JPH04267873A - Centrifugal separator - Google Patents

Abstract

PURPOSE:To provide a centrifugal separator capable of efficiently separating animal cells in an intact live state from a suspension culture solution of the animal cells. CONSTITUTION:A centrifugal separator is composed of a rotor body (2), a detachable cassette module (1) and a cover (3) having a structure in which the aforementioned module can be inserted. The above-mentioned module (1) is formed into the shape of an earthenware mortar and arranged so as to form a spiral or concentric flow.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a centrifugal separator for separating animal cells. More specifically, the present invention relates to an animal cell separation device for culturing animal cells in suspension.

0002

[Prior Art] In cell suspension culture, fresh culture medium is generally supplied into a culture tank in order to prevent cell growth from stopping at a relatively low density and to culture cells in large quantities and at high density. A method has been proposed in which the old culture solution containing growth-inhibiting substances is drained out of the culture tank, which is commonly called the perfusion method (Annual Report on Ferm.
Entation Processes Vol. 6
).

What is important when culturing using this method is to efficiently separate the living cells in the suspension solution from the old culture solution, take out the old culture solution out of the culture tank, and remove the cells in the culture tank. The goal is to maintain the growing environment under optimal conditions. Various filters and various types of filters have been proposed to separate living cells and old culture fluid from suspension culture fluid, but they are not suitable for industrial equipment due to filter clogging and the complexity of the device structure. It is hard to say that either of these is satisfactory.

[0004] In Japanese Unexamined Patent Publication No. 63-252558, the present inventor has proposed an animal cell separation device and a cell culture method using a perfusion method, which is suitable for culturing cells in large quantities and at high density.

Furthermore, in Japanese Patent Application No. 1-249269,
The present inventor relates to a centrifugal separator and separation system that improves the disadvantages of the above proposal: damage to cells due to pressure differences within the centrifugal separator and low volumetric efficiency due to the fact that only a part of the centrifugal separator body can be used. proposed by.

However, in the proposed centrifugal separator, the peripheral slot for separation has a welded and sealed structure, making it difficult to completely clean the inside of the slot after culturing, so the peripheral slot main body must be replaced after each culture. Therefore, it cannot be said that it is economically advantageous as an industrial device.

[0007]

OBJECTS OF THE INVENTION It is an object of the present invention to provide an advantageous separation device for animal cells.

Another object of the present invention is to provide a centrifugal separator for separating living animal cells from a suspension culture of animal cells extracted from a culture tank.

[0009] Still another object of the present invention is to efficiently separate animal cells, which are easily deformed and destroyed by external forces, unlike microorganisms, yeast, etc., from a culture solution containing them, while still alive, using a centrifugal separator. The purpose of the present invention is to provide a separation device at a low cost.

0010

SUMMARY OF THE INVENTION These objects and advantages of the present invention are achieved by the following centrifugal separator of the present invention.

[0011] That is, a centrifugal separator for separating animal cells and a culture solution from a suspension culture solution of animal cells, which includes (a) a rotor body (2) integrated with a rotating shaft, and a removable cassette. Consisting of a module (1) and a cover (3) having a structure in which the module can be inserted, (b) the module (1) has its upper and lower sides connected to the cover (3) and the rotor body (2).
By sealing with
forming a peripheral slot having an outer peripheral wall inclined at an angle of , the peripheral slot having a length of the outer wall corresponding to at least 360° around the central axis of the rotor, and forming a continuous flow of the liquid fluid; It has a space like
(c) A suspension culture solution supply port provided on the side of the slot with higher centrifugal force; (d) A port provided on the side of the slot with lower centrifugal force for discharging the culture mother liquor from which animal cells have been separated. mother liquor outlet, (e) separated animal cell outlet provided on the lower centrifugal force side of the slot, ((
A centrifugal separator equipped with d).

[0012] The above-mentioned centrifugal separator is preferred, in which the peripheral slot exists so as to form a continuous spiral flow without substantially changing the angle with respect to the direction perpendicular to the central axis of the rotor. . FIG. 2 is a specific example of a plan view of the upper side of this peripheral slot. An example of the procedure for manufacturing the spiral cassette module shown in FIG. 2 will be further explained with reference to FIG. First, as shown in A and B in Figure 5, the radii r1 and r2
Roll the board into a semicircle in the shape of a mortar. At this time, radius r1
, r2 are expressed as r2 = r1 + (slot depth d + plate thickness)/2. Next, make A and B eccentric, align the ends C, and connect them by welding from the outside of the plate. Also for the board ho, the radius r3
=r2+(d+plate thickness)/2, and connect the ends D in the same manner as above. By repeating these operations, a continuous spiral flow path can be formed. Furthermore, by sealing the ends with plates such as H and T, the manufacture of the spiral cassette module is completed.

[0013] The peripheral slot exists so as to form a concentric flow without substantially changing an angle with respect to a direction perpendicular to the central axis of the rotor, and at the end of the flow, the next concentric flow is formed. The above-mentioned centrifugal separator is suitable. 3 and 4 are plan views of the upper side of this peripheral slot.

[0014] Forming a continuous spiral flow can be achieved, for example, by winding a plate in a conical shape at equal intervals at equal intervals and sealing the ends. In addition, in order to form a concentric flow, for example, plates rolled up in a mortar shape are placed concentrically at equal intervals to block the concentric flow path and to ensure a smooth flow path from the outside to the inside. This is achieved by making a notch at an arbitrary position on the circumference.

[0015] Here, the depth of the spiral or concentric flow (the spacing when the thin plates are spread at equal intervals) is preferably 3 to 5 mm.

The cell separation device of the present invention is applied to suspension-type animal cell culture (suspension culture). Suspension culture refers to a method of culturing cells while suspending them in an aqueous medium.

In the present invention, the animal cells to be cultured are those that can grow or proliferate in suspension, and include not only natural animal cells but also cells that have been modified artificially or by genetic manipulation, such as hybridomas. interferon (I), a lymphocyte-derived cell that produces lymphokines such as IL-2,
They can be diploid cells that produce useful physiologically active substances such as FN) or cells that produce various monoclonal antibodies. The present invention is particularly suitable for culturing monoclonal antibody-producing cells for the purpose of obtaining high concentrations of monoclonal antibodies.

The features of the centrifugal separator are (a) a removable and reusable cassette module, (b) a peripheral slot having a mortar-like slope, (c) a suspension culture solution supply port, and (d) ) and a cell extraction port (e). The peripheral slot defined in (b) above refers to a structure that utilizes the entire circumference of the centrifugal separator and allows cells separated by centrifugal force to move along the sedimentation surface in the opposite direction to the culture mother liquor outlet. ing. Furthermore, the above (c) and (d) are
As proposed in Publication No. 249269, it has a structure in which a very small amount of high-density liquid exists inside the centrifugal separator during separation of animal cells, and this eliminates the disadvantages of conventional centrifugal separators. This structure allows for the reduction of damage to cells due to the pressure difference.

The above centrifugal separator will be further explained with reference to the attached drawings.

FIG. 1, FIG. 2, FIG. 3, and FIG. 4 of the attached drawings
1 shows a conceptual diagram of a rotor and a removable cassette module of a centrifugal separator suitable for use in the present invention. FIG. 1 is a sectional view taken in a plane parallel to the axis of rotation. FIG. 2 is a plan view of the upper side of the spiral peripheral slot as seen from the "A" direction in FIG. 1, and FIGS. 3 and 4 are plan views of the upper side of the concentric peripheral slot as seen from the "A" direction in FIG. . Arrows indicate the direction of flow.

As can be well understood from FIG. 1, the centrifugal separator main body consists of a cassette module 1, a rotor main body 2, and a cover 3, which are made of plates having a conical slope and wound spirally or concentrically at equal intervals. A peripheral slot is formed in the rotor, and by fixing it to the rotor body 2 with a tightening bolt 4,
The upper and lower sides of the cassette module 1 are packed with packings 5 and 6.
It is completely sealed with O-rings 7 and 8, and double-sealed with O-rings 7 and 8 to prevent bacterial contamination.

Reference numeral 9 serves as a supply port for the suspension culture solution and a take-out port for the high density liquid, and is connected to the flow path 11 of the rotor body 2 via a flow path 10 . The connecting portion 12 is sealed with an O-ring 13. 14
is an outlet for the culture mother liquor containing no cells and the culture solution containing separated cells, and is connected to the flow path 15 of the rotor body 2. The rotor body is supported by bearings 16, 17 and bearing fixing plates 18, 19, and 20, 21 are mechanical seals. 22 is a rotation transmission pulley.

Angle α between the slot surface and the centrifugal direction shown in FIG.
is preferably in the range of 30° to 80°, and the slot depth d is 3
A range of ~5 mm is preferable. Further, the width L of the plate of the cassette module for forming the peripheral slot is the length of the slot shown in FIG. Furthermore, the inner wall surface of the slot (cassette module) may be subjected to hydrophobic treatment to prevent adhesion of cells.

[0024] Hydrophobic treatment is preferably a method of coating the wall surface with a fluororesin such as PTEF (tetrafluoride resin), for example.

A baffle plate may also be provided within the peripheral slot to further improve separation efficiency.

According to the present invention, the centrifugal separator has a centrifugal effect Z during the centrifugation operation in order to efficiently separate the animal cells from the culture medium in which they are suspended. A range of 5 to 2000 is desirable. If it is less than 5, it will be difficult to carry out the cell separation operation efficiently, while if it exceeds 2000, the centrifugal force applied to the cells will become too large, resulting in significant cell destruction, which is not desirable.

The centrifugal effect Z changes the distance from the rotation axis to r (cm
), the rotational angular velocity is expressed as ω (radian/sec), and the gravitational acceleration is expressed as g (cm/sec2), then rω2
/g. Preferably 10-1000, particularly preferably 20-30
A range of 0 is advantageous.

[0028]

[Effects of the Invention] According to the present invention, the cassette module can be replaced and reused in a centrifugal separator for efficiently separating living cells and old culture fluid in a suspension culture solution without damaging the cells. This makes it possible to provide the product at a low cost. Furthermore, it becomes possible to install the optimal cassette module depending on cell density, cell size, and throughput, making it possible to efficiently separate animal cells.

[Brief explanation of the drawing]

FIG. 1 is an example of a centrifugal separator according to the present invention.

FIG. 2 shows the peripheral slot of the centrifugal separator in FIG.
This is an example of a view seen from the A'' direction.

FIG. 3 shows the peripheral slot of the centrifugal separator of FIG.
This is an example of a view seen from the A'' direction.

FIG. 4 shows the peripheral slot of the centrifugal separator of FIG.
This is an example of a view seen from the A'' direction.

FIG. 5 shows an example of a method for manufacturing the spiral peripheral slot of FIG. 2;

Claims (3)

[Claims] 1. A centrifugal separator for separating animal cells and a culture solution from a suspension culture solution of animal cells, comprising: (a) a rotor body (2) integrated with a rotating shaft;
It is composed of a detachable cassette module (1) and a cover (3) having a structure in which the module can be inserted, and (b) the module (1) has its upper and lower sides connected to the cover (3) and the rotor body (2). ) to form a peripheral slot having an outer peripheral wall inclined in a direction toward the central axis of the rotor at an angle of 30 to 80° with respect to a direction perpendicular to the central axis of the rotor, the peripheral slot , 360° around the central axis of the rotor
(c) a suspension culture solution supply provided on the side of the slot where the centrifugal force is high; a port (9), (d) a mother liquor outlet (14) for discharging the culture mother liquor from which animal cells have been separated, which is provided on the side of the slot where the centrifugal force is low; (e) a mother liquor outlet (14) provided on the side of the slot where the centrifugal force is low; A centrifugal separator equipped with an outlet for removing separated animal cells (which may also be used as (d)) provided on the side. 2. The centrifuge according to claim 1, wherein the peripheral slot exists so as to form a continuous flow in a spiral shape without substantially changing an angle with respect to a direction perpendicular to the central axis of the rotor. Device. 3. The peripheral slot exists so as to form a concentric flow without substantially changing an angle with respect to a direction perpendicular to the central axis of the rotor, and at the end of the flow, the peripheral slot forms a concentric flow in a direction perpendicular to the central axis of the rotor. 2. The centrifugal separator according to claim 1, wherein the centrifugal separator is continuous with the flow of.