylinder.

Example 2 This embodiment is shown in FIG.

Instead of the piping mechanism that moves up and down in the first embodiment, the flow path of the fluid moving inside the tubular body is communicated by a flexible tube 23 that is parallel to the tubular body. The other configurations are exactly the same as in the first embodiment.

Example 3 This embodiment is shown in FIG.

In place of the flexible tube of the piping mechanism that moves up and down in the second embodiment, a helical piping 14 made of a rigid material is used. The other configurations are exactly the same as in the first embodiment.

Example 4 This embodiment is shown in FIG.

Instead of the piping mechanism 6 that moves up and down in the first embodiment, a solenoid valve 16 is inserted into the flow path of the inner cylinder to open and close the flow path. The inner cylinder can be lowered into the rotor by introducing fluid, such as sterile air or liquid, from the outer cylinder with the solenoid valve closed.

Introducing the culture solution or medium into the rotor with the inner cylinder lowered,
The centrifugal supernatant and cell suspension from the rotor can be discharged. The other configurations are exactly the same as in the first embodiment.

Example 5 This embodiment is shown in FIG.

Instead of Embodiment 1, (1) the tubular body is such that the tube with the baffle 5 constitutes the inner cylinder 4, the tube connected to the rotor chamber 1 constitutes the inner cylinder 4, and the sliding between the two cylinders , has a structure in which the outer cylinder moves up and down while maintaining airtightness, and (2) an electromagnet or solenoid 18 is arranged in contact with the outer cylinder wall, and a magnetic metal 7' is attached to the outer wall of the inner cylinder, so that the outer cylinder 1 is connected to the outside of the rotor chamber 1. The structure is such that the inner cylinder 4 is driven in the vertical direction by turning the power on and off.

Example 6 Regarding the magnet drive unit (power transmission unit), Examples 1 to 5
An alternative plan is shown in Figure 6. The tubular body portion of the centrifugal separator shown in FIG. 6 is that of Example 1. In the magnet drive unit of this embodiment, the neck side surface between the rotor chamber side magnetic metal 7' and the rotor bottom is supported by a bearing 2o supported from the rotor chamber wall surface.

Example 7 An example of a process for using a centrifugal separator is shown in a flowchart in FIG.

At least seven pipes that pass through the rotor chamber of the centrifugal separator 106 of the present invention and transfer liquid into or out of the rotor include a culture tank 101, a culture medium storage tank 102, a waste liquid storage tank (cell removal culture liquid storage tank) 103, and An apparatus system that is connected to a steam generator 104 through piping, and opens and closes the pulp inserted into each piping using a process sequencer 105 to execute the process of centrifugation of biological cells shown in the eighth article.

Example 8 The centrifugal separator of Example 1 was used with the system of Example 7,
Biological cells are centrifuged in the following process.

An outline of the process is shown in FIG.

1) Steam is introduced into the rotor room from the boiler.

2) Hold at 120°C or higher for 10 minutes or more.

3) Close all valves on the pipes exiting the rotor chamber.

4) Cool to around 37°C.

5) The rotor chamber pressure is equilibrated with the external pressure by introducing sterile air into the rotor chamber.

6) Discharge the drain inside the rotor to the outside of the rotor chamber via the tubular body of the rotor chamber.

7) Pull up the inner cylinder of the tubular body above the fixed liquid level inside the rotor.

8) Introduce the culture solution from the culture tank into the rotor from the tubular body.

9) Centrifuge by rotor rotation.

10) Stop the rotor.

11) Lower the rotor of the inner cylinder to the bottom.

12) Discharge the centrifugal supernatant to the outside of the rotor chamber via the tubular body.

13) Introduce fresh culture medium from the culture medium storage tank into the rotor chamber via the tubular body.

14) Resuspend the cells by slow rotation of the rotor.

15) Return the cell resuspension in the rotor to the culture tank via the tubular body.

16) Pull the tubular body up to the top of the rotor.

17) Wait for a certain period of time.

18) Return to l).

[Effects of the Invention] With the centrifugal separator and method of using the same of the present invention, cells can be repeatedly separated and collected from a cell-containing solution in a sterile manner within a short period of time without damaging the cells or impairing their physiological activity. I can do it.

[Brief explanation of the drawing]

1 to 6 schematically show the structure of the centrifugal separator according to the present invention. FIG. 7 shows the flow of a culture system including the present centrifugal separator. FIG. 8 shows the operation flow of this centrifugal separator. 1... Rotor chamber, 2... Rotor, 3... Outer cylinder, 4
... Inner tube, 5... Baffle, 6... Outer cylinder vertical mechanism, 7... Permanent magnet, 8... Motor, 9... Cell-containing liquid, 10... For cell suspension. Medium liquid, 11...Air, 1
2...Steam, 13...0 ring, 14...Spiral piping, 15...Support rod, 16...Solenoid valve, 1
7... Solenoid valve electrical wiring, 18... Solenoid, 19
...Solenoid electrical wiring, 20...Bearing, 2
1... Bear link support, 22... Rotor neck, 23
···Flexible Tube

Claims (1)

[Claims] 1. Consists of a rotor that is rotatable in a sealed rotor chamber made of a steam pressure-resistant wall and a baffle that is movable up and down below and above the liquid level in the rotor, and the baffle is 1. A centrifugal separator for biological cells, characterized in that it can move above a liquid surface at least during centrifugation. 2. The centrifugal separator according to claim 1, wherein the rotor is rotated by a magnetic force transmission mechanism installed outside the rotor chamber wall. 3. In the rotor chamber, the following functions are combined: cell-containing liquid introduction pipe, centrifugation supernatant liquid discharge pipe, cell suspension baffle, cell suspension medium introduction pipe, and cell resuspension liquid discharge pipe. Claim 1, further comprising: a tubular body which is vertically expandable and retractable within the rotor chamber below and above the liquid level within the rotor, and which is fixed within the rotor chamber and communicates with a pipe leading to the outside of the rotor chamber.
Or the centrifugal separator according to 2. 4. The centrifugal separator according to claim 3, wherein the fluid moving between the tubular body and a pipe fixed in the rotor chamber and leading to the outside of the rotor chamber communicates with the tubular body through a flexible tube. 5. The centrifugal separator according to claim 3, wherein one of the outer cylinder and the inner cylinder of the tubular body is fixed in the vertical direction, and the other is movable in the vertical direction. 6. The centrifugal separator according to any one of claims 3 to 5, further comprising a plate-shaped baffle horizontally fixed to the lower portion of the tubular body. 7. It has an outer cylinder whose upper end is connected to the upper wall of the rotor chamber and an electromagnet or solenoid is attached to the lower part of the tube outer wall, and an inner cylinder which has a magnetic metal or permanent magnet on the upper part and a baffle on the lower part, and is connected to the electromagnet or solenoid. A centrifugal separator characterized in that the outer wall of the inner cylinder can slide vertically against the inner wall of the outer cylinder by turning on and off the current. 8. The centrifugal separator according to claim 1, wherein the rotor has a permanent magnet arranged on its bottom surface. 9. The centrifugal separator according to claim 1, wherein the side portion of the rotor is concave with respect to the central axis of rotation, and the diameter of the inner surface of the rotor side wall increases in a downward direction. 10. The centrifugal separator according to claim 1 or 8, further comprising a lubrication mechanism mounted on the outside of the bottom surface of the rotor in contact with the inner surface of the bottom wall of the rotor chamber. 11. Claim 1, characterized in that the rotor has a rotor holding mechanism on the bottom surface of the rotor chamber so that the rotation axis of the rotor does not move.
or the centrifugal separator according to any one of items 8 to 10. 12. A neck is configured between the rotor-side magnet, and the rotor is supported by a support mechanism that can slide on the side surface of the neck and is fixed to the inner wall of the rotor, and whether the support mechanism side or the sliding surface of the rotor neck The centrifugal separator according to claim 11, characterized in that one of the centrifugal separators is provided with a bearing. 13. The centrifugal separator according to claim 1 or any one of claims 8 to 11, characterized in that the rotor chamber has a vertically separable structure. 14. The centrifugal separator according to claim 1 or any one of claims 8 to 13, wherein at least the bottom wall of the rotor chamber wall is made of a non-magnetic or low-magnetic material. 15. The centrifugal separator according to claim 1, further comprising a constant-temperature water leakage jacket for adjusting the temperature inside the rotor chamber attached to at least a side surface of the rotor chamber. 16. The centrifugal separator according to any one of claims 1 to 15 is connected to a culture tank or a cell-containing liquid storage tank, a centrifugal supernatant liquid storage tank, and a liquid medium storage tank, and each element is connected on the piping by a process sequencer. A cell culture system characterized by opening and closing a valve. 17. A method for separating cells, which comprises separating cells by the following centrifugation operation using the centrifugal separator according to any one of claims 1 to 16. 1) Quantitative supply of cell-containing solution from the tubular body into the rotor 2) Centrifugation by rotor rotation 3) Stopping the rotor 4) Lowering the inner cylinder to the bottom of the rotor 5) Transferring the centrifugal supernatant from the tubular body to the outside of the rotor chamber Discharge 6) Quantitative supply of cell suspension into the rotor 7) Resuspension of cells by low speed rotor rotation 8) Discharge of cell resuspension from the tubular body to the outside of the rotor 9) Inner cylinder of the tubular structure 18. The cell separation method according to claim 17, wherein the following steps include lifting 10) standby 18 and steam sterilization of the centrifugal separator. 1) Introducing steam into the rotor chamber 2) Holding the temperature above 120°C for 10 minutes or more 3) Closing the valve on the piping exiting the rotor chamber 4) Cooling 5) Introducing sterile air into the rotor chamber to equilibrate the pressure in the rotor chamber with the external pressure 6 ) Drainage inside the rotor 7) Pull up the inner cylinder of the tubular structure above the set liquid level inside the rotor.