JPH0738954B2 - Centrifuge, method for separating animal cells and method for suspension culture of animal cells - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a centrifuge, a method for separating animal cells from a suspension containing animal cells using the centrifuge, and a method for suspension culture of animal cells. More specifically, the present invention relates to a centrifuge suitable for efficiently separating animal cells from a suspension containing animal cells and the use thereof.

[0002]

2. Description of the Related Art In suspension culture of animal cells, in order to prevent the growth and proliferation of cells from stopping at a relatively low density and to culture cells in a large amount and at a high density, a new culture medium is generally introduced into a culture tank. A method of culturing while supplying an old culture solution containing a growth-inhibiting substance to the outside of the culture tank while supplying it, that is, a method commonly called a perfusion method has been proposed (Annual Reports on Fermentation P
rocesses Vol.6).

What is important in culturing using this method is to efficiently separate the living cells in the suspension from the old culture solution, and remove the old culture solution from the culture tank.
To maintain the growth environment of the cells in the culture tank under optimum conditions. Various filters and various formats have been proposed to separate cells from old culture solutions from suspension culture solutions, but for large-scale culture in terms of filter clogging and the complexity of the device structure, etc. It is hard to say that all industrial equipment is satisfactory.

Japanese Patent Laid-Open No. 252558/1988 proposes an animal cell separating apparatus and cell culturing method by a perfusion method suitable for culturing a large number of cells at a high density. The proposal is composed of a centrifuge and a separation system for continuously or intermittently separating living cells and old culture medium from a suspension culture medium by utilizing centrifugal force. It is difficult to say that it is suitable for all kinds of cells because there is a concern that the cells will be damaged by the pressure difference in the centrifugal separator that occurs when the cells pass through the liquid.

Further, the above-mentioned proposed device has a drawback that it is not suitable for separating a large amount of animal cells within a certain period of time because it can use only a part of the main body of the centrifugal separator and therefore has a low volumetric efficiency. On the other hand, the separation of animal cells from a suspension containing animal cells is an important technique not only for the suspension culture described above, but also widely used, for example, for the separation of cells from mammalian blood.

[0006]

SUMMARY OF THE INVENTION Therefore, a first object of the present invention is to provide a centrifuge which can be applied to the separation of animal cells from a suspension containing animal cells alive without being substantially damaged. It is in.

A second object of the present invention is to provide a centrifuge which can be applied to the above-mentioned separation of animal cells and has a high volume efficiency.

Another object of the present invention is to provide a method for operating and operating a centrifuge for separating animal cells from the suspension.

Still another object of the present invention is to provide an industrially advantageous method for suspension culture of animal cells using the above centrifuge.

Still another object of the present invention is to carry out suspension culture of animal cells at high density using the above centrifuge,
It is intended to provide a suspension culture method suitable for obtaining a useful active protein produced by the animal cell.
Still other objects of the present invention will become more apparent from the following description.

[0011]

According to the research conducted by the present inventors, the object and advantages of the present invention are that the centrifugal separation space in the centrifuge is (i) in the direction toward the central axis of the rotor, and in the central axis of the rotor. Consisting of a peripheral slot having an outer peripheral wall inclined at an angle of 30-80 ° with respect to the right angle direction,
(Ii) The peripheral slot is 360 degrees around the center axis of the rotor.
A length of the outer wall equal to or more than (°), (iii) the peripheral slot has a space for forming a continuous flow of the liquid fluid, (iv) and the peripheral slot is a rotor A liquid fluid supply port or discharge port is provided at a position farthest from the central axis or in the vicinity thereof, and (v) the peripheral slot is a liquid fluid discharge port or a supply port at a position closest to or near the central axis of the rotor. It has been found to be achieved by a centrifuge characterized in that

Hereinafter, the centrifuge of the present invention will be described in more detail with reference to the drawings. FIG. 2 shows a schematic partial perspective view of an example of the centrifuge of the present invention.
In FIG. 2, both the inner peripheral wall 21 and the outer peripheral wall 22 form a peripheral slot 23. 24 is a supply port for a liquid fluid such as a suspension culture solution, and 25
Is an outlet for the separated liquid fluid. 26 and 27
Is a nozzle for supplying and discharging these liquid fluids to the rotor body.

In FIG. 2, the angle α between the slot surface and the centrifugal direction is kept in the range of 30 to 80 °. The depth d of the peripheral slot is usually in the range of 2 to 10 mm, for example, up to a throughput of about 200 ml / min.

The depth d may naturally become larger than this value as the processing amount further increases. The peripheral slots in this FIG. 2 are characterized in that they are present so as to form a spiral flow inclined at an angle in the direction of the central axis of the rotor.

FIG. 10 shows an example in which the peripheral slots are partially overlapped with each other to form a spiral flow. With such a structure, the separation area can be easily increased without increasing the length of the centrifuge in the central axis direction.

FIG. 3 shows another schematic partially transparent perspective view of the centrifuge of the present invention. In FIG. 3, the peripheral slots are characterized in that they exist so as to form a spiral continuous flow in the direction of the center axis of the rotor without substantially changing the angle.

That is, in FIG. 3, a plate 38 and flat plates 30 and 39 having a slope (α) and spirally wound at equal intervals.
A peripheral slot is formed by closing the peripheral slot. Reference numeral 32 is a supply port for a liquid fluid such as a suspension culture solution, which is connected to the rotary shaft 34. 33 is an outlet for the separated liquid fluid, which is connected to the rotary shaft 35. 36 and 37 are nozzles for supplying and discharging these liquid fluids to and from the rotor body.

In FIG. 3, the angle α between the slot surface and the centrifugal direction is in the range of 30 to 80 °, and the width d of the spiral peripheral slot is usually in the range of 2 to 10 mm until the throughput is about 200 ml / min. Is appropriate. The width d may naturally become larger than this value as the processing amount further increases. Further, the structure of the peripheral slot in FIG. 3 may be the structure shown in FIG.

FIG. 4 shows another schematic partially transparent perspective view of the centrifuge of the present invention. In this FIG. 4, the peripheral slots are characterized in that they are present so as to form a continuous stream of inverted spirals, without substantially changing the angle in the direction of the rotor central axis. That is, in FIG. 4, a cut-away plan view in the centrifugal direction (longitudinal direction of the central axis) is shown in FIG.

In FIG. 4, a plate 48, a partition plate 50 and a flat plate 4 having a mesh (α) and wound in multiple layers at equal intervals.
A peripheral slot is formed by closing at 0,49. Reference numeral 42 is a supply port for a liquid fluid such as a suspension culture solution, and is connected to a rotary shaft 44. This supply port 42 is indicated by A in FIG.

Reference numeral 43 denotes an outlet for the separated liquid fluid, which is connected to the rotary shaft 45. The discharge port 43 is indicated by B in FIG. As is apparent from FIGS. 4 and 5, in this centrifuge, the peripheral slots are 42
The liquid fluid supplied from (or B) forms a continuous flow of 360 ° (almost one round) along the rotation direction without changing the angle in the direction of the central axis, and the partition plate provided in the vicinity of the supply port The flow is reversed inward, and a continuous flow of 360 ° (almost one round) is formed again in the direction opposite to the rotation direction so that the liquid fluid is discharged from the discharge port 43 (or B). There is.

FIGS. 4 and 5 show a structure in which the peripheral slots are doubled for simplification of description, that is, a structure in which the flow of the liquid fluid is reversed once. The invention is not limited to this structure, and may be a structure in which the fluid is inverted twice to 10 times. 46 and 47 are nozzles for supplying and discharging the liquid fluid to and from the rotor body, respectively.

In FIG. 4, the angle α between the slot surface and the centrifugal direction is in the range of 30 to 80 °, and the peripheral slot width d is
For example, it is usually 2 to 10 when the throughput is about 200 ml / min.
A range of mm is suitable. This width d may naturally become larger than this value as the processing amount further increases.

In FIGS. 8 and 9, the peripheral slots are inclined at an angle with respect to the direction orthogonal to the central axis of the rotor and form a flow parallel to a plane substantially perpendicular to the central axis, Figure 3 shows an example of a centrifuge according to the invention, which is connected to the next parallel stream at the end of the stream.

In FIG. 8, the flow of liquid (indicated by an arrow) is
In a plane almost perpendicular to the central axis, the fluid flows in the tangential direction of a circle centered on the central axis. At the end of the flow, it is connected to the next parallel flow by slits, tubes, etc. These parallel streams can be repeated and increased as many times as needed. FIG. 9 is an example in which the flow is reversed when transitioning to the next parallel flow.

FIG. 11 shows a schematic view of a cross section of the above centrifuge. As can be seen from this cross-sectional view, a plurality of peripheral slots of the same shape are overlaid. FIG. 12 is a cross-sectional view of a case where a separating plate is placed in each peripheral slot.

In the centrifuge having such a shape, it is possible to have a structure in which a plurality of peripheral slots having the same shape are pinched in the central axis direction, and the number of peripheral slots is adjusted depending on the amount of liquid to be treated, the type of cells and the like. It has the advantage that it can be done easily. Further, there is an advantage that the throughput can be increased by pinching the peripheral slot in the central axis direction without increasing the radius of gyration.

As described above with reference to FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG. 8, FIG. 9 and FIG. 30-80 relative to the direction perpendicular to the central axis of the rotor in the direction toward the axis
The outer peripheral wall of the peripheral slot has a slanting outer peripheral wall at an angle of 0 °, the outer peripheral wall of the peripheral slot having a length of the outer wall corresponding to 360 ° or more around the center axis of the rotor, and the liquid fluid. Has a space for forming a continuous flow, and the peripheral slot is provided with a feed port at a position farthest from or near the central axis of the rotor, and at a position closest to or near the central axis of the rotor. It is characterized by having a discharge port in the vicinity.

The case of separating animal cells from a suspension culture solution containing animal cells in the above-mentioned centrifuge will be described. The centrifugal separation space for receiving this suspension culture solution consists of a peripheral slot provided in the rotor. The peripheral slots are 30 to 8 in the direction perpendicular to the center axis of the rotor in the direction toward the center axis of the rotor.
With an angle of 0 °, preferably 45-75 °,
It has an inclined outer peripheral wall. By having such an outer peripheral wall sloping toward the central axis of the rotor, it becomes possible for living animal cells separated from the mother liquor to move smoothly on the outer peripheral wall in the direction away from the central axis of the rotor. It can be prevented from spreading again by the flow of.

Further, by having the inclined outer peripheral wall, even when the accumulated animal cells are extruded and separated using the liquid carrier described later, the inner peripheral wall of the peripheral slot, particularly the inner peripheral wall of the peripheral slot, is separated. It is possible to take out animal cells that are easily left behind in the peripheral slot without leaving them. The angle of the outer peripheral wall may be uniform throughout, or may be a combination of two or more angles within the above range of angle values.

On the other hand, the inner peripheral wall of the peripheral slot does not necessarily have to be inclined toward the center of the rotor like the outer peripheral wall. For example, the inner peripheral wall can be parallel to the central axis of the rotor or can be sloped. The inner peripheral wall is preferably inclined toward the central axis of the rotor at an angle of 30 to 80 °, more preferably 35 to 75 ° with respect to the direction orthogonal to the central axis of the rotor, like the outer peripheral wall. ing. Further, the inner peripheral wall can draw a spiral with respect to the center axis of the rotor in an imaginary plane that intersects the center axis of the rotor.

The peripheral slots also exist for an outer wall length of more than 360 ° around the central axis of the rotor. The preferred outer wall length depends on the size of the rotor diameter, but is generally 720 ° around the center axis of the rotor.
The length is 3600 ° or less, particularly preferably 1080 ° or more and 2520 ° or less. That is, the peripheral slots may exist around the center axis of the rotor in the same imaginary plane, ie without changing the angle in the direction of the center axis of the rotor, in a spiral shape (for example, FIG. 3), and It may exist spirally around the central axis at an angle in the central axis direction of the rotor (for example, FIG. 2). The length of the peripheral slot can be increased by providing a peripheral slot having a length of more than 360 ° around the central axis of the rotor, as described above, thereby slowing the in-vivo separation of animal cells. It enables smooth, efficient and efficient operation. In the peripheral slot, a separation plate for animal cells, that is, a separation plate for shortening the sedimentation distance of animal cells to facilitate easy and reliable separation can be provided. Such separators are known per se in the art.

In the centrifuge, the mother liquor separated from the animal cells is supplied to the above-mentioned centrifuge void, that is, the peripheral slot, while the centrifuge is operating, that is, while the rotor is rotating. It has a discharge port for withdrawing. The supply port is provided at a position farthest from the central axis of the rotor of the peripheral slot or in the vicinity thereof,
The discharge port is provided at a position closest to the central axis of the rotor of the peripheral slot or in the vicinity thereof.

Here, the discharge port or the supply port of the liquid fluid, which is provided at a position closest to the central axis of the rotor of the peripheral slot or in the vicinity thereof, is located at 1/3 or more of the radius of the rotor from the central axis of the rotor. preferable.

Thus, in the centrifuge according to the present invention, the above-mentioned peripheral slot is formed in the void, and the peripheral slot has an outer peripheral wall inclined at an angle with respect to the rotation axis, and has a long continuous shape. It has such a structure that a continuous flow is formed. Therefore, this centrifuge is very suitable for treating a suspension containing animal cells to separate animal cells. That is, the substantial area of the peripheral slot can be made relatively large with respect to a centrifuge of the same scale, and furthermore, the suspension liquid can be smoothly flowed by forming a laminar flow in the peripheral slot. Therefore, damage to animal cells can be extremely reduced.

In other words, the centrifuge of the present invention has a structure in which the peripheral slot for separation has a large substantial area and forms a smooth flow even if the rotor has a relatively small turning radius. It can be said that they are doing.
In other words, the centrifuge of the present invention processes a large amount of suspension liquid containing animal cells, which are easily damaged by weighting or physical manipulation, per unit time and unit volume (substantial volume of the centrifuge), and is kept alive. It has an excellent structure for separating animal cells.

According to the studies by the present inventors, the following [I] method for separating animal cells and aqueous liquid from a suspension containing live animal cells and [ II] A method for suspension culture of animal cells is provided.

[I] Suspension containing live animal cells
Method for separating animal cells and aqueous liquid from John liquid; (A) Liquid fluid supply port of (iv) in the centrifuge of the present invention, wherein (B) an aqueous suspension liquid containing live animal cells is rotated. From the liquid fluid outlet of (v) and accumulate the animal cells in the centrifuge cavity, (C) then with water. A liquid carrier that is not mixed and has a higher density than both animal cells and an aqueous liquid and does not inhibit the growth of animal cells, and is supplied from the liquid fluid supply port (iv) in the rotating centrifuge, ( From the liquid fluid outlet of v), live animal cells are extruded with the residual aqueous liquid through the liquid carrier and extracted, thus obtaining live animal cells, and (D) further rotating the aqueous liquid. The living animal comprising: supplying from the liquid fluid supply port of (v) in the centrifuge, extracting the liquid carrier from the liquid fluid discharge port of (iv), and separating the liquid carrier. A method for separating animal cells and an aqueous liquid from an aqueous suspension containing cells.

[0039][II] Suspension culture method for animal cells
Law; (A) Suspension of live animal cells in a culture tank
Culturing, and (b) containing live animal cells from the culture tank.
Extract one part of the suspension culture solution and remove (c).
The suspension culture solution, which was spun out, was rotated.
Is (iv) the liquid fluid supply port in the centrifuge of the present invention?
From a living animal cell for a certain time
The culture solution thus prepared is taken out from the liquid medium outlet of (v),
And accumulating animal cells in the centrifuge cavity, (d)
It is then immiscible with water, neither animal cells nor culture
Is denser than and does not interfere with the growth of animal cells
Place the liquid carrier in the rotating centrifuge.
Liquid from the liquid fluid supply port (iv)
Living animal cells from the fluid outlet together with the culture fluid
Extruded with a rectangular carrier, extracted (e)
Culturing at least a part of the released animal cells according to the above (a)
Back into the bath, and (f) further spin the aqueous liquid
(V) Liquid fluid supply port in the centrifuge
From the liquid fluid outlet of (iv).
Remove the rear and place the liquid carrier in a centrifuge.
Suspension of animal cells
Culture method.

In the above-mentioned separation method [I], the aqueous suspension containing living animal cells to be separated may be any liquid containing animal cells in a suspended state, and its origin is particularly Not limited.
For example, it may be a suspension liquid taken out from a culture tank in suspension culture of animal cells, or may be mammalian body fluid, especially blood. As the aqueous liquid in (f), a fresh culture solution or a culture solution taken out from the above (c) can be used.

The method for culturing [II] of the present invention will be described below.
The operation method will be described. Steps (c), (d) and (f) in this method correspond to steps (B), (C) and (D) in the method [I].

The method for culturing animal cells of the present invention of the above [II] is applied to culture of suspension type animal cells (suspension culture). Suspension culture refers to a method of culturing cells in an aqueous medium while suspending the cells themselves.

In the culturing method of the present invention, the animal cells to be cultivated are those that can grow or proliferate in a suspended state, and are not only natural animal cells but also cells that have been artificially or genetically modified. For example, it can be a hybridoma, and can also produce useful bioactive substances such as interferon (IFN), cells derived from lymphocytes that produce lymphokines such as IL-2.
It can be a polyploid cell, or a cell that produces various monoclonal antibodies. The present invention is particularly suitable for culturing cells producing monoclonal antibodies for the purpose of obtaining high concentrations of monoclonal antibodies.

The step (a) of [II] of the present invention is a step of subjecting animal cells to suspension culture in a culture tank as described above. The culture medium used for suspension culture is an aqueous medium consisting essentially of water. Is. The aqueous medium contains various additives usually used for culturing animal cells, for example, various inorganic salts, vitamins, coenzymes, glucose, amino acids, antibiotics, growth promoting factors and the like.

Serum can be added to the culture medium, but so-called serum-free medium without serum can also be used as the culture medium. It is economically advantageous and desirable to use a serum-free medium.

As the culture tank, one having at least an animal cell inlet, a new culture medium inlet, an air introduction tube, a stirrer and a suspension culture solution withdrawal conduit is used. Such a culture tank can be a conventional culture tank.

The step (b) is a step of withdrawing a part of the suspension culture solution containing animal cells from the culture tank, for example, through a culture solution withdrawal conduit from the culture tank. The suspension culture solution can be extracted continuously or intermittently (intermittently). It is desirable to continuously introduce a new culture solution into the culture tank in an amount commensurate with the amount of the suspension culture solution containing the extracted animal cells. Thus, the concentration of substances that inhibit the growth of animal cells (which adversely affect the growth of animal cells) in the suspension culture solution in the culture tank, which are mainly composed of metabolites of animal cells, can be maintained constantly and at a fairly low level. It is possible to increase the growth density of animal cells in the suspension culture medium and carry out the culture.

In this way, the production amount of useful metabolites accompanying the growth of the animal cells can be increased by the increase in the growth density of the animal cells.

Of course, by maintaining the concentration of the substance that inhibits the growth of animal cells at a low level and under sufficient control as described above, the increased growth density of animal cells can be maintained for a long period of time. Cultivation can be continued and an increased production of useful metabolites can be secured.

In the above culturing method, live animal cells are accumulated in the centrifugation space of the centrifuge in step (c), and then, in step (d), the accumulated live animal cells are treated with water. It is characterized in that it is extruded and extracted together with the mother liquor in a liquid medium that is immiscible and has a density higher than that of both animal cells and culture medium and does not inhibit the growth of animal cells.

In order to accumulate live animal cells in the step (c), the suspension culture solution extracted in the step (b) is continuously supplied to the centrifugation space of the centrifuge for a certain period of time and kept alive. It is necessary to continuously remove the mother liquor separated from living animal cells for a certain period of time. That is, since the suspension culture solution extracted in step (b) has a low concentration of animal cells, until the desired amount of animal cells is accumulated,
Supplied to the centrifuge void. The operating conditions of the centrifugal separator at that time are: (1) θ ≦ 300 (2) Z × θ ≦ 3 × 10 ⁴ (3) Q / S · Z ≦ 0.3 and (4) 5 ≦ Z ≦ 2000 [In the above formula, θ is the average residence time (minutes) of animal cells in the centrifuge, Z is the centrifugal effect, and Q is the amount of suspension culture solution supplied to the centrifuge per unit time (ml / min) and S is the sedimentation area (cm ² ) under the action of centrifugal force. ] Is preferable.

The average residence time (θ, minutes) of animal cells in the centrifuge should be kept below 300 minutes, as shown in the above formula (1). When it exceeds 300 minutes, the survival rate of animal cells is remarkably reduced due to the cause of oxygen deficiency in the centrifuge. The average residence time (θ) is preferably 150 minutes or less, especially 60 minutes or less. Most preferably, it is 30 minutes or less and 5 minutes or more.

The average residence time (θ) of the animal cells in the centrifuge is, for example, a continuous method, that is, a method in which the suspension culture solution is continuously charged in the centrifuge and the separated animal cells are continuously taken out. The volume of the space (V · cm ³ ) in which the animal cells in the suspension culture medium can exist under the centrifugal conditions, the removal rate of the components containing the separated animal cells from the centrifuge (Qc · cm ³ / It is calculated as the value divided by (min).

Z is a centrifugal effect when the centrifugal separation operation is performed, and the distance from the rotation axis is γ (cm) and the rotation angular velocity is ω.
(Radian / sec) and the acceleration of gravity is g (cm / sec ² )
When expressed by, it is expressed by γω ² / g. The centrifugal effect indicates, so to speak, the magnitude of the centrifugal force applied to animal cells. Therefore, the position of the culture solution supply port for supplying the suspension culture solution to be separated to the centrifuge (distance from the rotation axis γ).

Z is in the range of 5-2000. When it is less than 5, it is difficult to efficiently carry out the cell separation operation, while when it exceeds 2000, the centrifugal force applied to the cells is too large and the cells are significantly destroyed, which is not desirable. The centrifugal effect (Z) is preferably in the range from 10 to 1000, particularly preferably in the range from 20 to 300. Further, the value (Z · θ) obtained by multiplying Z and θ is 3 × 10
Must be kept below ⁴ If centrifugation is performed under conditions exceeding this value, the viability will gradually decrease due to the compaction of the cells themselves, which is disadvantageous. The value of Z · θ is particularly preferably 2 × 10 ⁴ or less.

Further, S (cm ² ) is the sedimentation area (cm ² ) when the centrifugal force is applied. S (cm ² ) is defined as the effective area involved in the separation at the position of the inlet of the suspension culture to be separated to the centrifuge (distance γ (cm) from the axis of rotation). The effective area is the position (γ) of the supply port and the height of the suspension culture liquid level at that position when the virtual circle at the position of the supply port at a distance of γ from the rotation axis does not intersect the sedimentation surface. By (h),
It is obtained as the value of πγ ² h.

When the virtual circle intersects the subsidence surface, the effective area is reduced to the area of the portion until the virtual circle intersects the subsidence surface, and πγ ² h is defined as the angle ratio until the intersection. It is calculated as the multiplied value. In this case, the liquid level height (h) should be understood to mean the vertical distance from the deepest position of the separation layer of the centrifuge.

A value S · Z obtained by multiplying the effective area S and the centrifugal effect Z is a parameter indicating the separation capacity of the centrifuge. In the method of the present invention, a value obtained by dividing the amount Q (ml / min) of the suspension culture solution supplied to the centrifuge per unit time by this parameter, that is, the value of Q / S · Z is 0.
It must be 3 or less. The value of Q / S · Z is preferably 0.2 or less, particularly preferably 0.1 or less.

Thus, by ensuring the above operating conditions (1) to (4), according to the above step (c) of the present invention, the animal culture cells can be efficiently kept alive from the suspension culture solution at an extremely high survival rate. It becomes possible to separate.

The live animal cells separated by the centrifuge as described above and accumulated in the centrifuge void are then subjected to the above-mentioned method of the present invention in the following step (d):
A liquid carrier that is immiscible with water, has a higher density than both animal cells and culture medium, and does not hinder the growth of animal cells is supplied from the above-mentioned supply port to the centrifugal separator in operation, instead of suspension culture medium. Then, the liquid carrier is extruded from the discharge port together with the mother liquor.

As the liquid carrier, for example, perfluorocarbon is preferably used.

As such a fluorocarbon, one that is liquid at room temperature is advantageous, and commercially available ones can be widely used. For example, various heat carriers, fluorocarbons used as electrical insulating materials, and various fluorocarbons used as artificial blood can be used. Specific examples thereof include, for example, perfluoroalkanes having 8 or more carbon atoms, perfluorocycloalkanes (eg, perfluorodecalin, perfluoromethyldecalin, perfluoroalkylcyclohexane having an alkyl substituent having 3 to 5 carbon atoms). , Perfluoroalkyltetrahydrofurans having an alkyl substituent having 5 to 7 carbon atoms,
Perfluoroalkyltetrahydropyrans having 4 to 6 carbon atoms and perfluoroadamantanes (for example, perfluoroadamantane, perfluoromethyladamantane, perfluorodimethyladamantane, perfluoromethylethyladamantane, perfluorodiethyladamantane, etc.) Can be given. The fluorocarbon may contain various groups such as a tertiary amino group. These may be used alone or as a mixture of two or more.

By the combination of the above steps (c) and (d), not only the animal cells can be separated alive, but also the inside of the centrifugation space is constantly washed with the liquid carrier, and thus the centrifugation is performed. It is possible to maintain an environment suitable for living animal cells in the separation space at all times and create a situation suitable for industrially advantageous continuous operation.

According to the culture method of the present invention, at least a portion of the proliferative, viable animal cells removed in step (d) is then added in step (e) to step (a).
Returned to the fermentor.

Further, in the present invention, the fresh culture solution or the culture solution taken out from the above (c) in the step (f) is supplied from the liquid fluid supply port (v) in the rotating centrifugal separator. , (Iv) is taken out from the liquid fluid outlet, and the liquid carrier is separated and recovered from the centrifuge.

The above culturing method of the present invention comprises steps (b) to
By repeating (f), an industrially advantageous method that exhibits the above-mentioned advantages is provided.

A preferred series of devices for carrying out the culture method of the present invention will be specifically described with reference to FIG.

FIG. 1 shows a conceptual diagram of another apparatus suitable for carrying out the above-mentioned culture method of the present invention. In FIG. 1, the suspension culture solution containing the animal cells extracted from the culture tank T1 by the pump P1 is continuously supplied from the supply port (iv) provided on the high centrifugal side of the centrifuge S,
The animal cells are separated from the suspension by centrifugal force, and the culture mother liquor from which the animal cells are separated is continuously and aseptically taken out to the tank T2 from the outlet (v) provided on the low centrifugal force side. In that case, it is desirable to allow the suspension liquid to flow in a laminar flow through the peripheral slots.

Thereafter, a liquid carrier having no affinity for cells and culture liquid from the bottom of the culture tank T1 and having a high density is extracted by the pump P2 and provided on the side of the centrifugal separator S having a high centrifugal force. The culture solution containing the separated cells in the peripheral slot is aseptically taken out from the outlet (v) provided on the low centrifugal force side of the centrifuge by supplying a fixed amount from the inlet (iv) Return to tank T1.

Thereafter, the cell-free medium is extracted from the tank T3 by the pump P3 and supplied from the discharge port (v) of the centrifuge S to return the liquid carrier having a high density in the centrifuge to the culture tank T1 again. At the same time, the inside of the peripheral slot is replaced with the medium. The tank T4 is a new medium containing no cells, and is continuously or intermittently supplied to T1 by the pump P4 so that the liquid level in the culture tank T1 becomes constant.

By performing the above operation continuously or intermittently without stopping the centrifuge, animal cells can be efficiently separated in a state of extremely high survival rate. Valve V1
It will not be necessary to explain that the V6 requires an opening / closing operation suitable for the above operation.

As is clear from the above description, the culturing method of the present invention uses the centrifuge having the above-mentioned structure, is immiscible with water, and has a higher density than both animal cells and aqueous liquids. It can be said that the use of a liquid carrier that does not inhibit growth is a method in which those functions are most effectively exerted by each other.

That is, in the culture method of the present invention shown in FIG. 1, the liquid carrier at the bottom of the culture tank is introduced into the centrifuge S through the pump P2 and stored in the centrifuge S. Animal cells are extruded and returned with the cells to the fermentor. By carrying out this operation while operating the centrifuge, the liquid carrier is retained in the centrifuge, and the cells retained until now are naturally discharged due to the difference in specific gravity.

At this time, some or all of the liquid carrier may be introduced into the culture tank T1, so that the phase of the liquid carrier is formed at the bottom of the culture tank as described above. It will be appreciated that the above cyclic use of the liquid carrier phase formed at the bottom of the fermentor is of great advantage for cultivation on an industrial scale.

By such an operation, the cells do not pass through the liquid carrier having a large specific gravity, so that unnecessary pressure can be prevented from being applied to the cells and damage to the cells can be prevented.

According to the present invention, the time for passing through the liquid carrier can be eliminated or extremely reduced. When the pressure applied to the cells is substantially 0.2 kgf / cm ² or less, the damage is small, and it is acceptable to pass through the liquid carrier corresponding to almost the same.

By continuing the operation of this apparatus in this manner, the concentration of the substance that inhibits the growth of the animal cells, which is gradually accumulated in the culture tank as the animal cells are cultured, can be maintained at a low level. .

[0078]

INDUSTRIAL APPLICABILITY The centrifuge of the present invention is extremely suitable for treating a suspension containing animal cells to separate animal cells. That is, the substantial area of the peripheral slot can be made relatively large with respect to a centrifuge of the same scale, and furthermore, the suspension liquid can be smoothly flowed by forming a laminar flow in the peripheral slot. Therefore, damage to animal cells can be extremely reduced.

That is, the centrifuge of the present invention has a structure in which the peripheral slot for separation has a large substantial area and forms a smooth flow even if the rotor has a relatively small radius of gyration. It can be said that they are doing.
In other words, the centrifuge of the present invention processes a large amount of suspension liquid containing animal cells, which are easily damaged by weighting or physical manipulation, per unit time and unit volume (substantial volume of the centrifuge), and is kept alive. It has an excellent structure for separating animal cells.

[I] A method for separating animal cells and aqueous liquid from a suspension containing living animal cells using the centrifuge according to the present invention, and [II] Suspension culture methods are provided.

[0081]

Example 1 (1) Culture Device A culture system having a cell separation system shown in the attached FIG. 1 was used. However, the total volume of the culture tank (T1) is 7 made of stainless steel.
It is a 0-liter stirred culture tank, and the net amount of the culture solution charged is 40 liters.

At the bottom of the culture tank, 6 liters of fluorocarbon (FLUORINERT (registered trademark) FC-40 (registered trademark) manufactured by 3M, USA) was placed.

The centrifuge has a settling area S =
A centrifuge with a rotor of 590 cm ² , an effective volume of 230 ml and a turning radius of 13 cm was used. In Figure A d
= 0.4 cm, α = 75 °. Pump P1, P2, P
3 is a peristaltic pump.

(2) Medium As a basal medium, a mixture of RPMI1640 medium, Ham-12 medium and Dulbecco's modified Eagle medium at 2: 1: 1 (hereinafter referred to as RDF) was used. Insulin 9 μg / ml, transferrin 10 μg / in the above basal medium
ml, ethanolamine 10 μg / ml, selenious acid 2 × 10
^The medium to which ^-6 mole / liter was added was used as the medium.

(3) Culture Method and Results The culture system was autoclaved in advance. After that, 40 liters of medium sterilized by filtration was charged into the culture tank T1. Next, mouse myeloma P3U1 strain and human B
The mouse x human hybridoma x 87 strain obtained by fusing with the cells was inoculated at 8.1 x 10 ⁵ cells / ml. The highest attainable viable cell density in static culture of the x87 strain is 1.1 x 10 ⁶ cells / ml. This cell is IgG
Is to produce. In the culture tank, oxygen gas is automatically controlled and introduced through the blowing nozzle B so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. The culture solution in the culture tank is kept at 37 ° C. A marine type stirring blade is installed in the culture tank, and the stirring speed is 30 rp.
m.

That is, the centrifuge in which the culture solution sterilized by filtration was placed was driven, and the rotation speed was adjusted so that the centrifugal effect was 80 g.

Then 150 ml of 1500 ml culture mixture
The solution was sent to the centrifuge S at a speed of / mm for 10 minutes. The culture mother liquor separated from the cells by the centrifugal separator flowed out and was stored in the old culture medium storage tank T2. The viable cell density in this was about 3 × 10 ⁴ cells / ml or less. The total cell density was 2.5 × 10 ⁵ cells / ml.

After the above operation is completed, the pump P2 is driven to drive the fluorocarbon 30 at the bottom of the culture tank T1.
0 ml was sent to the centrifuge S at a speed of 150 ml / min for 4 minutes. By this operation, all the cells staying in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1.

Then, the pump P2 is stopped and the pump P3 is driven, the new medium in T3 is sent to the centrifuge S, and the fluorocarbon in the centrifuge is added to the culture tank T.
Returned to 1.

This operation was automatically performed once every time shown in Table 1 to continue the culture. The new medium in T4 was continuously supplied to the culture tank so that the liquid level in the culture tank was constant on average. Table 1 shows a part of the experimental conditions and the experimental results.

In the above experiment, θ = 6 min Z = 80. Therefore, Z × θ = 480 min Q / S · Z = 0.0032 cm / min.

[0092]

[Table 1]

[0093]

Example 2 (1) Culture device The same device as in Example 1 was used except for the centrifuge rotor. A centrifuge rotor having a spiral multilayer separation zone as shown in FIG. 3 was used. The specifications are as follows.

R = 13 cm α = 60 ° d = 0.4 cm t = 0.4 cm h = 2.9 cm n ^* = 3 V = 300 ml S = 590 cm ² * Number of sedimentation zones

(2) Medium The same medium as in Example 1 was used. (3) Culture method and effect The culture system was autoclave sterilized in advance. After that, 40 liters of medium sterilized by filtration was charged into the culture tank T1. Then, mouse × human hybridoma × 87 strains were seeded at 2.1 × 10 ⁵ cells / ml. This cell produces IgG. In the culture tank, oxygen gas is automatically controlled and introduced through a blowing nozzle so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. The culture solution in the culture tank is kept at 37 ° C.
The stirring speed is 30 rpm. Batch culture was performed for 3 days after seeding.

As shown in Table 2, the cell density reached 6.3 × 10 ⁵ cells / ml on the first day after the start of culture. Drive the centrifuge in which the culture solution that has been sterilized by filtration is driven, and the centrifugal effect is 10
The rotation speed was adjusted so that it would be 0 g.

Then 200 ml of 2000 ml culture mixture
The solution was sent to the centrifuge S at a speed of / mm for 10 minutes. The culture mother liquor separated from the cells by the centrifugal separator flowed out and was stored in the old culture medium storage tank T2. The viable cell density in this was about 2 × 10 ⁴ cells / ml.

After the above operation is completed, the pump P2 is driven to drive the fluorocarbon 40 at the bottom of the culture tank T1.
0 ml was sent to the centrifuge S at a speed of 200 ml / min for 4 minutes. By this operation, all the cells staying in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1.

Then, the pump P2 is stopped and the pump P3 is driven, the new medium in T3 is sent to the centrifuge S, and the fluorocarbon in the centrifuge is added to the culture tank T.
Returned to 1.

This operation was automatically performed once every time shown in Table 2 to continue the culture. The new medium in T4 was continuously supplied to the culture tank so that the liquid level in the culture tank was constant on average. Table 2 shows a part of the experimental conditions and the experimental results.

In the above experiment, θ = 6 min Z = 100. Therefore, Z × θ = 600 min Q / S · Z = 0.0034 cm / min.

[0102]

[Table 2]

[0103]

Example 3 (1) Culture Device A culture system having a cell separation system shown in the attached FIG. 4 was used. However, the culture tank (T1) is a glass-made agitation-type culture tank having a total volume of 95 liters, and the net amount of the culture solution charged is 6
It is 0 liter.

10 liters of fluorocarbon was added to the bottom of the culture tank.

As the centrifuge rotor, one having a separation zone arranged in multiple layers on concentric circles as shown in FIG. 4 was used. The specifications are as follows.

R = 17 cm α = 45 ° d = 0.4 cm t = 0.4 cm h = 3 cm Number of turns n = 2 Sedimentation zone volume V = 230 ml Sedimentation area S = 890 cm ²

(2) Medium As a basal medium, a mixture of RPMI1640 medium, Ham-12 medium and Dulbecco's modified Eagle medium at 2: 1: 1 (hereinafter referred to as RDF) was used. Insulin 9 μg / ml, transferrin 10 μ in the above basal medium
g / ml, ethanolamine 10 μg / ml, selenious acid 2
The medium to which x10 ^-6 mole / liter was added was used as a medium.

(3) Culture Method and Results The culture system was autoclave sterilized in advance. After that, 60 liters of medium sterilized by filtration was placed in the culture tank T1. Next, mouse myeloma P3U1 strain and human B
The mouse x human hybridoma P-8 strain obtained by fusing with cells was inoculated at 1.06 x 10 ⁶ cells / ml. The highest attainable viable cell density in the stationary culture of the P-8 strain is 1.3 × 10 ⁶ cells / ml. This cell is Ig
It produces G. In the culture tank, oxygen gas is automatically controlled and introduced through a blowing nozzle so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. The culture solution in the culture tank is kept at 37 ° C. A marine type stirring blade is installed in the culture tank, and the stirring speed is 30 rpm.
Is.

Immediately after seeding the cells, perfusion culture using a centrifuge was started. That is, the centrifuge in which the culture solution sterilized by filtration was placed was driven, and the rotation speed was adjusted so that the centrifugal effect was 120 g.

Then 250 ml of 2500 ml culture mixture
The solution was sent to the centrifuge S for 10 minutes at a speed of / min. The culture mother liquor separated from the cells by the centrifugal separator flowed out and was stored in the old culture medium storage tank T2. The viable cell density in this was about 4 × 10 ⁴ cells / ml or less.

After the above operation is completed, the pump P2 is driven to drive the fluorocarbon 30 at the bottom of the culture tank T1.
0 ml was sent to the centrifuge S at a speed of 150 ml / min for 4 minutes. By this operation, all the cells staying in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1.

Then, the pump P2 is stopped and the pump P3 is driven, the new medium in T3 is sent to the centrifuge S, and the fluorocarbon in the centrifuge is added to the culture tank T.
Returned to 1.

This operation was automatically performed once every time shown in Table 3 to continue the culture. The new medium in T4 was continuously supplied to the culture tank so that the liquid level in the culture tank was constant on average. Table 3 shows a part of the experimental conditions and the experimental results.

In the above experiment, θ = 6 min Z = 120. Therefore, Z × θ = 720 min Q / S · Z = 0.0023 cm / min.

[0115]

[Table 3]

[0116]

[Comparative example]

(1) Cultivation device The same device as in Example 1 was used except for the centrifuge rotor. As the centrifuge rotor, the rotor having the structure shown in FIG. 5 was used. The volume of the separation zone is 220 ml, which is almost the same as that of the rotor used in Example 1. (2) Medium The same medium as in Example 1 was used.

(3) Culture Method and Effects Mouse-human hybridomas were cultured in the same manner as in Example 1. That is, 1.2 × 10 ⁵ cells /
The culture was started by seeding at a density of ml. The culture was performed in batches for the first 4 days. Cell density after 4 days is 6.8 × 10
It was ⁵ cells / ml. At this point, the centrifuge was driven, the centrifugal effect was set to 80 G, the cells and the culture solution were separated under the same conditions as in Example 1, and the culture solution replacement rate was 1.0 vol / vo.
Culture was performed at l / day. The results are shown in Table 4 together with the conditions. After the start of perfusion, the cell density in the culture tank began to decrease.

When the cell density in the culture supernatant obtained from the centrifuge was measured, it was almost the same as that in the culture tank, and it was revealed that almost no cells were separated by the centrifuge. The culture was stopped 8 days after the start of the culture.

[0119]

[Table 4]

[0120]

Example 4 (1) Culture Device The device shown in the attached FIG. 1 was used. However, culture tank (T1)
Is a stirring type culture tank made of stainless steel and having a total volume of 120 liters, and the net amount of the culture solution charged is 80 liters. Fluorocarbon (FL made by 3M USA
UORINERT (trademark registration) FC-40 (trademark registration)) 10
I put a liter.

As the centrifuge, the centrifuge having a seven-layer structure shown in FIG. 13, a settling area S = 5300 cm ² , an effective volume of 2300 ml and a rotor having a turning radius of 12.5 cm was used. In FIG. 13, d = 0.5 cm and α = 60 °. In FIG. 1, pumps P1, P2 and P3 are peristaltic pumps.

(2) Medium As a basal medium, a mixture of RPMI1640 medium, Ham-12 medium and Dulbecco's modified Eagle medium at 2: 1: 1 (hereinafter referred to as RDF) was used. Insulin 9 μg / ml, transferrin 10 μ in the above basal medium
g / ml, ethanolamine 10 μg / ml, selenious acid 2
The medium supplemented with × 10 ^-6 mole / liter was used as the medium.

(3) Centrifuge Performance Test and Results The culture system was autoclaved in advance. After that, 80 liters of the medium sterilized by filtration was charged into the culture tank T1. Next, mouse myeloma P3U1 strain and human B
A raw material mixture was prepared by suspending mouse x human hybridoma x 87 strains obtained by fusing cells with cells at a cell density of 1.0 x 10 ⁷ cells / ml in a medium. In the culture tank, oxygen gas is automatically controlled and introduced through the blowing nozzle B so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. The culture solution in the culture tank is kept at 37 ° C. A marine type stirring blade is installed in the culture tank,
The stirring speed is 30 rpm.

Then, a separation test was conducted as follows. At the time of the separation ability test, the centrifuge in which the culture solution sterilized by filtration is impregnated is driven, and the centrifugal effect is 50G, 75G, 100.
The number of revolutions was changed each time so that it became either G.
Then, the culture solution containing the cells in the culture tank is pumped by the pump P1
The solution was sent to the centrifugal separator S for minutes. The culture supernatant separated from the cells by the centrifuge was distilled and stored in the old culture medium storage tank T2. Further, the density of viable cells leaking into the supernatant was measured for 10 minutes after the solution was sent to the centrifuge S, and was used for evaluation of the separation ability.

After the above operation is completed, the pump P2 is driven to drive the fluorocarbon 30 at the bottom of the culture tank T1.
Centrifuge S for 00 ml at a speed of 750 ml / min for 4 minutes
It was sent to. By this operation, all the cells staying in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1.

Then, the pump P2 is stopped and the pump P3 is driven, and the new medium in T3 is sent to the centrifuge S for 4 minutes at a speed of 750 ml / min, and the fluorocarbon in the centrifuge is added to the culture tank T1. I returned to.

The above operation completes one test. Similarly, from the next time onward, the flow rate of the culture medium containing cells to the centrifuge S and the centrifugal effect were changed to measure the leaking viable cell density in the culture supernatant.

The results of the above separation ability test are shown in FIG. From this result, it was judged that the separation was sufficiently performed when the viable cell density was 1% or less of the viable cell density in the culture tank.

That is, in this case, the leaked cell density is 1 × 10 ⁵ cells / ml or less. Based on this result, it was found that the maximum centrifugal separation capacity as shown in Table 5 (however, 72 separation operations / day) was possible.

[0130]

[Table 5]

[0131]

Example 5 (1) Culture Device The same device as in Example 4 was used. The culture tank (T1) is a stainless steel agitation type culture tank having a total volume of 120 liters, and the net amount of the culture solution charged is 80 liters. Fluorocarbon (FLUORINE manufactured by 3M Inc.
10 liters of RT (registered trademark) FC-40 (registered trademark)) were added. (2) Medium The same medium as in Example 4 was used.

(3) Culture Method and Results The culture system was autoclaved in advance. After that, 80 liters of the medium sterilized by filtration was charged into the culture tank T1. Then, mouse × human hybridoma × 87 strains were seeded at 7.2 × 10 ⁵ cells / ml. ×
The highest attainable viable cell density in static culture of 87 strains was 1.1.
× 10 ⁶ cells / ml. This cell produces IgG. In the culture tank, oxygen gas is automatically controlled and introduced through the blowing nozzle B so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. The culture solution in the culture tank is kept at 37 ° C. A marine-type stirring blade is installed in the culture tank, and the stirring speed is 30 rpm. That is, the centrifuge in which the culture solution sterilized by filtration was placed was driven, and the rotation speed was adjusted so that the centrifugal effect was 80G.

Then 15 liters of the culture mixture was added to 1.5
The solution was sent to the centrifugal separator S for 10 minutes at a speed of liter / min. The culture mother liquor separated from the cells by the centrifugal separator flowed out and was stored in the old culture medium storage tank T2.

After the above operation was completed, the pump P2 was driven to feed 6 liters of fluorocarbon at the bottom of the culture tank T1 to the centrifugal separator S at a rate of 1.5 liters / min for 4 minutes. By this operation, all the cells staying in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1.

Then, the pump P2 is stopped and the pump P3 is driven, the new medium in T3 is sent to the centrifuge S, and the fluorocarbon in the centrifuge is added to the culture tank T.
Returned to 1.

This operation was automatically performed once every time shown in Table 6 to continue the culture. The new medium in T4 was continuously supplied to the culture tank so that the liquid level in the culture tank was constant on average. Table 6 shows a part of the experimental conditions and the experimental results.

In the above experiment, θ = 7 min Z = 80. Therefore, Z × θ = 560 min Q / (S · Z) = 0.0035 cm / min.

[0138]

[Table 6]

[Brief description of drawings]

FIG. 1 is a schematic view of a series of devices suitable for carrying out the method for suspension culture of animal cells according to the present invention.

FIG. 2 is a schematic perspective view of a roller portion of the centrifuge of the present invention.

FIG. 3 is a schematic perspective view of a rotor portion of the centrifuge of the present invention.

FIG. 4 is a schematic perspective view of a rotor portion of the centrifuge of the present invention.

5 is a plan view of a rotor unit of the centrifuge of FIG.

FIG. 6 is a plan view of a rotor portion of another centrifuge of the present invention.

FIG. 7 is a perspective view showing a structure of a rotor of a centrifuge which does not belong to the present invention.

FIG. 8 shows an example of a liquid flow in the centrifuge of the present invention.

FIG. 9 shows an example of a liquid flow in the centrifuge of the present invention.

FIG. 10 shows an example of a liquid flow in the centrifuge of the present invention.

FIG. 11 shows an example of a cross-sectional shape of the centrifuge of the present invention.

FIG. 12 shows a sectional view of a centrifugal separator according to the present invention in which a separation plate is placed in a peripheral slot.

FIG. 13 shows a cross section of the centrifuge used in Example 4.

FIG. 14 shows the result of a separation ability test of the centrifuge in Example 4.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12N 5/06 5/10 (72) Inventor Hiroyuki Takamatsu 4-3-2 Asahigaoka, Hino City, Tokyo Teijin Limited Tokyo Research Center (56) Bibliographic Reference Shokai 56-151663 (JP, U)

Claims (15)

[Claims] 1. A peripheral edge of a centrifugal separation space in a centrifuge having an outer peripheral wall (i) 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. Consisting of slots, (ii)
The peripheral slot has a length of the outer wall corresponding to 360 ° or more around the central axis of the rotor, and (iii) the peripheral slot has a space for forming a continuous flow of the liquid fluid. , (Iv) and the peripheral slot is provided with a liquid fluid supply port or a discharge port at or near the position farthest from the central axis of the rotor, and (v) the peripheral slot is located closest to the central axis of the rotor. A centrifuge characterized in that it is provided with a liquid fluid discharge port or a supply port in the vicinity thereof. 2. The centrifuge of claim 1, wherein the peripheral slots are present to form a spiral flow inclined at an angle to a direction orthogonal to the central axis of the rotor. 3. The centrifuge of claim 1, wherein the peripheral slots are present so as to form a continuous spiral flow, substantially unchanged in angle with respect to the direction orthogonal to the central axis of the rotor. Machine. 4. The peripheral slots are present so as to form a continuous stream of inverted spirals, substantially unchanged in angle with respect to the direction orthogonal to the central axis of the rotor.
The described centrifuge. 5. The peripheral slot is inclined at an angle with respect to the direction orthogonal to the central axis of the rotor and forms a flow substantially parallel to the central axis, and at the end of the parallel flow, The centrifuge according to claim 1, wherein the centrifuges are connected in parallel flow. 6. The (A) aqueous suspension containing live animal cells is (B) supplied for a certain time from the liquid fluid supply port of (iv) in the centrifuge of claim 1, which is rotating, while Removing the aqueous liquid separated from the live animal cells from the liquid fluid outlet of (v) and accumulating the animal cells in the centrifuge cavity, (C) then
A liquid carrier which is not mixed with water and has a density higher than that of both animal cells and an aqueous liquid and does not inhibit the growth of animal cells is supplied from the liquid fluid supply port (iv) of the rotating centrifuge. Then, the living animal cells are extruded from the liquid fluid outlet of (v) together with the residual aqueous liquid by the liquid carrier to obtain living animal cells, and (D) the aqueous liquid is further rotated. A living animal cell, comprising: supplying from the liquid fluid supply port of (v) in the centrifuge, extracting the liquid carrier from the liquid fluid discharge port of (iv), and separating the liquid carrier. A method for separating animal cells and an aqueous liquid from an aqueous suspension containing the same. 7. The separation method according to claim 6, wherein the aqueous suspension containing living animal cells is a suspension that has been artificially cultured. 8. The method according to claim 6, wherein the aqueous suspension containing living animal cells is blood from a mammal. 9. The separation method according to claim 6, wherein the liquid carrier is a fluorocarbon. 10. The centrifugal separator has the following operating conditions: (1) θ ≦ 300 (2) Z × θ ≦ 3 × 10 ⁴ (3) Q / S · Z ≦ 0.3 and (4) 5 ≦ Z ≦ 2000 [wherein, θ is the average residence time (minutes) of animal cells in the centrifuge, Z is the centrifugal effect, and Q is the amount of suspension culture solution supplied to the centrifuge per unit time. (Ml / min) and S is the sedimentation area (cm ² ) under the action of centrifugal force. ] The separation method according to any one of claims 6 to 9 operated at. 11. (a) Suspension culture of live animal cells in a culture tank, (b) withdrawing at least a portion of a suspension culture solution containing live animal cells from the culture tank, and (c) withdrawing. The suspension culture solution is supplied from the liquid fluid supply port of (iv) in the centrifuge according to claim 1 for a certain period of time, while the culture solution separated from a living animal cell is (v). From the liquid medium outlet of (1), and accumulate the animal cells in the centrifuge cavity, (d) then not miscible with water,
A liquid carrier having a density higher than that of the animal cells and the culture medium and not inhibiting the growth of the animal cells is supplied from the liquid fluid supply port of (iv) in the rotating centrifuge, Living animal cells are extruded from the liquid fluid outlet together with the culture solution by the liquid carrier, (e) at least a part of the animal cells thus extracted is returned to the culture tank of (a), and ( f)
Further, the aqueous liquid is supplied from the liquid fluid supply port (v) of the rotating centrifugal separator, and the liquid carrier is extracted from the liquid fluid discharge port (iv) to centrifuge the liquid carrier. A method for suspending and culturing animal cells, which comprises separating and recovering from. 12. The suspension culture method according to claim 11, wherein steps (b) to (f) are repeated. 13. The suspension culture method according to claim 11 or 12, wherein the animal cell is a hybridoma. 14. The suspension culture method according to claim 11, wherein the liquid carrier is fluorocarbon. 15. The centrifugal separator has the following operating conditions: (1) θ ≦ 300 (2) Z × θ ≦ 3 × 10 ⁴ (3) Q / S · Z ≦ 0.3 and (4) 5 ≦ Z ≦ 2000 [wherein, θ is the average residence time (minutes) of animal cells in the centrifuge, Z is the centrifugal effect, and Q is the amount of suspension culture solution supplied to the centrifuge per unit time. (Ml / min) and S is the sedimentation area (cm ² ) under the action of centrifugal force. ] It is driven by].
Suspension culture method according to any of the claims.